HRP920628A2 - Human granulocyte colony stimulating factor - Google Patents
Human granulocyte colony stimulating factor Download PDFInfo
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- HRP920628A2 HRP920628A2 HR920628A HRP920628A HRP920628A2 HR P920628 A2 HRP920628 A2 HR P920628A2 HR 920628 A HR920628 A HR 920628A HR P920628 A HRP920628 A HR P920628A HR P920628 A2 HRP920628 A2 HR P920628A2
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Description
Područje tehnike The field of technology
Izum je iz područja molekularne biologije. The invention is from the field of molecular biology.
Tehnički problem Technical problem
Ova izum odnosi se na faktor koji stimulira humanu koloniju granulocita. This invention relates to human granulocyte colony stimulating factor.
Točnije, ovaj izum se odnosi na kodirajući gen za polipeptid koji posjeduje aktivnost faktora stimuliranja kolonije (dalje označenu kao CSF) koji je specifični stimulirajući faktor potreban principjelno za stvaranje kolonija humanih granulocitnih stanica. Ovaj izum se također odnosi na rekombinantni vektor prisutan u spomenutom genu, transformant koji sadrži spomenuti vektor, polipeptid ili glikoprotein koji ima CSF aktivnost kada je dobiven iz spomenutog transformanta, te postupak za dobivanje polipeptida ili glikoproteina koji ima CSF aktivnost. More specifically, the present invention relates to a gene encoding a polypeptide possessing colony stimulating factor activity (hereinafter referred to as CSF) which is a specific stimulating factor required in principle for the formation of colonies of human granulocytic cells. This invention also relates to a recombinant vector present in said gene, a transformant containing said vector, a polypeptide or glycoprotein having CSF activity when obtained from said transformant, and a method for obtaining a polypeptide or glycoprotein having CSF activity.
Stanje tehnike State of the art
Kada se stanice koštane srži kao stanice mete i stanice bubrega ili stanice embrija kultiviraju metodom kultiviranja dvoslojnim mekim agarom, sa stanicama koštane srži u gornjem sloju i stanicama bubrega ili embrija u donjem sloju, dio stanica u gornjem sloju raste i diferencira se tako da stvara kolonije neutrofilnih granulocita (dalje jednostavno označenih kao granulociti) ili monocitne makrofage. Ovo opažanje navodi na pretpostavku prisutnosti in vivo faktora koji pomaže stvaranje kolonija /Pluznik i Sach, J. Cell. Comp. Physiol., 66, 319(1965); i Bradley i Metcalf, Aust. J. Exp. Biol. Med. Sco., 44, 287(1966)/. When bone marrow cells as target cells and kidney cells or embryo cells are cultured by the two-layer soft agar culture method, with bone marrow cells in the upper layer and kidney or embryo cells in the lower layer, some of the cells in the upper layer grow and differentiate to form colonies neutrophil granulocytes (further simply referred to as granulocytes) or monocyte macrophages. This observation suggests the presence of an in vivo factor that facilitates colony formation /Pluznik and Sach, J. Cell. Comp. Physiol., 66, 319(1965); and Bradley and Metcalf, Aust. J. Exp. Biol. Honey. Sco., 44, 287(1966)/.
Poznato je da ovi faktori, koji su zajednički označeni kao CSF mogu biti producirani stanicama, kao što su T-stanice, monocitni makrofagi, fibroblasti i endotelijske stanice koje su normalno široko rasprostranjene in vivo. CSF uključuje sljedeće podklase: granulocit-monocitni makrogaf-CSF (označen kao GM-CSF) koji djeluje na sustav stanice granulocita ili monocitne makrofage na takav način da stimulira rast takvih stem stanica i izaziva njihovu diferencijaciju tako da stvaraju kolonije granulocita ili monocitnih makrofaga; monocitni makrofag CSF (označen kao M-CSF) koji je principijelno sposoban stvarati kolonije monocitnih makrofaga; multipotentni CSF (označen kao multi-CSF) koji djeluje na slabo diferencirane multipotentne stem stanice; i granulocit CSF (označen kao G-CSF) tipa razmatranog ovim izumom koji je u načelu sposoban stvarati granulocitne kolonije. Nedavno je istaknuto da se stanja diferenciranja ciljanih stanica razlikuju od jedne podklase do druge /Asano, Taisha-Metabolism and Disease, 22, 249(1985); i Yunis et al., "Growth and Maturation Factors", izdavač Guroff, John Wiley and Sons, NY, vol. 1, 209(1983)/. It is known that these factors, collectively designated as CSF, can be produced by cells such as T-cells, monocyte macrophages, fibroblasts and endothelial cells that are normally widely distributed in vivo. CSF includes the following subclasses: granulocyte-monocyte macrophage-CSF (designated as GM-CSF) which acts on the granulocyte or monocyte macrophage cell system in such a way as to stimulate the growth of such stem cells and induce their differentiation to form colonies of granulocytes or monocyte macrophages; monocytic macrophage CSF (designated as M-CSF) which is in principle capable of forming monocytic macrophage colonies; multipotent CSF (denoted as multi-CSF) acting on poorly differentiated multipotent stem cells; and granulocyte CSF (designated as G-CSF) of the type contemplated by the present invention which is in principle capable of forming granulocyte colonies. It has recently been pointed out that the differentiation states of target cells differ from one subclass to another /Asano, Taisha-Metabolism and Disease, 22, 249(1985); and Yunis et al., "Growth and Maturation Factors", published by Guroff, John Wiley and Sons, NY, vol 1, 209(1983)/.
Stoga objašnjavanje pojedinih CSF podklasa i proučavanje njihovih kemijskih i bioloških svojstava je vrlo važno za proučavanje kematopoetskih mehanizama i analizu patomorfoloških aspekata raznih hematoloških bolesti. Biološka djelovanja G-CSF izazivaju povećanu pozornost istraživača zbog svoje sposobnosti izazivanja diferencijacije leukemijskih stanica koštane srži i pojačavanje funkcija zrelih granulocita, te potencijalno pruža mogućnost uspješne kliničke primjene u područjima tretiranja i prevencije leukemije. Therefore, the explanation of individual CSF subclasses and the study of their chemical and biological properties is very important for the study of hematopoietic mechanisms and the analysis of pathomorphological aspects of various hematological diseases. The biological actions of G-CSF are attracting increased attention from researchers due to its ability to induce the differentiation of leukemic bone marrow cells and enhance the functions of mature granulocytes, and potentially provide the possibility of successful clinical application in the areas of leukemia treatment and prevention.
Do sada su vršeni pokušaji za izoliranje i pročišćavanje G-CSF zasnovani na postupku kultiviranja stanice gdje je G-CSF izoliran uz supernatanta stanica kulture, ali homogeni G-CSF još nije proizveden u većim količinama jer se G-CSF može dobiti samo u malim koncentracijama i složenim postupcima pročišćavanja iz velikog volumena tekućine kulture dobivaju se samo tragovi G-CSF. Zato je poželjno usvojiti postupak rekombinantne DNA tehnologije za masovniju proizvodnju G-CSF. So far, attempts have been made to isolate and purify G-CSF based on a cell culture procedure where G-CSF is isolated from the culture cell supernatant, but homogeneous G-CSF has not yet been produced in large quantities because G-CSF can only be obtained in small concentrations. and complex purification procedures yield only traces of G-CSF from a large volume of culture fluid. That is why it is desirable to adopt the process of recombinant DNA technology for mass production of G-CSF.
Opis rješenja tehničkog problema s primjerima izvođenja Description of the solution to the technical problem with implementation examples
Predmet ovoga izuma je dobivanje kodirajućeg gena polipeptida koji ima humanu G-CSF aktivnost. The object of this invention is to obtain a coding gene for a polypeptide that has human G-CSF activity.
Sljedeći cilj ovoga izuma je dobivanje rekombinantnog vektora ugrađenog u spomenuti gen. The next goal of this invention is to obtain a recombinant vector incorporated in the mentioned gene.
Daljnji cilj ovoga izuma je dobivanje transformata koji se dobiva transformiranjem domaćina sa navedenim rekombinantnim vektorom, te polipeptida i glikoproteina koji se dobivaju pomoću spomenutog transformata. A further aim of this invention is to obtain a transformant which is obtained by transforming the host with the said recombinant vector, and polypeptides and glycoproteins which are obtained by means of said transformant.
Cilj ovoga izuma je i postavljanje postupka za dobivanje polipeptida ili glikoproteina koji ima humanu G-CSF aktivnost. The aim of this invention is to set up a procedure for obtaining a polypeptide or glycoprotein that has human G-CSF activity.
Slika 1 prikazuje nizove tri različite probe IWQ, A i LC; Figure 1 shows arrays of three different probes IWQ, A and LC;
Slika 2 prikazuje nukleotidni niz pHCS-1 dijela; Figure 2 shows the nucleotide sequence of the pHCS-1 portion;
Slika 3(A) prikazuje nukleotidni niz cDNA dijela u pBRG4; Figure 3(A) shows the nucleotide sequence of the cDNA portion in pBRG4;
Slika 3(B) prikazuje aminokiselinski niz humanog zrelog G-CSF izvedenog iz pBRG4 cDNA; Figure 3(B) shows the amino acid sequence of human mature G-CSF derived from pBRG4 cDNA;
Slika 4(A) prikazuje nukleotidni niz cDNA dijela u pBRV2; Figure 4(A) shows the nucleotide sequence of the cDNA portion in pBRV2;
Slika 4(B) I prikazuje aminokiselinski niz humanog G-CSF prethodnika izvedenog iz pBRV2 cDNA; Figure 4(B) I shows the amino acid sequence of the human G-CSF precursor derived from pBRV2 cDNA;
Slika 4(B) II prikazuje aminokiselinski niz humanog zrelog G-CSF izvedenog iz pBRV2 cDNA; Figure 4(B) II shows the amino acid sequence of human mature G-CSF derived from pBRV2 cDNA;
Slika 5 prikazuje nukleotidni niz humanog kromosomskog kodirajućeg gena za humani G-CSF; Figure 5 shows the nucleotide sequence of the human chromosomal coding gene for human G-CSF;
Slika 6 prikazuje restrikcijski enzim mjesta cijepanja pBRG4- ili pBRV2-izvedene humane G-CSF cDNA. Figure 6 shows the restriction enzyme cleavage site of pBRG4- or pBRV2-derived human G-CSF cDNA.
Slika 7 prikazuje restrikcijski enzim mjesta cijepanja humanog kromosomskog gena koji je kodirajući gen za humani G-CSF. Figure 7 shows the restriction enzyme cleavage site of the human chromosomal gene coding for human G-CSF.
Slika 8 je djelomično predstavljanje postupka za dobivanje tac promotora koji sadrži vektor (+VSE linija); Figure 8 is a partial representation of a procedure for obtaining a tac promoter containing vector (+VSE line);
Slika 9 je predstavljanje postupka za dobivanje PL promotora koji sadrži vektor (+VSE linija); Figure 9 is a representation of the procedure for obtaining a PL promoter containing a vector (+VSE line);
Slika 10 je predstavljanje postupka za dobivanje trp promotora koji sadrži vektor (+VSE linija); Figure 10 is a representation of the procedure for obtaining a trp promoter containing vector (+VSE line);
Slika 11 je djelomično predstavljanje postupka za dobivanje tac promotora koji sadrži vektor (-VSE linija); Figure 11 is a partial representation of a procedure for obtaining a tac promoter containing vector (-VSE line);
Slika 12 je predstavljanje postupka za dobivanje PL promotora koji sadrži vektor (-VSE linija); Figure 12 is a representation of the procedure for obtaining a PL promoter containing a vector (-VSE line);
Slika 13 je predstavljanje postupka za dobivanje trp promotora koji sadrži vektor (-VSE linija); Figure 13 is a representation of the procedure for obtaining a trp promoter containing vector (-VSE line);
Slika 14 prikazuje shematski strukturu pHGA410; Figure 14 shows the schematic structure of pHGA410;
Slika 15 je predstavljanje postupka za konstrukcijsku ekspresiju rekombinantnih vektora, pTN-G4, pTN-G4VA i pTN-α G4VAβ; Figure 15 is a representation of the procedure for the construct expression of the recombinant vectors, pTN-G4, pTN-G4VA and pTN-α G4VAβ;
Slike 16a i 16 b prikazuju dva postupka za nastajanje pHGG4-dhfr; Figures 16a and 16b show two procedures for the formation of pHGG4-dhfr;
Slika 16c prikazuje postupke za nastajanje PG4DR1 i pG4DR2; Figure 16c shows the procedures for generating PG4DR1 and pG4DR2;
Slika 17 prikazuje shematski strukturu pHGV2; Figure 17 shows the schematic structure of pHGV2;
Slika 18 je predstavljanje postupka za konstrukcijsku ekspresiju rekombinantnih vektora, pTN-V2, pTN-VAα i pTN-VAβ; Figure 18 is a representation of the procedure for the construct expression of the recombinant vectors, pTN-V2, pTN-VAα and pTN-VAβ;
Slike 19a i 19b prikazuju dva postupka za konstrukcijsku ekspresiju rekombinantnog vektora pHGV2-dhfr. Figures 19a and 19b show two procedures for constitutive expression of the pHGV2-dhfr recombinant vector.
Slika 19c prikazuje postupke za dobivanje pV2DR1 i pV2DR2; Figure 19c shows the procedures for obtaining pV2DR1 and pV2DR2;
Slika 20 prikazuje shematski strukturu pMLCE3α; Figure 20 shows the schematic structure of pMLCE3α;
Slika 21 shematski prikazuje strukturu pTNCE3α; Figure 21 schematically shows the structure of pTNCE3α;
Slika 22 shematski prikazuje strukture pD26SVCE3α i pDRCE3α. Figure 22 schematically shows the structures of pD26SVCE3α and pDRCE3α.
Kodirajući gen za polipeptid koji ima humanu G-CSF aktivnost prema ovome izumu je DNA (cDNA) koji je komplementaran s glasničkom RNA (mRNA) koji je dobiven kao 15-17 frakcija centrifugalnog gradijenta gustoće saharoze i koji kodira polipeptid koji ima humanu G-CSF aktivnost. The gene encoding a polypeptide having human G-CSF activity according to the present invention is a DNA (cDNA) that is complementary to a messenger RNA (mRNA) obtained as a 15-17 fraction of a centrifugal density gradient of sucrose and which encodes a polypeptide having human G-CSF activity.
Ovi izumitelji su dobili dvije linije cDNA. These inventors obtained two cDNA lines.
cDNA jedne linije ima sav cijeli ili dio kodirajućeg gena za polipeptid I ili II na slici 3(B). Točnije, ovaj cDNA ima nukleotidni niz ocrtan sa ATG na 32-34 nukleotidnim položajima od 5'-terminusa (vidi sliku 3(A)/ i CCC na 650-652 nukleotidnim položajima ili sa ACC na 122-124 položajima i CCC na 650-652 položajima. Alternativno, cDNA ima nukleotidni niz prikazan na slici 3(A) ili njegov dio. cDNA ove linije je ovdje niže označen kao cDNA (+VSE). The cDNA of one line has all or part of the gene coding for polypeptide I or II in Figure 3(B). Specifically, this cDNA has a nucleotide sequence delineated with ATG at 32-34 nucleotide positions from the 5'-terminus (see Figure 3(A)/ and CCC at 650-652 nucleotide positions or with ACC at 122-124 positions and CCC at 650- 652 positions. Alternatively, the cDNA has the nucleotide sequence shown in Figure 3(A) or a portion thereof. The cDNA of this line is designated below as cDNA (+VSE).
cDNA druge linije ima cijeli ili dio kodirajućeg gena za polipeptid I ili II prikazan na slici 4(B). Točnije, ovaj ima nukletidni niz odlikovan sa ATG na 31-33 nukleotidnim položajima od 5'-terminusa /vidi sliku 4(A)/ i sa CCC na 640-642 nukleotidnim položajima, ili sa ACC na 121-123 položajima i sa CCC na 640-642 položajima. Alternativno, ovaj cDNA može imati nukleotidni niz prikazan na slici 4(A) ili njegov dio. cDNA ove linije je ovdje niže označen kao cDNA (-VSE). The cDNA of the second strand has all or part of the gene coding for polypeptide I or II shown in Figure 4(B). More specifically, this has a nucleotide sequence characterized by ATG at 31-33 nucleotide positions from the 5'-terminus /see Figure 4(A)/ and with CCC at 640-642 nucleotide positions, or with ACC at 121-123 positions and with CCC at 640-642 positions. Alternatively, this cDNA may have the nucleotide sequence shown in Figure 4(A) or a portion thereof. The cDNA of this line is denoted below as cDNA (-VSE).
Gen opisan gore može biti dobiven sljedećom procedurom: mRNA kodiran G-CSF je prvo dobiven od animalni stanica sisavaca ili drugih stanica domaćina koji ima G-CSF aktivnost; mRNA je tada preveden u dvonitni cDNA nekim od poznatih postupaka; set rekombinanata koji sadrže ovaj cDNA (set je nadalje označen kao cDNA biblioteka) se zatim podvrgava prosijavanju poznatim postupcima. The gene described above can be obtained by the following procedure: mRNA encoding G-CSF is first obtained from mammalian animal cells or other host cells having G-CSF activity; The mRNA was then translated into double-stranded cDNA by known methods; a set of recombinants containing this cDNA (the set is further referred to as a cDNA library) is then subjected to screening by known methods.
Gen iz ovoga izuma također uključuje humani kromosomski kodirajući gen za polipeptid koji ima humanu G-CSF aktivnost. Ovaj humani kromosomski gen sadrži nukleotidni niz koji sudjeluje u transkripcijskoj kontroli i također sadrži cijeli ili dio nukleotidnog niza prikazanog na slici 5. The gene of the present invention also includes a human chromosomal gene encoding a polypeptide having human G-CSF activity. This human chromosomal gene contains a nucleotide sequence that participates in transcriptional control and also contains all or part of the nucleotide sequence shown in Figure 5.
Kromosomski gen može se tada dobiti pripremanjem iz humanih stanica rekombinanata koji sadrže humani kromosomski gen (set je dalje označen kao biblioteka humanog kromosomskog gena), a zatim podvrgavanjem spomenutog gena testiranjem pomoću poznatih postupaka. The chromosomal gene can then be obtained by preparing from human cells recombinants containing the human chromosomal gene (the set is hereinafter referred to as a human chromosomal gene library) and then subjecting said gene to testing using known methods.
Humani kromosomski gen može se dobiti iz bilo kojeg tipa humanih stanica takvih kao stanice ekstrahirane iz jetre ili bubrega ili kultiviranih stanica kao što su stanice tumora. Biblioteka humanog kromosomskog gena može se dobiti iz humanih stanica bilo kojim poznatim postupkom /vidi Maniatis et al., Cell, 15, 687(1978); i Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, str. 269 (1982)/, koji su ilustrirani niže: ekstrakt humanog kromosomskog DNA iz takvog izvora kao što su humana jetra embrija s fenolom ili drugi odgovarajućim kemikalijama; razaranje ekstrahirane DNA djelomično ili potpuno sa odgovarajućim restrikcijskim enzimom rako da se dobije DNA dio odgovarajuće dužine; ubacivanje DNA dijela u λ-fag vektora DNA fragmentu sa T4 ligazom ili drugim odgovarajućim ligazama, s vezivom koje sadrži restrikcijsko mjesto za odgovarajući enzim takav kao EcoRI koji biva po izboru pripojen; zatim, dobivanje λ-faga čestice postupkom pakiranja in vitro i transformiranje stanica domaćina takvih kao E. coli sa česticama dobivenih λ-faga. A human chromosomal gene can be obtained from any type of human cell such as cells extracted from the liver or kidney or cultured cells such as tumor cells. A human chromosomal gene library can be obtained from human cells by any known method / see Maniatis et al., Cell, 15, 687 (1978); and Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, p. 269 (1982)/, which are illustrated below: extract human chromosomal DNA from such a source as human embryonic liver with phenol or other appropriate chemicals; destruction of the extracted DNA partially or completely with a suitable restriction enzyme to obtain a DNA fragment of the appropriate length; inserting the DNA portion into the λ-phage vector DNA fragment with T4 ligase or other suitable ligases, with a linker containing a restriction site for a suitable enzyme such as EcoRI which is optionally attached; then, obtaining λ-phage particles by an in vitro packaging process and transforming host cells such as E. coli with the obtained λ-phage particles.
Primjeri λ-faga primjenjivi kao vektor u gornjim postupcima uključuju Charon 4A i EMBL-3 i EMBL-4. Examples of λ-phages applicable as a vector in the above methods include Charon 4A and EMBL-3 and EMBL-4.
Stanica sisavca koja može biti upotrebljena kao izvor mRNA opskrbe je humana Ca usne šupljine izvedena stanica vrste, CHU-2 (pohranjena u Collection Nationale de Cultures de Mikroorganismes, ili C.N.C.M., pod brojem I-483). treba međutim istaknuti da se umjesto ovih stanica tumora, mogu koristiti i bilo koje druge stanice vrsta izdvojenih iz sisavaca. Dobivanje mRNA može se izvesti pomoću bilo koje od poznatih metoda za kloniranje gena drugih fiziološki aktivnih proteina; na primjer, cijela RNA se prvo dobiva tretiranjem sa površinski aktivnom tvari i fenolom u prisutnosti ribonuklidnog inhibitora takvog kao vanadilribonukleozid kompleks /vidi Berger i BirkenMeier, Biochemistry, 18, 5143(1979)/ ili CsCl gradijentom gustoće centrifugiranja koji je praćen tretmanom sa guanidin tiocijanatom /vidi Chirgwm et al., Biochemistry, 18, 5294(1979)/, tada se dobiva poli(A+)RNA (mRNA) podvrgavanjem cijele RNA stupnjevitoj adsorpciji ili utjecaju kromatografije na koloni na oligo(dT)-celulozi ili poli-saharozi 2B korištenih kao nosač. Poli(A+) može dalje biti frakcioniran nekim odgovarajućim postupkom. A mammalian cell that can be used as a source of mRNA supply is a human Ca oral cavity derived cell, CHU-2 (deposited in the Collection Nationale de Cultures de Mikroorganismes, or C.N.C.M., under number I-483). however, it should be pointed out that instead of these tumor cells, any other cells of the species isolated from mammals can be used. Obtaining mRNA can be performed using any of the known methods for gene cloning of other physiologically active proteins; for example, whole RNA is first obtained by treatment with surfactant and phenol in the presence of a ribonuclide inhibitor such as vanadylribonucleoside complex (see Berger and BirkenMeier, Biochemistry, 18, 5143(1979)) or by CsCl density gradient centrifugation followed by treatment with guanidine thiocyanate /see Chirgwm et al., Biochemistry, 18, 5294(1979)/, poly(A+)RNA (mRNA) is then obtained by subjecting the entire RNA to stepwise adsorption or column chromatography on oligo(dT)-cellulose or poly-sucrose 2B used as a carrier. Poly(A+) can be further fractionated by some suitable procedure.
Sposobnost tako dobivene mRNA za kodiranje za polipeptid koji ima G-CSF aktivnost može biti potvrđena s više metoda; na primjer; mRNA se prevodi u protein i ispituju se njegove fiziološke aktivnosti; alternativno, identitet ovoga proteina određen je pomoću anti-G-CSF antitijela. Određenije, mRNA se uvodi u ocite Xenopus laevis-a radi vršenja translacije /vidi Gurdon et al., Nature, 233, 117(1972)/, ili translacijske se mogu izvesti sa retikulocitima zeca ili klicama pšenice /Schelif i Wensink, "Practical Methods m Molecular Biology", Springer-Verlag, NY (1981)/. G-CSF aktivnost može biti ispitana primjenom metode kultiviranja na mekom agaru korištenjem stanica koštane srži, i postupaka za izvođenje ove metode, što je opisano /Metcalf, "Hemopoietic Colonies", Springer-Verlag Berlin, Heidelberg, NY (1977)/. The ability of the thus obtained mRNA to code for a polypeptide having G-CSF activity can be confirmed by several methods; for example; mRNA is translated into protein and its physiological activities are examined; alternatively, the identity of this protein was determined using an anti-G-CSF antibody. More specifically, mRNA is introduced into Xenopus laevis eyes for translation (see Gurdon et al., Nature, 233, 117(1972)), or translation can be performed with rabbit reticulocytes or wheat germ (Schelif and Wensink, "Practical Methods in Molecular Biology", Springer-Verlag, NY (1981)/. G-CSF activity can be assayed using the soft agar culture method using bone marrow cells, and procedures for performing this method, as described /Metcalf, "Hemopoietic Colonies", Springer-Verlag Berlin, Heidelberg, NY (1977)/.
Jednostruka cDNA sintetizirana s tako dobivenom mRNA koristi se kao šablona; jednostruka cDNA je sintetizirana iz ove jednostruke cDNA; i jednostruka cDNA se uvodi u odgovarajući vektor DNA tako da stvara rekombinantni plazmid. Ovaj rekombmantni plazmid može biti upotrebljen za transformaciju pogodnog domaćina, recimo Escherichia coli, tako da se dobiva grupa DNAs-a u transformantima (cDNA biblioteka). A single-stranded cDNA synthesized with the thus obtained mRNA is used as a template; single-stranded cDNA is synthesized from this single-stranded cDNA; and the single-stranded cDNA is introduced into the appropriate DNA vector to generate a recombinant plasmid. This recombinant plasmid can be used to transform a suitable host, for example Escherichia coli, so that a group of DNAs in transformants (cDNA library) is obtained.
Dvostruka cDNA može se dobiti iz mRNA jednim od sljedeća dva postupka: mRNA se tretira sa reverznom transkriptazom sa oligo (dT) koje je komplementama sa poli(A)-lancem na 3'-terminusu uzetim kao primjer; ili oligonukleotidom koji odgovara dijelu aminokiselinskog niza G-CSF koji se sintetizira, a CDNA koja je komplementarna sa mRNA se sintetizira tretiranjem sa reverznom transkriptazom sa sintetiziranim oligonukleotidom korištenim kao inicijator. Dvostruka cDNA može se također dobiti sljedećim postupcima: mRNA se razlaže i uklanja tretiranjem s bazom i dobivena jednostruka cDNA se tretira prvo sa reverznom transkriptazom ili DNA polimerazom I (npr. Klenow fragment), zatim sa S1 nukleazom; alternativno, mRNA može biti izravno tretirana sa RNAse H i DNA polimerazom (npr. E. coli polimeraza I). Za više informacija pogledati Maniatis et al., "Molecular Cloning", Cold Pring Harbor Laboratoiy (1982); i Guber i Hoffman, Gene, 25, 263(1983). Double-stranded cDNA can be obtained from mRNA by one of the following two procedures: the mRNA is treated with reverse transcriptase with an oligo (dT) that is complementary to the poly(A)-strand at the 3'-terminus taken as an example; or an oligonucleotide corresponding to the part of the amino acid sequence of G-CSF that is synthesized, and a cDNA that is complementary to the mRNA is synthesized by treatment with reverse transcriptase with the synthesized oligonucleotide used as an initiator. Double-stranded cDNA can also be obtained by the following procedures: mRNA is degraded and removed by base treatment and the obtained single-stranded cDNA is treated first with reverse transcriptase or DNA polymerase I (eg Klenow fragment), then with S1 nuclease; alternatively, mRNA can be directly treated with RNAse H and DNA polymerase (eg, E. coli polymerase I). For more information see Maniatis et al., "Molecular Cloning", Cold Spring Harbor Laboratory (1982); and Guber and Hoffman, Gene, 25, 263(1983).
Tako dobivena dvostruka cDNA se uvodi u odgovarajući vektor takav kao, primjerice, jedan od EK-tipa plazmid vektora označenih kao pSC101, pDF41, Co1E1, pMB9, pBR322, pBR327 i pACYC1 ili jedan od faga vektora označenih sa λgt, λc, λgt10 i λgtWES, a zatim, rekombinantni vektor se koristi da transformira vrstu E. coli (npr. X1776, HB101, DH1 ili C600) tako da se dobije biblioteka cDNA (vidi, primjerice, "Molecular Cloning", također). The double cDNA thus obtained is introduced into a suitable vector such as, for example, one of the EK-type plasmid vectors designated as pSC101, pDF41, Co1E1, pMB9, pBR322, pBR327 and pACYC1 or one of the phage vectors designated as λgt, λc, λgt10 and λgtWES , and then the recombinant vector is used to transform the E. coli strain (eg, X1776, HB101, DH1 or C600) to obtain a cDNA library (see, for example, "Molecular Cloning", also).
Dvostruka cDNA može biti pripojena vektoru sljedećim postupcima: terminus cDNA-a je opskrbljen pripojivim krajem pripajanjem odgovarajućeg kemijski sintetiziranog DNA fragmenta; vektor DNA podijeljen sa ograničavajućim enzimom se pripaja spomenutoj cDNA tretiranjem sa T4 fagom DNA ligaze u prisutnosti ATP. Alternativno, dG-lanci (ili dT, dA-lanci) se pripajaju, respektivno dvostrukoj cDNA i vektor DNA koji je prekinut sa restrikcijskim enzimom i prekaljena je otopina koja sadrži obje DNA (vidi "Molecular Cloning", isto). The double-stranded cDNA can be attached to the vector by the following procedures: the terminus of the cDNA is provided with an attachable end by attaching the appropriate chemically synthesized DNA fragment; vector DNA cleaved with a restriction enzyme is joined to said cDNA by treatment with T4 phage DNA ligase in the presence of ATP. Alternatively, the dG-strands (or dT, dA-strands) are ligated, respectively, to a double-stranded cDNA and vector DNA that has been cut with a restriction enzyme and quenched in a solution containing both DNAs (see "Molecular Cloning", ibid.).
Stanica domaćina može biti prevedena tako dobivenom rekombinantom DNA pomoću neke od poznatih metoda. Ako je stanica domaćina E. coli, može se primijeniti postupak koji je opisao Hanahan (J. Mol. Biol. 166, 557(1983)/, tako što se rekombinant DNA dodaje u odgovarajuću stanicu dobivenu u prisutnosti CaCl2, MgCl2 ili RbCl. The host cell can be transduced with the thus obtained recombinant DNA using any of the known methods. If the host cell is E. coli, the procedure described by Hanahan (J. Mol. Biol. 166, 557(1983)/) can be applied by adding the recombinant DNA to the appropriate cell obtained in the presence of CaCl 2 , MgCl 2 or RbCl.
Testiranje za stanice koje uzgajaju željene gene može se izvesti pomoću više metoda koje uključuju: plus-minus metodu primjenjivu u klonirajućem interferonu cDNA /Taniguchi et al., Proc. Jpn. Acad., 55, Ser. B, 464(1979)/, metodu ispitivanja hibridizacije-translacije /Nagata et al., Nature, 284, 316 (1980)/ i kolonija ili metoda hibiridizacijskog zaražavanja koja koristi oligonukleotidni uzorak sintetiziran kemijski na osnovi aminokiselinskog niza proteina koji ima humanu G-CSF aktivnost /Wallace et al., Nucleic Acids, Res., 9, 879(1981)/; i Benton & Davis, Science, 196, 180(1970)/. Testing for cells expressing the desired genes can be performed using a number of methods including: the plus-minus method applicable in cloning interferon cDNA /Taniguchi et al., Proc. Jpn. Acad., 55, Ser. B, 464(1979)/, the hybridization-translation test method /Nagata et al., Nature, 284, 316 (1980)/ and the colony or hybridization infection method using an oligonucleotide template synthesized chemically on the basis of the amino acid sequence of a protein having a human G- CSF activity /Wallace et al., Nucleic Acids, Res., 9, 879(1981)/; and Benton & Davis, Science, 196, 180(1970)/.
Fragment koji uzgaja tako kloniran kodirajući gen za polipeptid ima humanu G-CSF aktivnost može biti ponovo ubačen u odgovarajući DNA vektor za svrhu prevođenja drugih prokariotskih ili eukariotskih stanica domaćina. Uvođenjem odgovarajućeg promotora i izraza povezanog niza u vektor, gen može biti izražen u individualnoj stanici domaćina. The fragment growing the gene encoding the polypeptide thus cloned has human G-CSF activity can be reintroduced into a suitable DNA vector for the purpose of translation in other prokaryotic or eukaryotic host cells. By introducing the appropriate promoter and associated expression sequence into the vector, the gene can be expressed in an individual host cell.
Ilustrativne prokariotske stanice domaćina uključuju Escherichia coli, Bacillus subtillis i Bacillus thermophilus. Gen od interesa može biti izražen u ovim stanicama domaćina transformirajući ih sa replikonom (npr. plazmid vektor koji uzgaja početak i regulator niza) koji se izvodi iz domaćinu kompatibilnih vrsta. Poželjni vektor je onaj koji ima niz sposoban za transformiranje stanice sa selektivnošću za izraženu osobnost (fenotip). Illustrative prokaryotic host cells include Escherichia coli, Bacillus subtillis, and Bacillus thermophilus. The gene of interest can be expressed in these host cells by transforming them with a replicon (eg, a plasmid vector that grows the primer and a sequence regulator) derived from a host-compatible species. A preferred vector is one that has a sequence capable of transforming a cell with selectivity for an expressed personality (phenotype).
Na primjer, E. coli može biti prevedeno sa pBR322 koji je vektor sposoban za kopiranje E. coli /vidi Bolivar, Gene 2, 95(1975)/. Ovaj vektor sadrži oba ampicilin- i tetraciklin-otporne gene čije se osobine mogu iskoristiti za identifikaciju transformirane stanice. For example, E. coli can be transformed with pBR322 which is a vector capable of replicating E. coli (see Bolivar, Gene 2, 95(1975)). This vector contains both ampicillin- and tetracycline-resistance genes whose properties can be used to identify the transformed cell.
Primjeri promotora koji su neophodni za genetsko izražavanje u prtokariotskim domaćinima uključuju promotor δ-laktoze gen /Chang et al., Nature, 275, 615(1978)/, laktoza promotor /vidi Goeddel et aL, Nature, 281, 544(1979)/ i triptofan promotor /vidi Goeddel et aL, Nucleic Acid Res., 8, 4057(1980)7 i tako dalje. Bilo koji od ovih promotora može biti upotrebljen u produkciji polipeptida koji ima humanu G-CSF aktivnost prema ovome izumu. Examples of promoters that are essential for gene expression in prokaryotic hosts include the δ-lactose gene promoter /Chang et al., Nature, 275, 615(1978)/, lactose promoter /see Goeddel et al, Nature, 281, 544(1979)/ and the tryptophan promoter /see Goeddel et al, Nucleic Acid Res., 8, 4057(1980)7 et seq. Any of these promoters can be used in the production of polypeptides having human G-CSF activity according to the present invention.
Eukariotski mikroorganizam takav kao Saccharomyces cerevisiae može biti upotrijebljen kao stanica domaćin i transformiran vektorom takvim kao što je plazmid YRp7 /vidi Stinchcomb et al., Nature, 282, 39(1979)/. Ovaj plazmid ima TRP1 gen kao selekcijski obilježivač za vrste kvasca koje posjeduju sposobnost produkcije triptofana, tako tranformanti mogu biti odabrani izrastanjem u odsutnosti triptofana. Primjeri promotora koji mogu biti korišteni za ekspresiju gena uključuju kiseli fosfatazni gen promotor /Miyanohara et al., Proc. Natl. Acad. Sci., USA, 80, 1(1983)/ i alkohol dehidrogenazni gen promotor /Valenzuela et al., Nature, 298, 347(1982)/. A eukaryotic microorganism such as Saccharomyces cerevisiae can be used as a host cell and transformed with a vector such as the YRp7 plasmid (see Stinchcomb et al., Nature, 282, 39(1979)). This plasmid has the TRP1 gene as a selection marker for yeast species capable of producing tryptophan, so transformants can be selected by growth in the absence of tryptophan. Examples of promoters that can be used for gene expression include the acid phosphatase gene promoter /Miyanohara et al., Proc. Natl. Acad. Sci., USA, 80, 1(1983)/ and alcohol dehydrogenase gene promoter /Valenzuela et al., Nature, 298, 347(1982)/.
Stanica domaćin također može biti izvedena iz stanica sisavaca takvih kao COS stanice, stanice jajnika kineskog hrčka (CHO), C-127 stanica i Hela stanica. Ilustrativni vektor koji može biti upotrijebljen radi transformiranja ovih stanica je pSV2-gpr /vidi Mulligan i Berg; Proc. Natl. Acad. Sci., USA, 78, 2072(1981)/. Vektori korišteni za transformiranje ovih stanica sadrže početak, selekcijski obilježivač, promotor koji prethodi u položaju gdje će gen biti izražen, TNA koji spaja mjesto, poliadenilacijski signal itd. The host cell can also be derived from mammalian cells such as COS cells, Chinese Hamster Ovary (CHO) cells, C-127 cells and Hela cells. An illustrative vector that can be used to transform these cells is pSV2-gpr /see Mulligan and Berg; Proc. Natl. Acad. Sci., USA, 78, 2072(1981)/. The vectors used to transform these cells contain a primer, a selectable marker, a promoter preceding the position where the gene will be expressed, a TNA joining site, a polyadenylation signal, etc.
Ilustrativni promotori koji se mogu koristiti za ekspresiju gena u stanicama sisavaca uključuju promotore retrovirusa, polyoma virus, adenovirus, simian virus 40 (SV40), itd. Ako se koristi promotor SV40, željena ekspresija gena može biti lako postignuta prema metodi Mulligan et al. opisanoj u Nature, 277, 108(1979). Illustrative promoters that can be used for gene expression in mammalian cells include retrovirus promoters, polyoma virus, adenovirus, simian virus 40 (SV40), etc. If the SV40 promoter is used, the desired gene expression can be easily achieved according to the method of Mulligan et al. described in Nature, 277, 108(1979).
Ilustrativni počeci koji mogu biti korišteni uključuju one izvedene iz SV40, polyoma virus, adenovirus, papiloma virus goveda (BVP), itd. Ilustrativni selekcijski obilježivač koji može biti korišten uključuje fosfotransferazu APH (3') II ili I (neo) gen, timidin kinazu (TK) gen, E. coli ksantin guanin fosforiboziltransferazu (Ecogpt) gen, dihidrofolat reduktazu (DHFR) gen, itd. Illustrative primers that may be used include those derived from SV40, polyoma virus, adenovirus, bovine papilloma virus (BVP), etc. Illustrative selectable markers that may be used include the phosphotransferase APH (3') II or I (neo) gene, thymidine kinase (TK) gene, E. coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase (DHFR) gene, etc.
U cilju dobivanja polipeptida koji ima humanu G-CSF aktivnost iz gore navedenih domaćin-vektor sustava, mogu se koristiti sljedeći postupci: kodirajući gen za peptid koji ima humanu G-CSF aktivnost se ubacuje na pogodno mjesto u jedan od vektora spomenutih naprijed; stanica domaćina se transformira uz dobivanje rekombinanta DNA; i dobiveni transformati se kultiviraju. Željeni polipeptid može biti izoliran i pročišćen iz stanice ili otopine kulture bilo kojom od poznatih tehnika. In order to obtain a polypeptide having human G-CSF activity from the host-vector systems mentioned above, the following procedures can be used: the coding gene for a peptide having human G-CSF activity is inserted into a suitable place in one of the vectors mentioned above; the host cell is transformed to obtain recombinant DNA; and the obtained transformants are cultivated. The desired polypeptide can be isolated and purified from the cell or culture solution by any of the known techniques.
Eukariotski geni se općenito smatra da pokazuju polimorfizam kao što je poznato za slučaj humanog interferon gena /vidi Nichi et al., J. Biochem., 97, 153(1985)/. I ovaj fenomen može izazvati supstituciju jedne ili više aminokiselina u nukleotidnom nizu ali ne i promjenu u cijelom aminokiselinskom nizu. Eukaryotic genes are generally considered to exhibit polymorphism as is known for the case of the human interferon gene (see Nichi et al., J. Biochem., 97, 153(1985)). And this phenomenon can cause the substitution of one or more amino acids in the nucleotide sequence, but not a change in the entire amino acid sequence.
G-CSF aktivnost može posjedovati također i polipeptid kojemu nedostaje jedna ili više aminokiselina u aminokiselinskom nizu na slikama 3(B) ili 4(B) ili onaj koji ima dodane takve aminokiseline, ili polipeptid koji ima jednu ili više ovih kiselina zamijenjenih sa jednom ili više aminokiselina. Također je poznato da polipeptid dobiven prevođenjem svakog od cistein kodona u humani interleukin-2 (IL-2) gen u serin kodon ima aktivnost interleukina-2 /Wang et al., Science, 224, 1431(1984)/. Stoga, sve dok polipeptidi bilo prirodni ili kemijski sintetizirani imaju humanu G-CSF aktivnost, svi geni koji su kodirajući za ove polipeptide, rekombinantni vektori koji sadrže ove gene, transformate dobivene pomoću takvih rekombinantnih vektora, i polipeptidi ili glikoproteini koji su dobiveni kultiviranjem takvih transformanata ulazi u obim ovoga izuma. G-CSF activity may also be possessed by a polypeptide that is missing one or more amino acids in the amino acid sequence of Figures 3(B) or 4(B) or that has such amino acids added, or a polypeptide that has one or more of these acids replaced by one or more amino acids. It is also known that the polypeptide obtained by translating each of the cysteine codons in the human interleukin-2 (IL-2) gene to a serine codon has interleukin-2 activity /Wang et al., Science, 224, 1431(1984)/. Therefore, as long as the polypeptides, whether natural or chemically synthesized, have human G-CSF activity, all genes encoding these polypeptides, recombinant vectors containing these genes, transformants obtained using such recombinant vectors, and polypeptides or glycoproteins obtained by culturing such transformants falls within the scope of this invention.
Niže su dani postupci za dobivanje gena ovoga izuma kodirajućih za polipeptid koji ima humanu G-CSF aktivnost, rekombinantni vektor koji ima spomenuti gen i transformant koji ima ovaj rekombinantni vektor, i polipeptid ili glikoprotein koji ima humanu G-CSF aktivnost izraženu u ovom transformantu. Below are the procedures for obtaining the gene of this invention coding for a polypeptide having human G-CSF activity, a recombinant vector having said gene and a transformant having this recombinant vector, and a polypeptide or glycoprotein having human G-CSF activity expressed in this transformant.
(1) Priprema probe (1) Preparation of rehearsal
Homogeni humani CSF protein je pročišćen iz supernatanta kulture stanice tumora linije CHU-2 i određen je njegov aminokiselinski niz od N terminusa. Fragmenti su dobiveni razlaganjem sa bromcijanom i tretmanom sa tripsinom i određeni su aminokiselinski nizovi ovih fragmenata (primjer 3(i), (ii) i (iii)/. Homogeneous human CSF protein was purified from the culture supernatant of the tumor cell line CHU-2 and its amino acid sequence from the N terminus was determined. The fragments were obtained by digestion with cyanide bromide and treatment with trypsin, and the amino acid sequences of these fragments were determined (example 3(i), (ii) and (iii)/.
Od određenih aminokiselinskih nizova, sintetizirane su tri nukleotidne probe, (A), (LC) i (IWQ), koje imaju nizove prikazane na slici (1) (primjer 4). Proba (A) bila je od smjese tipa sastavljenog od 14 sukcesivnih nukleotida. Proba (IWQ) je sastavljena od 30 sukcesivnih nukletida sa deoksinozinom i bila je proba tipa korištenog u kloniranju humanog kolcistokinin gen /Takahashi et al., Proc. Natl. Acad. Sci, USA 82, 1931(1985)/. Proba (LC) bila je 24-nukleotidna proba koja je sintetizirana iz nukleotida na 32-39 položajima od N terminusa aminokiselinskog niza prikazanog u primjeru 3(i) na osnovu nukleotidnog niza prikazanog na slici 3. From the specified amino acid sequences, three nucleotide probes, (A), (LC) and (IWQ), having the sequences shown in Figure (1) (Example 4), were synthesized. Sample (A) was of the type mixture composed of 14 successive nucleotides. The probe (IWQ) was composed of 30 successive nucleotides with deoxynosine and was a probe of the type used in the cloning of the human cholecystokinin gene /Takahashi et al., Proc. Natl. Acad. Sci, USA 82, 1931(1985)/. The probe (LC) was a 24-nucleotide probe that was synthesized from nucleotides at positions 32-39 from the N terminus of the amino acid sequence shown in Example 3(i) based on the nucleotide sequence shown in Figure 3.
Kemijske sinteze nukleotida mogu se vršiti primjenom poboljšane fpsfortriester metode u krutoj fazi koju je opisao Narang /Tetrahedron, 39, 3-22(1983)/. Chemical syntheses of nucleotides can be performed using the improved fpsfortriester method in the solid phase described by Narang /Tetrahedron, 39, 3-22 (1983)/.
Probe zasnovane na aminokiselinskim nizovima na položajima različitim od onih u naprijed navedenim probama također se mogu koristiti. Probes based on amino acid sequences at positions other than those in the above probes can also be used.
(2) Stvaranje cDNA biblioteke (2) Creation of cDNA library
CHU-2 stanice su homogenizirane nakon dodatka otopine guanidin tiocijanata i ukupne RNA dobivene pomoću CsCl centrifugirajućeg gradijenta gustoće. CHU-2 cells were homogenized after addition of guanidine thiocyanate solution and total RNA obtained by CsCl density gradient centrifugation.
Poli(A+) RNA je izolirana iz ukupne RNA kromatografijom na koloni od oligo (dT)-celuloze. Zatim je sintetizirana jednostruka cDNA sa reverznom transkriptazom, i RNAza H i E. coli DNA polimeraze I su dodani tako da se dobije dvostruka. dC lanac je pripojen tako dobivenoj dvostrukoj cDNA, koja je pripojena vektoru, pBR322, na je pripojen lanac dG na Pst I mjesto pripajanja. Dobiveni rekombinant DNA je upotrebljen za transformiranje vrste E. coli, XI776, i stvorena je pBR322-linija cDNA biblioteke (primjeri 5 i 6). Poly(A+) RNA was isolated from total RNA by chromatography on an oligo (dT)-cellulose column. A single-stranded cDNA was then synthesized with reverse transcriptase, and both RNAse H and E. coli DNA polymerase I were added to form a double-stranded cDNA. The dC chain was joined to the thus obtained double cDNA, which was inserted into the vector, pBR322, and the dG chain was joined to the Pst I splice site. The obtained recombinant DNA was used to transform the E. coli strain, XI776, and the pBR322 cDNA library line was created (examples 5 and 6).
Na sličan način, dvostruka cDNA je pripojena na λgt10 vektor sa EcoRI vezivom i stvorena je λ-fag linija cDNA biblioteke (primjer 7). In a similar manner, the double-stranded cDNA was ligated to the λgt10 vector with an EcoRI linker and a λ-phage cDNA library line was generated (Example 7).
(3) Testiranje (3) Testing
Rekombinanti izvedeni iz pBR322-linije cDNA biblioteke su fiksirani na Whatman 541 filtar papiru i jednostruki klon može biti izdvojen pomoću hibridizacije kolonije sa 32P-obilježenom probom (IWQ). Daljnje proučavanje sa Southern upijajućom metodom /Southern, J. Mol. Biol, 98, 503(1975)/ pokazuje da je ovaj klon također hidrolizirao sa probom (A). Nukleotidni niz ovoga klona je određen pomoću dideoksi metode /Sanger, Science, 214, 1205(1981)/. Recombinants derived from the pBR322-line cDNA library were fixed on Whatman 541 filter paper and a single clone could be isolated by colony hybridization with a 32P-labeled probe (IWQ). Further study with the Southern blotting method /Southern, J. Mol. Biol, 98, 503(1975)/ shows that this clone also hydrolyzed with probe (A). The nucleotide sequence of this clone was determined using the dideoxy method /Sanger, Science, 214, 1205(1981)/.
Nukleotidni niz dobivenog cDNA dijela je prikazan na slici 2, iz koje se može vidjeti da ovaj dio sadrži 308 osnovnih parova koji uključuju probe (IWQ) i (A), i ima otvoreni čitajući okvir kodirajući za 83 aminokiselina koje sadrže aminokiselinski niz prikazan u primjeru 3(iii). pBR322 izvedeni plazmid koji sadrži 308 osnovnih parova niže je naznačen kao pHCS-1 (primjer 8). The nucleotide sequence of the resulting cDNA fragment is shown in Figure 2, from which it can be seen that this fragment contains 308 base pairs including probes (IWQ) and (A), and has an open reading frame coding for 83 amino acids containing the amino acid sequence shown in the example 3(iii). The pBR322 derived plasmid containing 308 base pairs is designated below as pHCS-1 (Example 8).
DNA fragment koji sadrži 308 osnovnih parova dobiven iz pHCS-1 je radioaktivno obilježen pomoću metode translatornog ureza (vidi Molecular Cloning, ibid) i ovaj fragment je korišten kao proba, λgt10-izvedena cDNA biblioteka je prosijana pomoću zarazne hibridizacije /Benton i Davis, Science, 196, 180(1977)/ tako da se dobije 5 klonova. Nukleotidni niz klona za kojega se vjeruje da sadrži cDNA je određen pomoću iste metode kao što je opisano naprijed /(Slika 3(A)/. A DNA fragment containing 308 base pairs obtained from pHCS-1 was radiolabeled using the translational nick method (see Molecular Cloning, ibid) and this fragment was used as a probe, a λgt10-derived cDNA library was screened by infectious hybridization /Benton and Davis, Science , 196, 180(1977)/ so that 5 clones are obtained. The nucleotide sequence of the clone believed to contain cDNA was determined using the same method as described above /(Figure 3(A)/).
Kao što je pokazano u slici 3(A), ovaj cDNA dio ima jednostruki široko otvoren čitajući okvir. As shown in Figure 3(A), this cDNA portion has a single wide open reading frame.
Aminokiselinski niz kodiran ovim cDNA može se prikazati kao na slici 3(A). The amino acid sequence encoded by this cDNA can be shown as in Figure 3(A).
Uspoređivanje sa N-terminalnim aminokiselinskim nizom G-CSF proteina prikazanog u primjeru 3(i) pokazuje da ovaj cDNA sadrži nukleotidni niz koji odgovara signalnom peptidu kodiranom pomoću 90 osnovnih polaznih parova sa ATG nizom na 32-34 nukleotidnim položajima od 5'-terminusa i završenim sa GCC nizom na 119-121 položajima, i zrelom G-CSF polipeptidu kodiranom pomoću 531 osnovna polazna para sa ACC nizom na 122-124 položajima i koji se završava CCC nizom na 650-652 položajima. Stoga, polipeptid aminokiselinskog niza I prikazanog na slici 3(B) je sastavljen od 207 aminokiselina i izračunata mu je molekularna masa kao 22292,67 daltona. Polipeptid aminokisleinskog niza II je sastavljen od 177 aminokiselina i izračunata mu je molekularna masa kao 18986,74 daltona (primjer 9). Comparison with the N-terminal amino acid sequence of the G-CSF protein shown in Example 3(i) shows that this cDNA contains a nucleotide sequence corresponding to a signal peptide encoded by 90 base pairs with an ATG sequence at 32-34 nucleotide positions from the 5'-terminus and ending with a GCC sequence at positions 119-121, and a mature G-CSF polypeptide encoded by a 531 base pair primer with an ACC sequence at positions 122-124 and ending with a CCC sequence at positions 650-652. Therefore, the polypeptide of amino acid sequence I shown in Figure 3(B) is composed of 207 amino acids and has a calculated molecular weight of 22292.67 daltons. Amino acid sequence II polypeptide is composed of 177 amino acids and has a calculated molecular weight of 18986.74 daltons (Example 9).
Treba istaknuti da ATG na 32-34 položajima ili na 68-70 položajima može također biti razmatrana kao početno mjesto proteina. Escherishia coli vrsta X1776 koja uzgaja pBR322 koji ima cDNA + (VSE) na EcoRI mjestu cijepanja je pohranjen u "Fermentation Research Institute, Agency of Industrial Science and Technology" (FERM BP.954). It should be noted that ATG at positions 32-34 or at positions 68-70 can also be considered as the starting site of the protein. Escherichia coli strain X1776 growing pBR322 having a cDNA + (VSE) at the EcoRI cleavage site is deposited in the "Fermentation Research Institute, Agency of Industrial Science and Technology" (FERM BP.954).
Slika 6 prikazuje mjesta cijepanja resktrikcijskog enzima gena. Figure 6 shows the restriction enzyme cleavage sites of the gene.
Ova cDNA je pripojena na pBR327 /Sorberon et al., Gene, 9, 287(1980)/ na EcoRI položaju i dobiveni plazmid je niže označen kao pBRG4. This cDNA was ligated to pBR327 /Sorberon et al., Gene, 9, 287(1980)/ at the EcoRI site and the resulting plasmid is designated below as pBRG4.
Tako dobiveni pBRG4 je tretiran sa restrikcijskim enzimom, EcoRI, tako da se dobiva DNA fragment koji sadrži cDNA od oko 1500 osnovnih parova. Ovaj fragment je radioizotopski obilježen pomoću metode translatornog ureza (vidi Molecular Cloning, također) i, sa ovim radioizotopski obilježenim DNA fragmentom korištenim kao proba, λgt10-izvedena biblioteka je testirana ponovo pomoću hibridizacije zaraze (vidi također Benton i Darvis). U ovoj hibridizaciji zaraze, pripremljene su dvije trake nitroceluloznog papira na kojima je fiksirana λ-faga DNA; jedna od ovih traka je upotrebljena za gore spomenutu hibridizaciju zaraze, a druga je podvrgnuta hibridizaciji zaraze sa već opisanom probom (LC). Odabrane su fage koje su bile pozitivne za obje probe. Klon koji ima "punu dužinu" cDNA je odabran i nukleotidni niz dijela cDNA koji je određen dideoksi metodom je prikazan na slici 4(A). pBRG4 thus obtained was treated with the restriction enzyme, EcoRI, so that a DNA fragment containing a cDNA of about 1500 base pairs is obtained. This fragment was radiolabeled using the translational incision method (see Molecular Cloning, also) and, with this radiolabeled DNA fragment used as a probe, the λgt10-derived library was tested again by infection hybridization (see also Benton and Darvis). In this infection hybridization, two strips of nitrocellulose paper were prepared on which λ-phage DNA was fixed; one of these strips was used for the above-mentioned infection hybridization, and the other was subjected to infection hybridization with the already described probe (LC). Phages that were positive for both tests were selected. A clone having the "full length" cDNA was selected and the nucleotide sequence of the portion of the cDNA determined by the dideoxy method is shown in Figure 4(A).
Ova cDNA ima jednostruki široko otvoreni čitaju okvir i aminokiselinski niz koji treba biti kodiran ovom cDNA je izveden kao što je prikazano na slici 4(A). This cDNA has a single wide open reading frame and the amino acid sequence to be encoded by this cDNA is derived as shown in Figure 4(A).
Uspoređivanje sa N-terminalnim aminokiselinskim nizom G-CSF proteina prikazanog u primjeru 3(i) pokazuje da ova cDNA sadrži nukleotidni niz koji odgovara i signal peptidu kodiranom sa 90 baznih parova koji počinje sa ATG nizom na 31-32 nukleotidnim položajima od 5'-terminala i završenog sa GCC nizom na 118-120 položajima i zrelom G-CSF peptidu kodiranom sa 522 bazna para koji počinje sa ACC nizom na 121-123 položajima i koji se završava sa CCC nizom na 640-642 položajima. Stoga, polipeptid aminokiselinskog niza I prikazan na slici 4(B) je sastavljen od 204 aminokiseline i izračunata je njegova molekularna masa kao 21977,35 daltona. Polipeptid aminokiselinskog niza II je sastavljen od 174 aminokiseline i njegova molekularna masa je izračunata i iznosi 18671,42 daltona (primjer 10). Comparison with the N-terminal amino acid sequence of the G-CSF protein shown in Example 3(i) shows that this cDNA contains a nucleotide sequence corresponding to a signal peptide encoded by 90 base pairs beginning with an ATG sequence at 31-32 nucleotide positions from the 5'- terminal and ending with the GCC sequence at positions 118-120 and the mature G-CSF peptide encoded by 522 base pairs starting with the ACC sequence at positions 121-123 and ending with the CCC sequence at positions 640-642. Therefore, the polypeptide of amino acid sequence I shown in Figure 4(B) is composed of 204 amino acids and its molecular weight is calculated as 21977.35 daltons. The polypeptide of amino acid sequence II is composed of 174 amino acids and its molecular mass is calculated to be 18671.42 daltons (Example 10).
Treba istaknuti da ATG na 58-60 položajima ili na 67-69 položajima također može biti razmatran kao proteinsko početno mjesto. It should be noted that ATG at positions 58-60 or at positions 67-69 can also be considered as a protein start site.
Escherichia coli vrste X1776 koja uzgaja pBR322 koja ima ovu cDNA (-VSE) na EcoRI mjestu cijepanja je pohranjena u "Fermentation Research Insdtute, Agency of Industrial Science and Technology" (FERM BP.955). Escherichia coli strain X1776 growing pBR322 having this cDNA (-VSE) at the EcoRI cleavage site is deposited in the "Fermentation Research Insdtute, Agency of Industrial Science and Technology" (FERM BP.955).
Slika 6 prikazuje mjesta cijepanja restrikcijskog enzima gena. Ova cDNA je pripojena na pBR327 na EcoRI mjestu tako da nastaje plazmid koji je niže označavan kao pBRV2. Figure 6 shows the restriction enzyme cleavage sites of the gene. This cDNA was ligated into pBR327 at the EcoRI site to generate a plasmid designated below as pBRV2.
(4) Testiranje biblioteke humanog kromosomskog gena (4) Human chromosomal gene library testing
Biblioteka humanog kromosomskog gena koja je dobivena prema postupcima koje su opisali Maniatis et al. (Molecular Cloning, također) je podvrgnuta testiranju sa pHCS-1 prikazanim naprijed. Probe koje mogu biti upotrebljene za testiranje uključuju: pHCS-1 izveden 308-bp DNA fragment, pBRG4-izveden sa 1500-bp DNA fregment, pBRV2-izveden sa 1500-bp DNA fragment, DNA fragment odgovarajuće dužine sadrži dio jednog ili više DNA fragmenata, kao i gore spomenutih nukleotidnih proba /npr. (IWQ), (A) i (IC)/. Niže je opisan slučaj korištenja pHCS -1 DNA fragmenta. The human chromosomal gene library obtained according to the procedures described by Maniatis et al. (Molecular Cloning, too) was tested with pHCS-1 shown above. Probes that can be used for testing include: pHCS-1-derived 308-bp DNA fragment, pBRG4-derived 1500-bp DNA fragment, pBRV2-derived 1500-bp DNA fragment, DNA fragment of appropriate length containing part of one or more DNA fragments , as well as the above-mentioned nucleotide probes / e.g. (IWQ), (A) and (IC)/. A case of using the pHCS -1 DNA fragment is described below.
Ovaj DNA fragment je radioizotopski obilježen sa 32P prema metodi translatornog zasijecanja /vidi Roop et al., Cell, 15, 431(1978)/. S dobivenim 32P-obilježenim fragmentom korištenim kao proba, biblioteka humanog kromosomskog gena je podvrgnuta testiranju pomoću hibridizacije zaraze (vidi također Benton i Davis), tako da se dobiva deset-sporednih klonova. This DNA fragment was radioisotope labeled with 32P according to the translational cleavage method /see Roop et al., Cell, 15, 431(1978)/. With the resulting 32 P-labeled fragment used as a probe, the human chromosomal gene library was screened by infection hybridization (see also Benton and Davis), yielding ten-second clones.
Nakon izdvajanja DNA iz klonova, pripremljena je mapa enzima poznatim postupcima /Fritsch et al., Cell, 19, 959(1980)/. After extracting the DNA from the clones, an enzyme map was prepared using known procedures /Fritsch et al., Cell, 19, 959 (1980)/.
Sa istom probom izvršeno je Southeron upijanje (vidi također Southeron) i nađeno je da je DNA fragment od oko 4 kb isječen van sa AcoRI i Khol koji potencijalno sadrže područje za kodiranje humanog G-CSF polipeptida. Stoga, 4-kb DNA fragment je ubačen u pBR327 na EcoRI mjestu koristeći EcoRI vezivo tako da se dobiva pBRCE3β. Sa ovim plazmidom korištenim kao baznim nizom DNA, određen je pomoću dideoksi metode nukleotidni niz 3-kb dijela 4-kb DNA fragmenta. Nađeno je da je spomenuti fragment kodirajući gen za humani G-CSF polipeptid (slika 5). Southeron blotting was performed with the same probe (see also Southeron) and a DNA fragment of about 4 kb was found to be excised with AcoRI and Khol potentially containing the coding region of the human G-CSF polypeptide. Therefore, a 4-kb DNA fragment was inserted into pBR327 at the EcoRI site using the EcoRI linker to give pBRCE3β. With this plasmid used as the DNA base sequence, the nucleotide sequence of the 3-kb part of the 4-kb DNA fragment was determined using the dideoxy method. The said fragment was found to be the gene coding for the human G-CSF polypeptide (Figure 5).
E. coli vrsta X1776 koja uzgaja pBRCE3β (tj. plazmid pBR327 koji spomenuti 4-kb DNA fragment ubačen u EcoRI mjesto) je pohranjena u "Fermentation Research Institute, Agency of Industrial Science and Technology" (FERM BP.956). E. coli strain X1776 growing pBRCE3β (ie, plasmid pBR327 having the aforementioned 4-kb DNA fragment inserted into the EcoRI site) is deposited in the "Fermentation Research Institute, Agency of Industrial Science and Technology" (FERM BP.956).
Uspoređivanje između pBRG4 cDNA dijela prikazanog na slici 3 i pBRV2 cDNA dijela prikazanog na slici 4 pokazuje da diskutirani dio DNA sadrži pet dijelova eksona zbog čega kodira samo aminokisleinske nizove izvedene iz pBRG4 i pBRV2. A comparison between the pBRG4 cDNA part shown in Figure 3 and the pBRV2 cDNA part shown in Figure 4 shows that the discussed DNA part contains five exon parts, therefore it encodes only the amino acid sequences derived from pBRG4 and pBRV2.
Slika 7 prikazuje mjesto cijepanja restrikcijskog enzima dobivenog gena. Ovaj DNA fragment sadrži kromosomski gen humanog G-CSF, ili područje koje mu prethodi sa transkribira u humani G-CSF mRNA, plus nukleotidni niz koji sudjeluje u transkripcijskoj kontroli /Benoist i Chambon, Nature, 290, 304(1981); i Breathnack i Chambon, Ann. Rev. Biochem., 50, 349(1981)/. Figure 7 shows the restriction enzyme cleavage site of the resulting gene. This DNA fragment contains the chromosomal gene for human G-CSF, or the region preceding it that is transcribed into human G-CSF mRNA, plus a nucleotide sequence that participates in transcriptional control /Benoist and Chambon, Nature, 290, 304(1981); and Breathnack and Chambon, Ann. Rev. Biochem., 50, 349(1981)/.
(5) Konstrukcija rekombinantnog vektora za izražavanje u E. coli (5) Construction of a recombinant vector for expression in E. coli
(A) -VSE linija rekombinantni vektor (A) -VSE line recombinant vector
Iz pBRG4 plazmida dobivenog u (3) (primjer 9), isječen je cDNA fragment G-CSF peptida sa restrikcijskim enzimom i rekombinantni vektor je načinjen jednom od sljedećih metoda: From the pBRG4 plasmid obtained in (3) (Example 9), the cDNA fragment of the G-CSF peptide was cut with a restriction enzyme and the recombinant vector was made by one of the following methods:
(i) koristeći kaljeno sintetsko vezivo, fragment je povezan sa fragmentom dobivenim iz tac promotora koji sadrži pKK223-3 (Pharmacia Fine Chemicals) (primjer 12 i slika 18); (i) using a quenched synthetic linker, the fragment was ligated to a fragment derived from the tac promoter containing pKK223-3 (Pharmacia Fine Chemicals) (Example 12 and Figure 18);
(ii) tri fragmenta dobivena iz PL promotora koji sadrži PPL-lambda ((Pharmacia Fine Chemicals) su povezana sa kaljenim sintetskim vezivom i produkt vezanja i fragment su podvrgnuti ponovnom postupku dobivanja tako da nastaje rekombinantni vektor (primjer 13, slika 9); ili (ii) three fragments obtained from the PL promoter containing PPL-lambda ((Pharmacia Fine Chemicals)) are ligated with a hardened synthetic linker and the ligation product and the fragment are subjected to a reassembly procedure to form a recombinant vector (Example 13, Figure 9); or
(iii) korištenjem kaljenog sintetskog veziva, fragment se povezuje s fragmentom dobivenim iz trp promotora koji sadrži pOYI plazmid (primjer 14 i slika 10). (iii) using a hardened synthetic linker, the fragment is ligated to a fragment derived from the trp promoter containing the pOYI plasmid (Example 14 and Figure 10).
(B) -VSE klinija rekombinantni vektor (B) -VSE clinical recombinant vector
Na isti način kao što je gore opisano, načinjena su tri rekombinantna vektora koristeći plazmid pBRV2 (primjer 10) kao što je prikazano u primjeru 19 i slikama 11, 12 i 13. In the same manner as described above, three recombinant vectors were made using plasmid pBRV2 (Example 10) as shown in Example 19 and Figures 11, 12 and 13.
(6) Dobivanje E. coli transformata i kultiviranje i njeno izražavanje (6) Obtaining the E. coli transformant and its cultivation and expression
Koristeći tri rekombinantna vektora svaki +VSE i -VSE linije, E. coli vrsta DH1, N4830 ili JM105 je transformirana kalcij kloridom ili rubidij-kloridom postupkom koje je opisan u Molecular Cloning, također (primjeri 12, 13, 14 i 19). Svaki od dobivenih transformata je kultiviran u Luria mediju koji sadrži ampicilin, sa indukcijom izvedenom kao što traži željeno izražavanje (primjeri 15 i 20). Using three recombinant vectors each of +VSE and -VSE lines, E. coli strains DH1, N4830 or JM105 were transformed with calcium chloride or rubidium chloride by the procedure described in Molecular Cloning, also (Examples 12, 13, 14 and 19). Each of the resulting transformants was cultured in Luria medium containing ampicillin, with induction performed as required for the desired expression (Examples 15 and 20).
(7) Odvajanje i pročišćavanje G-CSF polipeptida iz E. coli i analiza njegovih aminokiselina (7) Separation and purification of G-CSF polypeptide from E. coli and analysis of its amino acids
Otopina kultura transformanata je centrifugirana tako da se dobiva tableta stanica. Sakupljene stanice su tretirane sa lizozimom i zatim cikličkim zamrzavanjem i taljenjem dobiven je supernatant. Alternativno, stanice su tretirane sa guanidin-kloridom, centrifugirane i izdvojen je supernatant. The transformant culture solution was centrifuged to obtain a pellet of cells. The collected cells were treated with lysozyme and then the supernatant was obtained by cyclic freezing and thawing. Alternatively, the cells were treated with guanidine chloride, centrifuged and the supernatant was collected.
Supernatant je podvrgnut gel filtraciji na Ultrogel ACA54 koloni (LKB) i aktivne frakcije su koncentrirane sa ultrafiltracijskim aparatom. The supernatant was subjected to gel filtration on an Ultrogel ACA54 column (LKB) and the active fractions were concentrated with an ultrafiltration apparatus.
Zatim, vodena otopina trifluoroctene kiseline koja sadrži n-propanol je dodana koncentratu i ovaj je stavljen u led. Smjesa je centrifugirana i adsorbirana na reverzno-faznoj C18 koloni. Nakon eluiranja, ispitana je aktivnost frakcija. Aktivne frakcije su sakupljene i podvrgnute istom postupku pročišćavanja koji je opisan naprijed. Pročišćene frakcije su suho smrznute i prah je otopljen i podvrgnut HP tekućinskoj kromatografiji, zasnovanoj na veličini molekula. Dobiveni polipeptidi su podvrgnuti SDS poliakrilamid gel elektroforezi i pronađene su posebne vrpce za željeni G-CSF polipeptid (primjeri 16 i 20). Tako dobiveni polipeptid pokazuje humanu G-CSF aktivnost (primjeri 17 i 20). G-CSF polipeptid je analiziran postupkom analize aminokiselina sa Hitachi 835 Automatic Acid Analyzer (Hitachi, Ltd.). Za analize N-terminala aminokiselina, korišteni su plinsko-fazni ispitivač nizova (za Edman razgradnju), aparatura za tekućinsku kromatografiju na visokom tlaku i Ultrasferna-ODS kolona (primjeri 18 i 21). Then, an aqueous solution of trifluoroacetic acid containing n-propanol was added to the concentrate and it was placed in ice. The mixture was centrifuged and adsorbed on a reverse-phase C18 column. After elution, the activity of the fractions was tested. Active fractions were collected and subjected to the same purification procedure described above. The purified fractions were freeze-dried and the powder was thawed and subjected to molecular size-based HP liquid chromatography. The resulting polypeptides were subjected to SDS polyacrylamide gel electrophoresis and distinct bands for the desired G-CSF polypeptide were found (Examples 16 and 20). The resulting polypeptide shows human G-CSF activity (examples 17 and 20). G-CSF polypeptide was analyzed by the amino acid analysis procedure with a Hitachi 835 Automatic Acid Analyzer (Hitachi, Ltd.). For the analysis of N-terminal amino acids, a gas-phase array tester (for Edman decomposition), a high-pressure liquid chromatography apparatus and an Ultrasphere-ODS column (examples 18 and 21) were used.
(8) Stvaranje rekombinantnih vektora za životinjske stanice (8) Creation of recombinant vectors for animal cells
Rekombinantni vektori (izvedeni iz BPV) za upotrebu sa C127 i NiH3T3 stanicama domaćina su načinjem za svaku od +VSE i -VSE liniju cDNA i za svaki kromosomski gen. Rekombinantni vektori (sa dhfr) sa korištenje sa CHO stanicama su također načinjeni za svaku +VSE i -VSE liniju cDNA i za kromosomski gen. Rekombinantni vektori za upotrebu sa COS stanicama su također načinjeni. Niže su opisani reprezentativni primjeri detaljnije i sa naznakom radnih primjera. Recombinant vectors (derived from BPV) for use with C127 and NiH3T3 host cells are available for each of the +VSE and -VSE cDNA lines and for each chromosomal gene. Recombinant vectors (with dhfr) for use with CHO cells were also made for each +VSE and -VSE cDNA line and for the chromosomal gene. Recombinant vectors for use with COS cells have also been constructed. Representative examples are described below in more detail and with an indication of working examples.
(A) Stvaranje rekombinantnih vektora +VSE i -VSE linija (A) Generation of recombinant vectors of +VSE and -VSE lines
cDNA fragment dobiven u (3) je ubačen u vektor pdKCR tako da nastaje plazmid pHGA 410 (primjer 22 i slika 14), koji je djelomično razoren sa EcoRI a zatim tretiran sa polimerazom I (Klenow fragment) tako da nastanu otvoreni krajevi. Vezivo HindIII je vezano na DNA, koja je zatim tretirana sa HindIII i T4DNA ligazom. Tretirana DNA je upotrebljena za transformiranje E. coli vrste DH1 postupkom sa rubidij-kloridom (vidi također Molecular Cloning). Dobiveni plazmid je nazvan pHGA410(H) (slika 15). The cDNA fragment obtained in (3) was inserted into the pdKCR vector so that plasmid pHGA 410 (example 22 and figure 14) was created, which was partially digested with EcoRI and then treated with polymerase I (Klenow fragment) so that open ends were formed. The HindIII binder is bound to the DNA, which is then treated with HindIII and T4DNA ligase. The treated DNA was used to transform E. coli strain DH1 by the rubidium chloride method (see also Molecular Cloning). The resulting plasmid was named pHGA410(H) (Figure 15).
pHGA410(H) je tretiran sa SalI i, nakon čega su nastali otvoreni krajevi, a zatim je još jednom tretiran sa HindIII i izdvojen je HindIII-SalI fragment. Plazmid pdBPV-1 koji ima transformirani fragment papiloma virusa goveda je tretiran sa HindIII i PvuII i veliki DNA fragment su razdvojeni i pripojeni odvojeno pripremljenom HindIII-SalI fragmentu. Pripajanje fragmenata je primijenjeno radi transformiranja E. coli vrste DH1 tako da se dobije plazmid PTN-G4, koji ima pHGA410-izvedenu G-CSF-cDNA (slika 15 i primjer 23). pHGA410(H) was treated with SalI and, after which open ends were formed, it was then treated again with HindIII and the HindIII-SalI fragment was isolated. Plasmid pdBPV-1 having a transformed fragment of bovine papillomavirus was treated with HindIII and PvuII and the large DNA fragment was cleaved and ligated to a separately prepared HindIII-SalI fragment. Fragment ligation was used to transform E. coli strain DH1 to yield plasmid PTN-G4, which has a pHGA410-derived G-CSF-cDNA (Figure 15 and Example 23).
Plazmid pHGA410 ili pHGA410(H), kombinaciji sa plazmidom pAdD26SVpA je upotrebljen tako da nastane pHGG4-dhfr koji je bio rekombinantni vektor (+VSE) za upotrebu sa CHO stanicama (slike 16a i b, i primjer 25). Plasmid pHGA410 or pHGA410(H), combined with plasmid pAdD26SVpA was used to generate pHGG4-dhfr which was a recombinant vector (+VSE) for use with CHO cells (Figures 16a and b, and Example 25).
2-kb fragment koji sadrži dhfr gen je izdvojen iz pAdD26SVpA tretiranjem sa EcoRI i BamHI i izdvojeni fragment je ubačen u pHGA410(H) na mjestu tako da nastaje pG4DR1 i pG4DR2 (slika 16c i primjer 25). A 2-kb fragment containing the dhfr gene was excised from pAdD26SVpA by treatment with EcoRI and BamHI and the excised fragment was inserted into pHGA410(H) in situ to generate pG4DR1 and pG4DR2 (Figure 16c and Example 25).
(B) Stvaranje -VSE linije rekombinantnih vektora (B) Generation of the -VSE line of recombinant vectors
cDNA (-VSE) fragment dobiven u (3) ubačen je u vektor pdKCR tako da nastane plazmid pHGV2 (primjer 28), koji je djelomično razoren sa EcoRI a zatim tretiran sa DNA polimerazom I (Klenow fragment) tako da nastanu otvoreni krajevi. Vezivo HindIII je pripojeno na DNA, koja je zatim tretirana sa HindIII i T4DNA ligazom. Tretirana DNA je upotrebljena za transformiranje E. coli vrste DH1 postupkom sa rubidij-kloridom (vidi također Molecular Cloning). Dobiveni plazmid je nazvan pHGV2(H) (slika 18). The cDNA (-VSE) fragment obtained in (3) was inserted into the pdKCR vector to form the pHGV2 plasmid (example 28), which was partially digested with EcoRI and then treated with DNA polymerase I (Klenow fragment) to create open ends. The HindIII binder was attached to the DNA, which was then treated with HindIII and T4DNA ligase. The treated DNA was used to transform E. coli strain DH1 by the rubidium chloride method (see also Molecular Cloning). The resulting plasmid was named pHGV2(H) (Figure 18).
pHGV2(H) je tretiran sa SalI i, nakon čega se stvaraju otovoreni krajevi, te ponovnim tretiranjem još jednom sa HindIII izdvojen je HindIII-SalI fragment. Plaz-mid pdBPV-1 koji ima transformirani fragment papilom virusa goveda je tretiran sa HindIII i PvuII i veliki DNA fragment je izdvojen i pripojen na odvojeno pripremljen Hind-III-SalI fragment. Pripojeni fragmenti su upotrebljeni za transformiranje E. coli vrste DH1 tako da se dobije plazmid, pTN-V2, koji ima pHGV2-izvedenu CSF-cDNA (slika 18 i primjer 29). pHGV2(H) was treated with SalI and, after that, open ends were created, and the HindIII-SalI fragment was isolated by retreatment with HindIII. Plasmid pdBPV-1 having a transformed fragment of bovine papillomavirus was treated with HindIII and PvuII and the large DNA fragment was isolated and ligated to a separately prepared Hind-III-SalI fragment. The attached fragments were used to transform E. coli strain DH1 to yield a plasmid, pTN-V2, having a pHGV2-derived CSF-cDNA (Figure 18 and Example 29).
Sličnim postupcima pHGV2 ili pHGV2(H) plazmid, u kombinaciji sa plazmidom pAdD26SVpA je upotrebljen za dobivanje pHGV2-dhfr koji je bio rekombinantni vektor (-VSE) za primjenu sa CHO stanicama (slike 19a i b, i primjer 31). By similar procedures the pHGV2 or pHGV2(H) plasmid, in combination with the plasmid pAdD26SVpA was used to obtain pHGV2-dhfr which was a recombinant vector (-VSE) for use with CHO cells (Figures 19a and b, and Example 31).
DNA fragment od oko 2kb koji sadrži dhfr gen je izdvojen iz pAdD26SCpA tretmanom sa EcoRI i BamHI i izdvojeni fragment je ubačen u pHGV2(H) na HindIII mjestu tako da nastaje pV2DR1 i pV2DR2 (slika 19 c i primjer 31). A DNA fragment of about 2kb containing the dhfr gene was isolated from pAdD26SCpA by treatment with EcoRI and BamHI and the isolated fragment was inserted into pHGV2(H) at the HindIII site so that pV2DR1 and pV2DR2 were created (Figure 19 c and example 31).
(C) Stvaranje rekombinantnih vektora koji sadrže kromosomski gen (C) Creation of recombinant vectors containing the chromosomal gene
Plazmid pBRCE3 koji je dobiven u (4) i koji sadrži kromosomski gen prikazan na slici 5 je tretiran sa EcoRI. Plasmid pBRCE3 obtained in (4) and containing the chromosomal gene shown in Figure 5 was treated with EcoRI.
pSVH+K+ plazmid koji je opisao Banerji et al., Cell, 27, 299(1981) je tretiran sa KpnI tako da se ukloni globin gen. The pSVH+K+ plasmid described by Banerji et al., Cell, 27, 299(1981) was treated with KpnI to remove the globin gene.
Plazmid je dalje podvrgnut djelomičnom razlaganju sa HindIII tako da se ukloni dio zaostalog gena SV40. Fragmenti su ponovno pripojeni tako da nastane pML-E+ vektor ekspresije. The plasmid was further subjected to partial digestion with HindIII to remove part of the residual SV40 gene. The fragments were rejoined to form the pML-E+ expression vector.
Ovaj vektor je tretiran sa restrikcijskim enzimom EcoRI, i defosforiliziran sa alkalnim fosfatom (Takara Shuzo Co., Ltd.). Tako se dobije vektor DNA, koji je vezan na gore spomenuti kromosomski DNA fragment pomoću T4DNA ligaze (Takara Shuzo Co., Ltd.) tako da se dobije pMLCE3 koji je bio rekombinantni vektor za COS stanice (primjer 34). Kao što je prikazano na slici 20, ovaj plazmid sadrži pojačivač SV40 gena, replikator početka SV40, replikator početka pBR322 i pBR322-izvedeni p-laktamaza gen (Ampr) i ima humani G-CSF kromosomski gen spojen niže od pojačivača SV40 gena. This vector was treated with restriction enzyme EcoRI, and dephosphorylated with alkaline phosphate (Takara Shuzo Co., Ltd.). Thus, a DNA vector was obtained, which was linked to the above-mentioned chromosomal DNA fragment by T4DNA ligase (Takara Shuzo Co., Ltd.) to obtain pMLCE3, which was a recombinant vector for COS cells (Example 34). As shown in Figure 20, this plasmid contains the SV40 gene enhancer, the SV40 origin replicator, the pBR322 origin replicator and the pBR322-derived β-lactamase gene (Ampr) and has the human G-CSF chromosomal gene fused downstream of the SV40 gene enhancer.
Vektor ekspresije za C127 stanice je načinjen sljedećim postupkom. DNA fragment koji sadrži kromosomski CSF gen je izvučen van s odgovarajućim enzimom iz pMLCE3α koji ima vektor ekspresije za COS stanice. Ovaj fragment je pripojen, sa T4DNA ligazom, na DNA fragment koji sadrži početak papiloma virusa goveda (BPV) i DNA fragment koji sadrži promotor sa SV40. Dobiveni pTNCE3α je bio vektor ekspresije koji ima kromosomski CSF gen vezan ispod promotora SV40 koji sadrži 65% dijela BPV-a. An expression vector for C127 cells was made by the following procedure. The DNA fragment containing the chromosomal CSF gene was pulled out with the appropriate enzyme from pMLCE3α having an expression vector for COS cells. This fragment was ligated, with T4DNA ligase, to a DNA fragment containing the beginning of bovine papillomavirus (BPV) and a DNA fragment containing the SV40 promoter. The resulting pTNCE3α was an expression vector having the chromosomal CSF gene linked under the SV40 promoter containing 65% of the BPV portion.
Vektor ekspresije za CHO stanice ima dva DNA fragmenta povezana zajedno sa T4DNA ligazom; jedan fragment sadrži kromosomski CSF gen i raniji promotor SV40 u slučaju da ekspresijski vektor za C177 stanice, i drugi fragment koji sadrži pAdD26SVpA-izvedeni dhfr gen. Dobiveni pD26SVCE3α je bio ekspresijski vektor koji ima kromosomski CSF gen ispod SV40 promotora, dhfr gen ispod posljednjeg promotora adenovirusa. The expression vector for CHO cells has two DNA fragments linked together with T4DNA ligase; one fragment containing the chromosomal CSF gene and the earlier SV40 promoter in case of an expression vector for C177 cells, and the other fragment containing the pAdD26SVpA-derived dhfr gene. The resulting pD26SVCE3α was an expression vector that has the chromosomal CSF gene under the SV40 promoter, the dhfr gene under the last adenovirus promoter.
(9) Ekspresija u životinjskim stanicama (9) Expression in animal cells
Dva reprezentativna primjera su niže opisana, i za više detalja pogledaj odgovarajuće radne primjere. Two representative examples are described below, and for more details see the corresponding working examples.
(A) Ekspresija u stanicama C127 miša (A) Expression in mouse C127 cells
Plazmid pTN-G4 ili pTN-V2 je tretiran sa BamHI. Tretirani plazmid je upotreb-ljen za transformaciju C127 stanica (prethodno izrasle kultiviranjem) postupkom s kalcij-fosfatom. Transformirane stanice su kultivirane i odabrani su klonovi koji imaju veliku brzinu proizvodnje CSF-a. Glikoproteini koji sadrže izražen G-CSF su izdvojeni i pročišćeni iz otopine kulture transformiranih stanica i nađeno je da posjeduju humanu G-CSF aktivnost. Prisutnost željenog glikoproteina je također potvrđena analizom aminokiselina i šećera u uzorku. Plasmid pTN-G4 or pTN-V2 was treated with BamHI. The treated plasmid was used to transform C127 cells (previously grown by culturing) by the calcium-phosphate method. The transformed cells were cultured and clones were selected that had a high rate of CSF production. Glycoproteins containing expressed G-CSF were isolated and purified from the culture solution of transformed cells and found to possess human G-CSF activity. The presence of the desired glycoprotein was also confirmed by analyzing the amino acids and sugars in the sample.
Za analizu sadržaja šećera, CSF uzorak korišten za analizu aminokiselina podvrgnut je određivanju aminošećera pomoću Elson-Margan metode, određivanju neutralnih šećera pomoću orcinol-sulfat postupka, ili određivanju sialinske kiseline pomoću tiobarbituratnog postupka. Svi postupci određivanja su prikazani u 'Tohshitsu no Kagaku "Chemistry of Saccharides" (dio 2 drugog dijela), Chapter 13, vol. 4 A od "A Course in Biochemical Experiments", koju su objavili Tokyo Kagaku Dojin. Prevođenje izmjerenih vrijednosti u postotke mase pokazalo je da je dobiveni sadržaj šećera u području 1-20% ovisno o tipu stanica domaćina, vektora ekspresije i uvjeta kultiviranja. For the analysis of sugar content, the CSF sample used for amino acid analysis was subjected to the determination of amino sugars using the Elson-Margan method, the determination of neutral sugars using the orcinol-sulfate method, or the determination of sialic acid using the thiobarbiturate method. All determination procedures are described in 'Tohshitsu no Kagaku "Chemistry of Saccharides" (part 2 of the second part), Chapter 13, vol. 4 A of "A Course in Biochemical Experiments", published by Tokyo Kagaku Dojin. Translating the measured values into mass percentages showed that the obtained sugar content was in the range of 1-20% depending on the type of host cells, expression vector and cultivation conditions.
(B) Ekspresija u COS stanicama (B) Expression in COS cells
COS stanice, koje su izvedene iz CV-L stanica majmuma i koje su transformirane sa SV40-početkom deficijentnog mutanta tako da izraze veliki T antigen SV40 /vidi Gluzman et aL, Cell, 32, 175(1981)/ su transformirane vektorom pMLCE3α koji je dobiven u (5) (C) i koji sadrži humani kromosomski G-CSF gen. Supernatant kulture COS stanica pokazuje humanu G-CSF aktivnost (primjer 35). COS cells, which were derived from monkey CV-L cells and which were transformed with an SV40-start deficient mutant to express the SV40 large T antigen (see Gluzman et al, Cell, 32, 175 (1981)) were transformed with the pMLCE3α vector which obtained in (5) (C) and containing the human chromosomal G-CSF gene. COS cell culture supernatant exhibits human G-CSF activity (Example 35).
COS stanice su bile izdvojene i podvrgnute mRNA analizi, koja je pokazala prisutnost dva mRNA što odgovara aminokiselinskim nizovima prikazanim na slikama 3(A) i 4(A). COS cells were isolated and subjected to mRNA analysis, which showed the presence of two mRNAs corresponding to the amino acid sequences shown in Figures 3(A) and 4(A).
Primjeri Examples
Ovaj izum je naprijed detaljno opisan glede radnih primjera. Sljedeći referentni primjer je dan u cilju ilustriranja postupaka ispitivanje CSF aktivnosti. This invention is described in detail above with regard to working examples. The following reference example is given in order to illustrate the procedures for testing CSF activity.
Referentni primjer: Ispitivanje CSF aktivnosti Reference example: Examination of CSF activity
Sljedeći primjeri su korišteni radi određivanja CSF aktivnosti (niže označene kao CSA) u ovome izumu. The following examples were used to determine the CSF activity (referred to below as CSA) in the present invention.
CSA ispitivanja CSA tests
(a) sa humanim stanicama koštane srži (a) with human bone marrow cells
Jednostruki sloj mekog agara je korišten za kultiviranje prema metodi Bradley, T.R. i Metcalf, D. (Aust. J. Exp. Biol. Med. ScL, 44, 287-300(1966). Određenije, 0,2 ml seruma embriona goveda, 0,1 ml uzorka, 0,1 ml suspenzije humanih sta-nica koštane srži (1-2xl05 nukleusa stanica), 0,2 ml modificiranog MsCoy-eve 5A otopine kulture koja sadrži 0,75% agara, je uliveno u plastičnu posudu za kulturu tkiva (promjera 55 mm), koagulirano je i kultivirano na 37°C u 5% CO2/95% zraku i na 100% vlažnosti. Deset dana kasnije, određen je broj nastalih kolonija (jedna kolonija se sastoji od bar 50 stanica) i CSA je određena tako da je jedna jedinica aktivnosti potrebna za stvaranje jedne kolonije. A single layer of soft agar was used for cultivation according to the method of Bradley, T.R. and Metcalf, D. (Aust. J. Exp. Biol. Med. ScL, 44, 287-300(1966). More specifically, 0.2 ml bovine embryo serum, 0.1 ml sample, 0.1 ml suspension of human sta bone marrow (1-2x105 cell nuclei), 0.2 ml of modified MsCoy's 5A culture solution containing 0.75% agar, was poured into a plastic tissue culture dish (diameter 55 mm), coagulated and cultured on 37°C in 5% CO2/95% air and 100% humidity.Ten days later, the number of colonies formed (one colony consists of at least 50 cells) was determined and the CSA was determined so that one unit of activity was required to form one colonies.
(b) sa stanicama koštane srži miša (b) with mouse bone marrow cells
Serum konja (0,4 ml), 0,1 ml uzorka, 0,1 ml suspenzije stanica C3H/He koštane srži ženke miša (0,5-1x105 jezgara stanica), i 0,4 ml modificirane McCoyeve 5A otopine kulture koja sadrži 0,75% agara, je uliveno u plastičnu posudu za kulturu tkiva (promjera 55 mm), koagulirano je i kultivirano na 37°C u 5% CO2/95% zraku i na 100% vlažnosti. Horse serum (0.4 ml), 0.1 ml sample, 0.1 ml suspension of female mouse C3H/He bone marrow cells (0.5-1x105 cell nuclei), and 0.4 ml modified McCoy's 5A culture solution containing 0.75% agar was poured into a plastic dish for tissue culture (diameter 55 mm), coagulated and cultured at 37°C in 5% CO2/95% air and 100% humidity.
Deset dana kasnije, određen je broj nastalih kolonija (jedna kolonija se sastoji od bar 50 stanica) i CSA je određena tako da je jedna jedinica aktivnosti potrebna za stvaranje jedne kolonije. Ten days later, the number of formed colonies was determined (one colony consists of at least 50 cells) and the CSA was determined so that one unit of activity was required to form one colony.
Otopina modificirane McCoyeve 5A kulture korištena u svakoj od metoda (a) i (b) i suspenzija humanih stanica koštane srži upotrebljena u (a) dobiveni su sljedećim postupcima. The modified McCoy 5A culture solution used in each of methods (a) and (b) and the human bone marrow cell suspension used in (a) were obtained by the following procedures.
Otopina modificirane McCoyeve 5A kulture (dvostruko koncentrirane) Modified McCoy's 5A culture solution (double concentrated)
12 grama ofopine McCoyeve 5A kulture (Gibco), 2,55g MEM aminokiselinsko-vitaminskog medija (Nissui Seiyaku Co., Ltd.), 2,18 g natrij-bikarbonata i 50.000 jedinica kalij-penicilina G je otopljeno dva puta u 500 ml destilirane vode i otopina je aseptički profiltrirana kroz Millipore filter (0,22 µm). 12 grams of opopin McCoy's 5A culture (Gibco), 2.55 g of MEM amino acid-vitamin medium (Nissui Seiyaku Co., Ltd.), 2.18 g of sodium bicarbonate, and 50,000 units of potassium penicillin G were dissolved twice in 500 ml of distilled of water and the solution was aseptically filtered through a Millipore filter (0.22 µm).
Suspenzija humanih stanica koštane srži Suspension of human bone marrow cells
Tekućina koštane srži dobivena od zdrave osobe sterilnom punkcijom je razrijeđena 5 puta sa RPMI 1640 otopinom kulture i stavljena iznad otopine Fikol-zaraze (Pharmacia Fine Chemicals) i centrifugirana na 400 g 30 minuta na 25°C. Izdvojen je sloj interfacijalnih stanica gustoće manje od 1,077. Stanice su isprane, i koncentracija je načinjena da bude 5x106 stanica/ml sa RPMI 1640 otopinom kulture koja sadrži 20% seruma embrija goveda, ulivene u 25 cm3 plastičnu bocu za kulturu tkiva i inkubirane 30 minuta u CO2 inkubatoru. Neslijepljene stanice su izdvojene u supernatantu, izlivene u plastičnu bocu od 25 cm3 i inkubirane 2,5 sati. Neslijepljene stanice u supernatantu su skupljene i korištene u istraživanju. Bone marrow fluid obtained from a healthy person by sterile puncture was diluted 5 times with RPMI 1640 culture solution and placed over Ficol-infection solution (Pharmacia Fine Chemicals) and centrifuged at 400 g for 30 minutes at 25°C. A layer of interfacial cells with a density of less than 1.077 was isolated. Cells were washed, and the concentration was adjusted to 5x106 cells/ml with RPMI 1640 culture solution containing 20% fetal bovine serum, poured into a 25 cm3 plastic tissue culture flask and incubated for 30 minutes in a CO2 incubator. Non-adherent cells were separated in the supernatant, poured into a 25 cm3 plastic bottle and incubated for 2.5 hours. Non-adherent cells in the supernatant were collected and used in the study.
Primjer 1: Example 1:
Zasnivanje CHU-2 Establishment of CHU-2
Tumor pacijenta s oralnim Ca gdje je naglašen porast zapažen u broju neutrofilia je transplantiran u nu/nu miša. Oko 10 dana nakon transplantacije, izražen je porast mase tumora i broja neutrofilija. 12 dana nakon operacije, tumor je aseptički ekstrahiran, izrezan u kocke 1-2 mm3 i kultiviran na sljedeći način. The tumor of a patient with oral Ca, where a pronounced increase was observed in the number of neutrophils, was transplanted into a nu/nu mouse. About 10 days after transplantation, there was a marked increase in tumor mass and the number of neutrophils. 12 days after surgery, the tumor was aseptically extracted, cut into 1-2 mm3 cubes and cultured as follows.
10 do 15 kockica stavljeno je u plastičnu epruvetu za centrifugiranje od 50 ml. Nakon dodatka 5 ml otopine tripsina (koji sadrži 0,25% tripsina i 0,02% EDTA) epruveta je mućkana 10 minuta u toploj kupelji na 37°C i odbačen je supernatant. Drugih 5 ml iste otopine tripsina je dodano i cijepanje tripsinom je vršeno uz miješanje 15 minuta na 37°C. Suspenzija stanica supernatanta je izdvojena i stavljena u led pošto je tripsin deaktiviran dodatkom 1 ml seruma goveđeg embrija. 10 to 15 cubes were placed in a 50 ml plastic centrifuge tube. After adding 5 ml of trypsin solution (containing 0.25% trypsin and 0.02% EDTA), the tube was shaken for 10 minutes in a warm bath at 37°C and the supernatant was discarded. Another 5 ml of the same trypsin solution was added and cleavage by trypsin was performed with stirring for 15 minutes at 37°C. The supernatant cell suspension was separated and placed in ice, since the trypsin was deactivated by the addition of 1 ml of bovine embryo serum.
Nakon ponavljanja ovih postupaka još jednom, izdvojena je suspenzija stanica, kombinirana s prethodno dobivenom suspenzijom i centrifugirana na 15.000 okr/min tijekom 10 minuta tako da se dobije tableta stanica. Tableta je isprana dva puta sa F-10 koji sadrži 10% seruma embrija goveda i zatim prebačena u plastičnu bocu od 25 cm3 tako da koncentracija stanica bude 5x106 stanica/boci. Nakon inkubiranja preko noći u CO2 inkubatoru (5% CO2 i 100% vlažnosti) sa F-10 otopinom kulture koja sadrži 10% seruma embrija goveda, supernatant je uklonjen zajedno sa neslijepljenim stanicama, i kultiviranje je nastavljeno sa svježe dodanom otopinom kulture. Šest dana poslije početka kultiviranja, boca postaje ispunjena stanicama i otopina kulture se zamijeni svježom. Sljedećeg dana, otopina kulture je odbačena i boca je dopunjena sa 2 ml antitijela eritrocita anti-miša (Cappel) razrijeđenog 5 puta sa RPMI 1640 i 2 ml komplementa guinea svinje (Kyokuto Seyasku Co., Ltd.) razrijeđenog 2,5 RPMI 1640. Nakon inkubiranja tijekom 20 minuta na 37°C, kultura je dva puta isprana sa F-10 koji sadrži 10% seruma embrija goveda i uklonjeni su izvedeni fibroblasti nu/nu miša. Zatim je dodana F-10 otopina kulture koja sadrži 10% seruma embrija goveda i kultiviranje je nastavljeno još dva dana. Nakon ovoga, ista stanice su izdvojene i podvrgnute kloniranju pomoću metode graničnog razrijeđenja. After repeating these procedures once more, the cell suspension was separated, combined with the previously obtained suspension and centrifuged at 15,000 rpm for 10 minutes to obtain a cell pellet. The tablet was washed twice with F-10 containing 10% fetal bovine serum and then transferred to a 25 cm3 plastic bottle so that the cell concentration was 5x106 cells/bottle. After overnight incubation in a CO2 incubator (5% CO2 and 100% humidity) with F-10 culture solution containing 10% fetal bovine serum, the supernatant was removed along with non-adherent cells, and culturing was continued with freshly added culture solution. Six days after the start of culturing, the bottle becomes filled with cells and the culture solution is replaced with fresh. The next day, the culture solution was discarded and the bottle was supplemented with 2 ml of anti-mouse erythrocyte antibody (Cappel) diluted 5-fold with RPMI 1640 and 2 ml of guinea pig complement (Kyokuto Seyasku Co., Ltd.) diluted 2.5 times with RPMI 1640. After incubation for 20 minutes at 37°C, the culture was washed twice with F-10 containing 10% fetal bovine serum and the derived nu/nu mouse fibroblasts were removed. F-10 culture solution containing 10% bovine embryo serum was then added and cultivation was continued for another two days. After this, the same cells were isolated and subjected to cloning using the limiting dilution method.
Dobivenih 11 klonova je ispitano na CSF aktivnost i jedan klon (CHU-2) je pokazivao aktivnost oko 10 puta veću od one za druge klonove. The obtained 11 clones were tested for CSF activity and one clone (CHU-2) showed an activity about 10 times higher than that of the other clones.
Primjer 2: Example 2:
Izoliranje CSF Isolation of CSF
Stanice zasnovane u primjeru 1 su izrasle u sasvim gustoj populaciji u dvije boce za kultiviranje (150 cm3). Stanice su izdvojene, suspendirane u 500 ml F-10 otopine kulture koja sadrži 10% seruma goveđeg embrija, prenesene u staklenu rotirajuću bocu od 1580 cm3 (Delco), i obrtanjem kultivirane na 0,5 okr/min. Ka-da je nađeno da su stanice izrasle u sasvim gustoj populaciji na unutrašnjoj stijenci rotirajuće boce, otopina kulture je zamijenjena sa RPMI 1640 koji ne sadrži serum. Nakon 4 dana kultiviranja, supernatant kulture je izdvojen i kultiviranje je nastavljeno sa F-10 koji sadrži 10% serum goveđeg embrija koji je dodan. Nakon 3. dana kultiviranja, otopina kulture je ponovo zamijenjena sa RPMI 1640 koji ne sadrži serum i supernatant kulture je 4 dana kasnije uklonjen. Ponavlja-njem ovoga postupka, dobiveno je po 500 ml supernatanta koji ne sadrži serum svakog tjedna. Dalje ova metoda omogućava izdvajanje supernatanta kulture, sa stanicama održavanim iznad znatno produženog perioda. Cells based on example 1 were grown in a fairly dense population in two culture flasks (150 cm 3 ). Cells were isolated, suspended in 500 ml of F-10 culture solution containing 10% fetal bovine serum, transferred to a 1580 cm 3 glass rotary flask (Delco), and cultured by rotation at 0.5 rpm. When the cells were found to have grown in a fairly dense population on the inner wall of the rotating flask, the culture solution was replaced with serum-free RPMI 1640. After 4 days of cultivation, the culture supernatant was removed and cultivation was continued with F-10 containing 10% fetal bovine serum added. After the 3rd day of cultivation, the culture solution was replaced again with serum-free RPMI 1640 and the culture supernatant was removed 4 days later. By repeating this procedure, 500 ml of serum-free supernatant was obtained every week. Furthermore, this method enables extraction of the culture supernatant, with cells maintained over a significantly extended period.
Serija koja sadrži 5000 ml supernatanta kulture dobivena je miješanjem sa 0,1% Tween 20 i koncentrirana oko 1000 puta ultrafiltriranjem sa Hollow Fiber DC-4 i Amicon PM-10 (Amicon). Koncentrat je pročišćen u sljedećim stupnjevima: A batch containing 5000 ml of culture supernatant was obtained by mixing with 0.1% Tween 20 and concentrated about 1000 times by ultrafiltration with Hollow Fiber DC-4 and Amicon PM-10 (Amicon). The concentrate is purified in the following stages:
(i) Dio (5 ml) koncentriranog supernatanta kulture podvrgnuto je gel filtriranju na Ultrogen AcA54 koloni (promjera 4,6 cm i dužine 90 cm; LKB) pri protoku od oko 50 ml/sat sa 0,01 M Tris-HCl puferom (pH 7,4) koji sadrži 0,15 M NaCl i 0,01% Tween 20 (Nakai Kagaku Co., Ltd.). Kolona je kalibrirana sa albumin se-rumom goveda (M 67000), ovoalbuminom (M 45000) i citokromom C (M 12400). Nakon završetka filtriranja, 0,1 ml svake frakcije je razrijeđeno 10 puta i testirano na aktivnost gore opisanom metodom ispitivanja CSA (b). Frakcije 400-700 ml, nađeno je, pokazuju makrofaga-dominantnu CSA dok frakcije 800-1200 ml pokazuju granulocit-dominantnu CSA. Stoga, zadnje frakcije su skupljene i koncentrirane na volumen od oko 5 ml na ultrafiltracijskoj aparaturi sa PM-10 (Amico). (i) A portion (5 ml) of the concentrated culture supernatant was subjected to gel filtration on an Ultrogen AcA54 column (diameter 4.6 cm and length 90 cm; LKB) at a flow rate of about 50 ml/hr with 0.01 M Tris-HCl buffer ( pH 7.4) containing 0.15 M NaCl and 0.01% Tween 20 (Nakai Kagaku Co., Ltd.). The column was calibrated with bovine serum albumin (M 67000), ovalbumin (M 45000) and cytochrome C (M 12400). After completion of filtration, 0.1 ml of each fraction was diluted 10-fold and tested for activity using the CSA assay method described above (b). Fractions 400-700 ml were found to show macrophage-dominant CSA while fractions 800-1200 ml showed granulocyte-dominant CSA. Therefore, the last fractions were collected and concentrated to a volume of about 5 ml on an ultrafiltration apparatus with PM-10 (Amico).
(ii) U koncentrirane frakcije dodana je vodena otopina 0,1% trifluoroctene kiseline koja sadrži 30% n-propanola (za određivanje aminokiselinskog niza; dobivene od Tokyo Kasei K.K.). Smjesa je zatim ostavljena da stoji u ledu oko 15 minuta, talog je uklonjen centrifugiranjem tijekom 10 minuta na 15000 okretaja/min. Supernatant je adsorbiran na µ-Bondpak C18 koloni (8 mmx30 cm za polupreparacijsko korištenje; Waters (uravnotežen s vodenom otopinom koja sadrži n-propanol i trifluoroctenu kiselinu; kolona je kontinuirano eluirana s vodenom otopinom 0,1% trifluorocotene kiseline koja sadrži n-propanol koja ima linearni koncentracijski gradijent od 30-60%. Aparatura za tekućinsku kromatografiju na visokom tlaku, Hitachi Model 685-50 (Hitachi, Ltd.) i detektor, Hitachi Model 638-41 (Hitachi, Ltd.) su korišteni za određivanje adsorpcije na 220 nm i 280 nm, istovremeno. Nakon eluiranja, 10 µl svake frakcije je razrijeđeno 100 puta i testirano na aktiv-ne frakcije gore opisanom metodom ispitivanja CSA (b). Pikovi eluirani sa 40% n-propanolom nađeno je da imaju CSA aktivnost, tako da su oni prikupljeni i ponovno kromatografirani pod istim uvjetima, te ispitivani na CSA istom metodom. Ponovno, CSA aktivnost je opažena u pikovima na 40% n-propanolu. Stoga, ovi pikovi su skupljeni (4 frakcije = 4 ml) i suho zamrznuti. (ii) An aqueous solution of 0.1% trifluoroacetic acid containing 30% n-propanol (for amino acid sequence determination; obtained from Tokyo Kasei K.K.) was added to the concentrated fractions. The mixture was then allowed to stand in ice for about 15 minutes, the precipitate was removed by centrifugation for 10 minutes at 15000 rpm. The supernatant was adsorbed on a µ-Bondpak C18 column (8 mmx30 cm for semipreparative use; Waters (equilibrated with an aqueous solution containing n-propanol and trifluoroacetic acid; the column was continuously eluted with an aqueous solution of 0.1% trifluoroacetic acid containing n-propanol which has a linear concentration gradient of 30-60%. High pressure liquid chromatography apparatus, Hitachi Model 685-50 (Hitachi, Ltd.) and detector, Hitachi Model 638-41 (Hitachi, Ltd.) were used to determine adsorption on 220 nm and 280 nm, simultaneously. After elution, 10 µl of each fraction was diluted 100 times and tested for active fractions by the CSA assay method described above (b). Peaks eluted with 40% n-propanol were found to have CSA activity, so they were collected and rechromatographed under the same conditions, and assayed for CSA by the same method. Again, CSA activity was observed in peaks at 40% n-propanol. Therefore, these peaks were pooled (4 fractions = 4 ml) and dried with to freeze.
(iii) Suho zamrznuti prah je otopljen u 200 µl vodene otopine 0,1% trifluoroctene kiseline koja sadrži 40% n-propanol i otopina je podvrgnuta tekućinskoj kromatografiji na visokom tlaku na TSK-G 3000SW koloni (Toya Soda Manufacturing Co., Ltd.; 7,5 mmx60 cm). Eluiranje je vršeno istom vodenom otopinom pri protoku od 0,4 ml/min a frakcije su uzimane u porcijama od 0,4 ml sa sakupljačem frakcija, FRAC-100 (Pharmacia Fine Chemicals). Svaka frakcija je ispitivana na CSA istom metodom kao što je opisano naprijed i aktivnost je opažena u frakcijama za retencijska vremena od 37-38 minuta (koja odgovaraju molekularnoj masi od 2x104). Aktivne frakcije su izdvojene i pročišćene na analitičkoj µ-Bond-pak C18 koloni (4,6 mmx30 cm). Glavni pikovi su izdvojeni i suho zamrznuti. Dobiveni uzorak ispitivan je metodom CSA ispitivanja (a); nađeno je da ima humanu G-CSF aktivnost. (iii) The freeze-dried powder was dissolved in 200 µl of an aqueous solution of 0.1% trifluoroacetic acid containing 40% n-propanol and the solution was subjected to high pressure liquid chromatography on a TSK-G 3000SW column (Toya Soda Manufacturing Co., Ltd. ; 7.5 mmx60 cm). Elution was performed with the same aqueous solution at a flow rate of 0.4 ml/min and fractions were taken in portions of 0.4 ml with a fraction collector, FRAC-100 (Pharmacia Fine Chemicals). Each fraction was assayed for CSA by the same method as described above and activity was observed in fractions for retention times of 37-38 minutes (corresponding to a molecular weight of 2x10 4 ). Active fractions were separated and purified on an analytical µ-Bond-pak C18 column (4.6 mmx30 cm). The main peaks were separated and freeze-dried. The obtained sample was tested using the CSA test method (a); was found to have human G-CSF activity.
Primjer 3: Example 3:
Određivanje aminokisleinskog niza Determination of amino acid sequence
(i) Određivanje N-terminala aminokiselinskog niza (i) Determination of the N-terminal amino acid sequence
Uzorak je podvrgnut Edman razlaganju sa plinko-faznim sekvencerom (App. Biosystems) i dobivena PTH aminokiselina je analizirana rutinskim postupkom sa aparaturom za tekućinsku kromatografiju na visokom tlaku (Beckman Instruments) i Ultrasphere-ODS koloni (Beckman Instruments). Kolona (5 µm; 4,6 mmx250 mm) je uravnotežena s polaznim puferom (vodena otopina koja sadrži 15 mM natrij-acetatnog pufera (pH 4,5 i 40% acetonitrila) i ubačenim sa uzorkom (otopljenog u 20 µl polaznog pufera). Odvajanje je izvršeno jednokratnim eluiranjem s polaznim puferom. Protok je bio 1,4 ml/min a kolona je održavana na temperaturi od 40°C. Detekcija PTH aminokiseline je izvršena korištenjem apsorpcije u UV području na 260 nm i 320 nm. Standardni uzorci PTH aminokiseline (Sigma) u 2-nmol obrocima su razdvojeni na istoj liniji radi određivanja njenog retencijskog vremena, koje je usporedeno sa onim za uzorak koji je ispitivan. Nađeno je da uzorak ima sljedeći aminokiselinski niz od 40 ostataka N-terminala: The sample was subjected to Edman digestion with a plinko-phase sequencer (App. Biosystems) and the obtained PTH amino acid was analyzed by a routine procedure with a high-pressure liquid chromatography apparatus (Beckman Instruments) and an Ultrasphere-ODS column (Beckman Instruments). The column (5 µm; 4.6 mmx250 mm) was equilibrated with starting buffer (aqueous solution containing 15 mM sodium acetate buffer (pH 4.5 and 40% acetonitrile)) and loaded with sample (dissolved in 20 µl of starting buffer). The separation was performed by elution once with the starting buffer. The flow rate was 1.4 ml/min and the column was maintained at a temperature of 40°C. Detection of PTH amino acid was performed using absorption in the UV region at 260 nm and 320 nm. Standard samples of PTH amino acid (Sigma) in 2-nmol aliquots were separated on the same line to determine its retention time, which was compared with that of the sample under investigation.The sample was found to have the following N-terminal 40-residue amino acid sequence:
[image] [image]
(ii) Razlaganje sa bromcijanom (ii) Decomposition with cyanide bromine
Uzorak je otopljen u 70% mravljoj kiselini. Otopini je dodano 200 ekvivalentnih količina bromcijana koji je bio pročišćen sublimacijom. Smjesa je ostavljena preko noći na 37°C radi reakcije. Produkt reakcije je suho zamrznut i frakcioniran pomoću HPLC na TSK G3000SW koloni (Toyo Soda Manufacturing Co., Ltd.) tako da se dobivaju 4 pika. Pikovi su nazvani CN-1, CN-2, CN-3 i CN-4 sa opadajućim molekularnim masama. Prva dva pika (CN-1 i CN-2) imaju bolje prinose i njihovi aminokiselinski nizovi su analizirani sa automatskim sekvencerom (App. Biosystems) u istim uvjetima kao u (i). The sample was dissolved in 70% formic acid. 200 equivalent amounts of cyanide bromine, which had been purified by sublimation, were added to the solution. The mixture was left overnight at 37°C for reaction. The reaction product was freeze-dried and fractionated by HPLC on a TSK G3000SW column (Toyo Soda Manufacturing Co., Ltd.) to give 4 peaks. The peaks are named CN-1, CN-2, CN-3 and CN-4 with decreasing molecular masses. The first two peaks (CN-1 and CN-2) have better yields and their amino acid sequences were analyzed with an automatic sequencer (App. Biosystems) under the same conditions as in (i).
Nađeno je da je CN-1 peptid sastavljen od N-terminala G-CSF proteina, a CN-2 je sljedeći aminokiselinski niz: CN-1 was found to be a peptide composed of the N-terminal of the G-CSF protein, and CN-2 was the following amino acid sequence:
[image] [image]
(iii) Razlaganje sa tripsinom (iii) Digestion with trypsin
Uzorak je otopljen u 0,1 M Tris-HCl puferu (pH 7,4) koji sadrži 8M uree i otopina je izmješana sa 0,1 M Tris-HCl puferom (pH 7,4) koji sadrži 0,1% 2-merkaptoetanola tako da se dobiva završna koncentracija uree od 2 M. TPCK-tretirani tripsin (Sigma) tako je dodan da odnos uzorak:enzim bude 50:1. Smjesa je 4 sata držana na 25°C i nakon dodatka iste količine TPCK-tretiranog tripsina, smjesa je dodatno držana 16 sati na 25°C. Zatim, produkt reakcije je podvrgnut reverzno-faznoj brzoj kromatografiji na koloni (Yamamura Kagaku K.K.), sa eluiranjem izvedenim sa 0,1% THA koji sadrži n-propanol koji ima linearni gradijent gustoće od 5 do 60%. The sample was dissolved in 0.1 M Tris-HCl buffer (pH 7.4) containing 8 M urea and the solution was mixed with 0.1 M Tris-HCl buffer (pH 7.4) containing 0.1% 2-mercaptoethanol. to give a final urea concentration of 2 M. TPCK-treated trypsin (Sigma) was added so that the sample:enzyme ratio was 50:1. The mixture was kept at 25°C for 4 hours and after the addition of the same amount of TPCK-treated trypsin, the mixture was additionally kept at 25°C for 16 hours. Next, the reaction product was subjected to reverse-phase flash column chromatography (Yamamura Kagaku K.K.), with elution performed with 0.1% THA containing n-propanol having a linear density gradient from 5 to 60%.
Mada je dobiveno više pikova mjerenjem apsorpcije na 280 nm, glavni pik je analiziran na svoj aminokiselinski niz sa automatskim plinsko-faznim sekvencerom (App. Biosystems) u istim uvjetima kao u (i). Nađeno je da je glavni pik peptid koji ima sljedeći niz koji sadrži dio CN-2 fragmenta prikazanog u (ii): Although multiple peaks were obtained by measuring absorbance at 280 nm, the main peak was analyzed for its amino acid sequence with an automatic gas-phase sequencer (App. Biosystems) under the same conditions as in (i). The major peak was found to be a peptide having the following sequence containing part of the CN-2 fragment shown in (ii):
[image] [image]
Primjer 4: Example 4:
Pripremanje DNA probe Preparation of DNA test
(i) Sinteza probe (i) Probe synthesis
30 sukcesivnih nukleotida (vidi sliku 1) je dobiveno na bazi niza 10 aminokiselina (Ile-Trp-Gln-Met-Glu-Glu-Gly-Met) uključenih u aminokiselinski niz dobiven u primjeru 3(iii). Bit će potrebno dati jedan komentar o označavanju nukleotida prikazanih na slici 1; na primjer, nukleotid na 9-položaju od 5'-termmala je ekvimolama smjesa dA i dG. Polazni nukleotidi su najviše dimeri ali su također korišteni i monomeri ako je to traženo. Staklena filtar kolona napunjena je sa 20 mg polazne nukleotidne smole, Ap-d(G) (Yamasa Shoyu Co., Ltd). Nakon ponovljenog pranja s metilen kloridom 4,4'-dimetiloksitritil grupa je eliminirana tretiranjem sa otopinom metilen klorida koji sadrži 3% trikloroctenu kiselinu. Zatim je kolona isprana više puta sa 1 ml metilen klorida. Kolona je onda isprana sa anhidriranim piridinom radi istiskivanja otapala, 20 mg nukletidnog dimera (DMTr)ApTp(NH3), (Nippon Zeon; NH3=trietilamonij; DMTr=dimetoksitritil) i dodano je 0,2 ml piridina, i unutrašnjost kolone je vakuumski osušena sa vakuum pumpom. Zatim je dodano 20 mg 2,4,6-trimetilbenzolsulfonil-3-nitrotiazolida (MSNT Wako Pure Chemical Industries, Ltd.) i 0,2 ml anhidriranog piridina, i unutrašnjost kolone je isprana sa plinovitim dušikom. Nukleotidna smola je kondenzirana sa dimerom reakcijom u trajanju od 45 minuta na sobnoj temperaturi, sa povremenim mućka-njem. Po završetku reakcije kolona je isprana sa piridinom i neizreagirane OH grupe su acetilizirane sa otopinom piridina, koja sadrži višak acetanhidrida i 4-dimetilamino piridina. Poslije ispiranja kolone sa piridinom, kondenzirani su sljedeći dimeri ili monomeri, po napisanom poretku, ponavljanjem gore opisanih postupaka; (DMTr)Ip(NH3), (DMTr)GpGp(NH3), (DMTr)Ip(NH3), ekvimolarna smjesa (DMTr)CpTp(NH3) i (DMTr)TpTp(NH3), ekvimolarna smjesa (DMTr) ApAp(NH3) i (DMTr)ApGp(NH3), (DMTr)GpAp(NH3), (DMTr)TpGp(NH3), ekvimolarna smjesa (DMTr) ApAp(NH3) i (DMTr)GpAp(NH3), (DMTr)CpAp(NH3), ekvimolarna smjesa (DMTr)ApAp(NH3), (DMTr) ApGp(NH3), (DMTr)GpCp(NH3), (DMTr)TpGp(NH3), (DMTr)Ip(NH3) i (DMTr)ApTp(NH3), sa svim ovim nukleotidima raspoloživim od Nippon Zeon, osim za (DMTr)Ip(NH3), koji je bio raspoloživ od Yamasa Shoyu Co., Ltd. Po završetku reakcije u zadnjem stupnju, smola je isprana sa piridinom, a zatim sa metilen kloridom i najzad eterom bez acetiliranja, a onda osušena. Osušena smola je suspendirana u 1,7 ml smjese piridina (0,5 ml) vode (0,2 ml) i dioksana (1 ml), koja sadrži 1 M tetrametilguanidin i 1 M α-pikolinaldoksim. Suspenzija je ostavljena da stoji preko noći na sobnoj temperaturi, i onda koncentrirana na 100-200 µ1 pod vakuumom. Koncentrat je izmješan sa malom količinom (2-3 kapi) piridina i 2-3 ml koncentriranog otopljenog amonijaka, i smjesa je grijana na 55°C 6 sati. Koristeći ekstrakciju sa etil acetatom, vodeni sloj je odvojen i koncentriran pod vakuumom. Koncentrat je rastvoren u otopini 50 mM trietil amonij acetata (pH 7) i otopina je podvrgnuta kromatografiji na C-18 koloni (1,0x15 cm; Waters), sa eluiranjem koje je vršeno acetonitrilom (linearni gradijent gustoće od 10-30%) u otopini od 50 mM trietilamonij acetata (pH 7,0). Eluirana pik frakcija pri koncentraciji acetonitrila od oko 25% je koncentrirana pod vakuumom. 30 successive nucleotides (see Figure 1) were obtained based on a sequence of 10 amino acids (Ile-Trp-Gln-Met-Glu-Glu-Gly-Met) included in the amino acid sequence obtained in example 3(iii). It will be necessary to make one comment about the labeling of the nucleotides shown in Figure 1; for example, the nucleotide at the 9-position of the 5'-terminus is an equimolar mixture of dA and dG. Starting nucleotides are mostly dimers, but monomers were also used if requested. A glass filter column was loaded with 20 mg of starting nucleotide resin, Ap-d(G) (Yamasa Shoyu Co., Ltd). After repeated washing with methylene chloride, the 4,4'-dimethyloxytrityl group was eliminated by treatment with a methylene chloride solution containing 3% trichloroacetic acid. Then the column was washed several times with 1 ml of methylene chloride. The column was then washed with anhydrous pyridine to expel the solvent, 20 mg of nucleotide dimer (DMTr)ApTp(NH3), (Nippon Zeon; NH3=triethylammonium; DMTr=dimethoxytrityl) and 0.2 ml of pyridine was added, and the inside of the column was vacuum dried. with a vacuum pump. Then, 20 mg of 2,4,6-trimethylbenzenesulfonyl-3-nitrothiazolide (MSNT Wako Pure Chemical Industries, Ltd.) and 0.2 ml of anhydrous pyridine were added, and the inside of the column was flushed with nitrogen gas. The nucleotide resin was condensed with the dimer reaction for 45 minutes at room temperature, with occasional shaking. At the end of the reaction, the column was washed with pyridine and the unreacted OH groups were acetylated with a pyridine solution containing an excess of acetic anhydride and 4-dimethylamino pyridine. After washing the column with pyridine, the following dimers or monomers were condensed, according to the written order, by repeating the procedures described above; (DMTr)Ip(NH3), (DMTr)GpGp(NH3), (DMTr)Ip(NH3), equimolar mixture of (DMTr)CpTp(NH3) and (DMTr)TpTp(NH3), equimolar mixture of (DMTr) ApAp(NH3 ) and (DMTr)ApGp(NH3), (DMTr)GpAp(NH3), (DMTr)TpGp(NH3), an equimolar mixture of (DMTr) ApAp(NH3) and (DMTr)GpAp(NH3), (DMTr)CpAp(NH3 ), an equimolar mixture of (DMTr)ApAp(NH3), (DMTr)ApGp(NH3), (DMTr)GpCp(NH3), (DMTr)TpGp(NH3), (DMTr)Ip(NH3) and (DMTr)ApTp(NH3 ), with all these nucleotides available from Nippon Zeon, except for (DMTr)Ip(NH3), which was available from Yamasa Shoyu Co., Ltd. At the end of the reaction in the last step, the resin was washed with pyridine, then with methylene chloride and finally with ether without acetylation, and then dried. The dried resin was suspended in 1.7 ml of a mixture of pyridine (0.5 ml), water (0.2 ml) and dioxane (1 ml), containing 1 M tetramethylguanidine and 1 M α-picolinaldoxime. The suspension was allowed to stand overnight at room temperature, and then concentrated to 100-200 µl under vacuum. The concentrate was mixed with a small amount (2-3 drops) of pyridine and 2-3 ml of concentrated dissolved ammonia, and the mixture was heated at 55°C for 6 hours. Using extraction with ethyl acetate, the aqueous layer was separated and concentrated under vacuum. The concentrate was dissolved in a solution of 50 mM triethyl ammonium acetate (pH 7) and the solution was subjected to chromatography on a C-18 column (1.0x15 cm; Waters), eluting with acetonitrile (10-30% linear density gradient) in solution of 50 mM triethylammonium acetate (pH 7.0). The eluted peak fraction at an acetonitrile concentration of about 25% was concentrated under vacuum.
Koncentratu je dodana 80% octena kiselina i smjesa je ostavljena da stoji 30 minuta na sobnoj temperaturi. Koristeći ekstrakciju sa etil acetatom, odvojen je vodeni sloj i koncentriran pod vakuumom. Dobiveni koncentrat je dalje pročišćavan pomoću HPLC na C-18 koloni (od Senshu Kagaku K.K.; SSC-ODS-272; 6mmx 200 mm). Eluiranje je vršeno sa acetonitrilom (10-20% linearni gradijent brzine) u otopini 50 mM trietil amonij acetata (pH 7,0). Sintetska DNA je dobivena u prinosu ne manjem od 10A260 jedinica. 80% acetic acid was added to the concentrate and the mixture was left to stand for 30 minutes at room temperature. Using extraction with ethyl acetate, the aqueous layer was separated and concentrated under vacuum. The resulting concentrate was further purified by HPLC on a C-18 column (from Senshu Kagaku K.K.; SSC-ODS-272; 6mmx200mm). Elution was performed with acetonitrile (10-20% linear velocity gradient) in a solution of 50 mM triethyl ammonium acetate (pH 7.0). Synthetic DNA was obtained in a yield of no less than 10A260 units.
Analiza Maxim-Gilbert sekventnom metodom /Math. Enzym., 65, 499(1980)/ pokazuje da dobiveni oligonukleotid ima nukleotidni niz prikazan na slici 1. Maxim-Gilbert analysis using the sequential method /Math. Enzym., 65, 499(1980)/ shows that the resulting oligonucleotide has the nucleotide sequence shown in Figure 1.
(ii) Sinteza probe (A) (ii) Synthesis of test (A)
15 sukcesivnih nukleotida (vidi sliku 1) je dobiveno na osnovi niza 5 amino-kiselina (Met-Pro-Ala-Phe-Ala) uključenih u aminokiselinski niz dobiven u primjeru 3(m). 15 successive nucleotides (see Figure 1) were obtained on the basis of a sequence of 5 amino acids (Met-Pro-Ala-Phe-Ala) included in the amino acid sequence obtained in example 3(m).
Sinteza je slična onoj upotrebljenoj za pripremanje probe (IWQ) i sljedeći nukleotidi su kondenzirani na nukleotidnoj smoli, Ap-d(T) (Yamasa Shoyu Co., Ltd.) u sljedećem poretku: The synthesis is similar to that used for sample preparation (IWQ) and the following nucleotides are condensed on a nucleotide resin, Ap-d(T) (Yamasa Shoyu Co., Ltd.) in the following order:
(DMTr)CpAp(NH3), (DMTr)GpAp(NH3), ekvimolarna smjesa (DMTr)CpAp(NH3), (DMTr)CpTp(NH3), (DMTr)CpGp(NH3), i (DMTr)CpCp(NH3), ekvimolarna smjesa (DMTr)ApGp(NH3), (DMTr)TpGp(NH3), (DMTr)GpGp(NH3), (DMTr)CpGp(NH3), (DMTr)ApAp(NH3), ekvimolarna smjesa (DMTr)CpAp(NH3), i (DMTr)CpGp(NH3), i (DMTr)Gp(NH3), sa svim nukleotidima dobivenim od Nippon Zeon. Sintetska DNA je tako dobivena u prinosu od oko 10A260 jedinica. Analiza pomoću Maxim-Gilbert sekvencijske metode pokazuje da dobiveni oligonukleotid ima nukleotidni niz prikazan na slici 1. (DMTr)CpAp(NH3), (DMTr)GpAp(NH3), an equimolar mixture of (DMTr)CpAp(NH3), (DMTr)CpTp(NH3), (DMTr)CpGp(NH3), and (DMTr)CpCp(NH3) , equimolar mixture of (DMTr)ApGp(NH3), (DMTr)TpGp(NH3), (DMTr)GpGp(NH3), (DMTr)CpGp(NH3), (DMTr)ApAp(NH3), equimolar mixture of (DMTr)CpAp( NH3), and (DMTr)CpGp(NH3), and (DMTr)Gp(NH3), with all nucleotides obtained from Nippon Zeon. Synthetic DNA was thus obtained in a yield of about 10A260 units. Analysis using the Maxim-Gilbert sequencing method shows that the resulting oligonucleotide has the nucleotide sequence shown in Figure 1.
(iii) Sinteza probe (LC) (iii) Trial Synthesis (LC)
Izvršena je automatska DNA sinteza sa DNA sintetizatorom, model 380A Applied Biosystems. Ova tehnika je zasnovana na principima koje su dali Caratherus et al., /J. Am. Chem. Soc., 103, 3185(1981)/, i općenito je označena kao fosforamiditni postupak. Automatic DNA synthesis was performed with a DNA synthesizer, model 380A Applied Biosystems. This technique is based on the principles given by Caratherus et al., /J. Am. Chem. Soc., 103, 3185(1981)/, and is generally referred to as the phosphoramidite process.
Fosforamidit iz (DMTr)-dT prethodno aktiviran sa tetrazolom je kondenziran u dG-S (S:nosač) gdje se 5'-dimetoksinitrilna grupa (DMTr) deblokira. Zatim se neizreagirane hidroksilne grupe aciliraju i oksidiraju jodom u prisutnosti vode tako da nastaje fosforilna grupa. Nakon deblokiranja DMTr grupe, kondenzacija je ponovljena na isti način nisu sintetizirana 24 nukleotida koji imaju niz prikazan na slici 1. Ovi nukleotidi su odvojeni od nosača, deblokirani i pročišćeni reverzno-faznom HPLC na C-18 koloni (Senshu Kagaku Co., Ltd.; SSC-ODS-272). Phosphoramidite from (DMTr)-dT previously activated with tetrazole is condensed to dG-S (S:support) where the 5'-dimethoxynitrile group (DMTr) is deblocked. Then the unreacted hydroxyl groups are acylated and oxidized with iodine in the presence of water so that a phosphoryl group is formed. After unblocking the DMTr group, the condensation was repeated in the same way, and 24 nucleotides with the sequence shown in Figure 1 were not synthesized. These nucleotides were separated from the support, deblocked and purified by reverse-phase HPLC on a C-18 column (Senshu Kagaku Co., Ltd. ; SSC-ODS-272).
Primjer 5: Example 5:
Kultiviranje i odvajanje CHU-2 stanica Cultivation and isolation of CHU-2 cells
1) 1)
Zasnovane CHU-2 stanice su izrasle u potpuno gustoj populaciji u dvije boce za kultiviranje (150 cm3), izdvojene, suspendirane u 500 ml RPMI 1640 otopine kulture koja sadrži 10% seruma embrija goveda, prebačene su u staklenu rotirajuću tikvicu od 1580 cm3 (Belco) i kultivirane uz miješanje 4 dana pri 0,5 okr/min. Kada je utvrđeno da izrasle stanice na unutrašnjima stijenkama tikvice imaju sasvim gustu populaciju, otopina kulture je uklonjena iz rotirajuće tikvice, koja je dopunjena sa 100 ml fiziološke slane otopine zagrijane na 37°C koja sadrži 0,02% EDTA. Nakon grijanja 2 minute na 37°C, stanice su odvojene od unutrašnje stijenke tikvice pipetiranjem. Dobivena suspenzija stanica je centrifugirana na 1500 okr/min 10 minuta tako da se dobiva tableta stanica. Stanice su ponovo suspendirane u 5 ml fiziološke slane otopine koja ne sadrži EDTA. Suspenzija je centrifugirana na 1500 okr/min 10 minuta tako da se dobije tableta stanica (mase oko 0,8 g). Tako dobivene stanice su ostavljene zamrznute na -80°C dok se ne podvrgnu postupku ekstrakcije RNA. Established CHU-2 cells were grown to full density in two culture flasks (150 cm3), separated, suspended in 500 ml RPMI 1640 culture solution containing 10% fetal bovine serum, transferred to a 1580 cm3 glass rotary flask (Belco ) and cultivated with stirring for 4 days at 0.5 rpm. When it was determined that the grown cells on the inner walls of the flask had quite a dense population, the culture solution was removed from the rotating flask, which was supplemented with 100 ml of physiological saline solution heated to 37°C containing 0.02% EDTA. After heating for 2 minutes at 37°C, the cells were separated from the inner wall of the flask by pipetting. The resulting cell suspension was centrifuged at 1500 rpm for 10 minutes to obtain a cell pellet. The cells were resuspended in 5 ml of EDTA-free physiological saline. The suspension was centrifuged at 1500 rpm for 10 minutes to obtain a pellet of cells (weight about 0.8 g). The cells thus obtained were left frozen at -80°C until they were subjected to the RNA extraction procedure.
2) Pročišćavanje mRNA 2) Purification of mRNA
Izoliranja mRNA iz CHU-2 stanica dobivenih u 1) su temeljena postupcima koji su bili bitno isti s onima opisanima u "Molecular Cloning", Maniatis et al., Cold Spring Harbor, str. 196, 1982. Zamrznute CHU-2 stanice (vlažne mase, 3,8 g) su suspendirane u 20 ml otopine 6M guanidina /(M guanidinij izocijanata, 5 mM natrij-citrata (pH 7,0), 0,1 M β-merkaptoetanola i 0,5 natrij-sarkozil sulfata/ i suspenzija je dobro miješana 2-3 minute. Smjesa je podvrgnuta 10-strukom izvlačenju i ubacivanju štrcaljkom (kapaciteta 20 ml) sa 18G iglom. Oko 6 ml viskozne guanidinij otopine koja sadrži razorene stanice je rašireno na 6 ml podloge 5,7 M CsCl u 0,1M EDTA (pH 7,5) u Beckman SW40 Ti poliallomer epruveti za centri-fugiranje tako da epruveta bude sasvim puna. Pripremljene su 4 epruvete postupcima opisanim naprijed i centnfugirane su na 30000 okr/min 15 sati na 20°C. Dobivene tablete su tri puta isprane vodom s malom količinom 70% etanola. Isolations of mRNA from CHU-2 cells obtained in 1) were based on procedures that were essentially the same as those described in "Molecular Cloning", Maniatis et al., Cold Spring Harbor, p. 196, 1982. Frozen CHU-2 cells (wet weight, 3.8 g) were suspended in 20 ml of a solution of 6M guanidine/(M guanidinium isocyanate, 5 mM sodium citrate (pH 7.0), 0.1 M β- mercaptoethanol and 0.5 sodium sarcosyl sulfate/ and the suspension was mixed well for 2-3 minutes. The mixture was withdrawn and injected 10 times with a syringe (capacity 20 ml) with an 18G needle. About 6 ml of the viscous guanidinium solution containing the disrupted cells was spread on 6 ml medium of 5.7 M CsCl in 0.1 M EDTA (pH 7.5) in a Beckman SW40 Ti polyallomer centrifuge tube so that the tube is completely full. 4 tubes were prepared by the procedures described above and centrifuged at 30000 rpm for 15 hours at 20° C. The resulting tablets were washed three times with water and a small amount of 70% ethanol.
Tablete dobivene iz posebnih epruveta su kombinirane, otopljene u 560 µl vode i obrađene tako da se postigne koncentracija NaCl 0,2 M. Nakon tretiranja sa 1:1 smjesom fenola i kloroforma i sa samim kloroformom, dodano je 2,5 volumena etanola tako da se precipitira ukupna RNA (oko 10,1 mg ukupne RNA je dobiveno iz 3,8 g lažnih stanica). Tablets obtained from special test tubes were combined, dissolved in 560 µl of water and processed to reach a NaCl concentration of 0.2 M. After treatment with a 1:1 mixture of phenol and chloroform and with chloroform itself, 2.5 volumes of ethanol were added so that total RNA is precipitated (about 10.1 mg of total RNA was obtained from 3.8 g of mock cells).
Poli(A+)RNA je pročišćena iz ukupne RNA pomoću sljedećih postupaka afinitetne kromatografije koja koristi prednost vezanja poli(A) lanca na 3' terminalu mRNA. Adsorpcija na oligo(dT)-celulozi (Tip 7 P-L Biochem.) je postignuta propuštanjem kroz oligo(dT)-celuloznu kolonu ukupne RNA u nosećem puferu (koji sadrži 10 mM Tris-Hd (pH 7,5), 0,5 M NaCl, 1 mM EDTA i 0,1% SDS otopine) pošto je otopina grijana 5 minuta na 65°C. Poly(A+)RNA was purified from total RNA using the following affinity chromatography procedures that take advantage of binding of the poly(A) strand at the 3' terminus of the mRNA. Adsorption on oligo(dT)-cellulose (Type 7 P-L Biochem.) was achieved by passing total RNA through an oligo(dT)-cellulose column in carrier buffer (containing 10 mM Tris-Hd (pH 7.5), 0.5 M NaCl, 1 mM EDTA and 0.1% SDS solution) as the solution was heated for 5 minutes at 65°C.
Kolona je uravnotežena sa istim nosećim puferom. Eluiranje poli(A+)RNA je vršeno sa TE otopinom (koja sadrži 10 mM Tris-HCl (pH 7,5) i 1 mM EDTA). Neadsorbirani efluent se ponovno propušta kroz kolonu i eluat dobiven ponavljanjem istih postupaka se miješa sa eluatom dobivenim u prvom eluiranju. Na ovaj način, dobiveno je 400 µg poli(A+)RNA. The column is equilibrated with the same carrier buffer. Elution of poly(A+)RNA was performed with TE solution (containing 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA). The non-adsorbed effluent is again passed through the column and the eluate obtained by repeating the same procedures is mixed with the eluate obtained in the first elution. In this way, 400 µg of poly(A+)RNA was obtained.
Tako dobivena mRNA je frakcionirana po veličini čestica pomoću centrifugalnog gradijenta gustoće saharoze prema postupcima opisanim u laboratorijskom priručniku Schleif i Wensink "Practical Methods in Molecular Biology", Springer-Verlag, New York, Heidelberg, Berlin (1981). The thus obtained mRNA was fractionated by particle size using a centrifugal sucrose density gradient according to the procedures described in the laboratory manual Schleif and Wensink "Practical Methods in Molecular Biology", Springer-Verlag, New York, Heidelberg, Berlin (1981).
Određenije, gradijent gustoće 5-25% saharoze je stvoren u Beckman SV40 Ti centrifugalnoj cijevi. Napravljene su dvije otopine saharoze otapanjem 5% i 25% otopine saharoze koje ne sadrže RNase (Schwarz/Mann) u otopini koja sadrži 0,1 M NaCl, 10 mM Tris-HCl (pH 7,5), 1 mM EDTA i 0,5% SDS. Specifically, a 5-25% sucrose density gradient was created in a Beckman SV40 Ti centrifuge tube. Two sucrose solutions were made by dissolving 5% and 25% RNase-free sucrose solutions (Schwarz/Mann) in a solution containing 0.1 M NaCl, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 0. 5% SDS.
800 µg m RNA /poli (A+)-RNA/ dobivene prema već opisanoj metodi, otopljeno je u 200-500 µl TE otopine. Otopina je grijana 5 minuta na 65°C, poslije čega je naglo ohlađena i stavljena u gradijent gustoće otopina saharoze, koje su centrifugirane na 30000 obr/min 20 sati. Frakcije od 0,5 ml su sakupljane i izmjerena im je adsorpcija na 260 nm. Veličine frakcionirane RNA su određene na osnovu položaja standarda RNA (ribosa RNAs 28S, 18S, i 5S). U isto vrijeme G-CSF aktivnost svake frakcije je ispitivana oocitima Xenopus laevis-a sljedećim postupcima. Prvo, mRNA svake frakcije je preveden u vodenu otopinu, koja ima koncentraciju od 1 µg/µl; oociti su uzeti od Xenopus (starog oko 1 godine) i mRNA otopina je ubačena na takav način da je 50 ng mRNA ubačeno u jedan oocit; deset takvih oocita je stavljeno u svaki od 96 otvora mikrotitar plitice; oociti su kultivirani 48 sati na sobnoj temperaturi u 100 µ1 Barth sredine (88 mM NaCl; 1 mM KCl; 2,4 mM NaHCO3 0,82 mM MgSO4 0,33 mM Ca(NO3)2; 0,41 mM CaCl2; 7,5 mM Tris-HCl (pH 7,6); 10 mg/1 penicilina i 10 mg/1 streptomicin sulfata); izdvojen je supernatant kulture, koncentriran i pročišćen do pogodnog stupnja za ispitivanje G-CSF aktivnosti. 800 µg m RNA /poly (A+)-RNA/ obtained according to the already described method, was dissolved in 200-500 µl of TE solution. The solution was heated for 5 minutes at 65°C, after which it was suddenly cooled and placed in a density gradient of sucrose solutions, which were centrifuged at 30,000 rpm for 20 hours. Fractions of 0.5 ml were collected and their adsorption was measured at 260 nm. Sizes of fractionated RNA were determined based on the position of RNA standards (ribose RNAs 28S, 18S, and 5S). At the same time, the G-CSF activity of each fraction was tested with Xenopus laevis oocytes by the following procedures. First, the mRNA of each fraction was translated into an aqueous solution, which has a concentration of 1 µg/µl; oocytes were taken from Xenopus (about 1 year old) and the mRNA solution was injected in such a way that 50 ng of mRNA was injected into one oocyte; ten such oocytes were placed in each of the 96 wells of the microtiter plate; oocytes were cultured for 48 hours at room temperature in 100 µl of Barth medium (88 mM NaCl; 1 mM KCl; 2.4 mM NaHCO3 0.82 mM MgSO4 0.33 mM Ca(NO3)2; 0.41 mM CaCl2; 7, 5 mM Tris-HCl (pH 7.6); 10 mg/1 penicillin and 10 mg/1 streptomycin sulfate); the culture supernatant was separated, concentrated and purified to a level suitable for testing G-CSF activity.
G-SCF aktivnost nađeno je da je prisutna u 17S frakcija. G-SCF activity was found to be present in the 17S fraction.
Primjer 6: Example 6:
Sinteza cDNA (stvaranje pBR-linije cDNA biblioteke) Synthesis of cDNA (creation of pBR-line cDNA library)
Od poli(A+) RNA dobivene u primjeru 6 sintetizirana je cDNA postupkom Land-a et al. /Nucleic Acids Res., 9, 2251 (1981)/ kojega su modificirali Gubler i Hoffman /Gene, 25, 263(1983)/. From the poly(A+) RNA obtained in example 6, cDNA was synthesized by the procedure of Land et al. /Nucleic Acids Res., 9, 2251 (1981)/ modified by Gubler and Hoffman /Gene, 25, 263 (1983)/.
(1) Sinteze jednostruke cDNA (1) Synthesis of single-stranded cDNA
Eppendorfova cijev (kapaciteta 1,5 ml) je napunjena reagensima sljedećim redoslijedom: 80 µl reakcijskog pufera (500 mM KCl, 50 mM MgCl2 250 mM Tris-HCl, pH 8,3); 20µ1 200 mM ditiotrietiola, 32 µ1 12,5 mM dNTP (koji sadrži po 12,5 mM od dATP, dGTP, dCTP i dTTP), 10 µl 32P-dCTP (PB 10205 Amersham), 32 µl oligo(dT)12-18 (od P-L Biochem.; 500 µg/ml), 20 µl poli(A+) RNA (2,1 µg/µl) i 206 µ1 destilirane vode. Ukupno 400 µl reakcijske otopine je grijano 5 minuta na 65°C, a zatim 5 minuta na 42°C. Toploj otopini, dodano je 120 jedinica reverzne transkriptaze (Takara Shuzo Co., Ltd.). Prateći reakciju još dva sata na 42°C, dodano je 2 µl RNase inhibitora (Bethesda Research Labo-ratories), 20 µl TE otopine, 16 µ1 100 mM natrij-pirofosfata i 48 jedinica (4 µl) reverzne transkriptaze, i reakcija je dalje vršena 2 sata na 46°C. Reakcija je naglo zaustavljena dodatkom 0,5 M EDTA (8 µl) i 10% SDS (8 µl). Tretiranjem dalje sa fenol/kloroformom i taloženjem sa etanolom (dva puta) dobivena je jednostruka cDNA. An Eppendorf tube (capacity 1.5 ml) was filled with reagents in the following order: 80 µl reaction buffer (500 mM KCl, 50 mM MgCl2 250 mM Tris-HCl, pH 8.3); 20 µl 200 mM dithiotrietiol, 32 µl 12.5 mM dNTP (containing 12.5 mM each of dATP, dGTP, dCTP and dTTP), 10 µl 32P-dCTP (PB 10205 Amersham), 32 µl oligo(dT)12-18 (from P-L Biochem.; 500 µg/ml), 20 µl poly(A+) RNA (2.1 µg/µl) and 206 µl of distilled water. A total of 400 µl of the reaction solution was heated for 5 minutes at 65°C and then for 5 minutes at 42°C. To the warm solution, 120 units of reverse transcriptase (Takara Shuzo Co., Ltd.) were added. Following the reaction for another two hours at 42°C, 2 µl of RNase inhibitor (Bethesda Research Laboratories), 20 µl of TE solution, 16 µl of 100 mM sodium pyrophosphate, and 48 units (4 µl) of reverse transcriptase were added, and the reaction continued performed for 2 hours at 46°C. The reaction was stopped abruptly by the addition of 0.5 M EDTA (8 µl) and 10% SDS (8 µl). By further treatment with phenol/chloroform and precipitation with ethanol (twice), a single-stranded cDNA was obtained.
(2) Pripajanje dC-lanca na jednostruku cDNA (2) Attachment of dC-strand to single-stranded cDNA
Jednostruka cDNA dobivena u (1) je otopljena u destiliranoj vodi. Otopini je dodano 60 µ1 dC-lanca dodavanjem pufera /400 mM kalij-kakodilata, 50 mM Tris-HCl (pH 6,9), 4 mM ditiotrietola, 1 mM CoCl2 1 mM dCTP/ i smjesa je 5 minuta grijana na 37°C. Reakcijskoj otopini je dodano 3 µ1 terminalne transferaze (27 jedinica/µl; P-1 Biochem.) i smjesa je 2,5 minute grijana na 37°C. Tretiranjem dalje sa fenol/kloroformom (jednom) i taloženjem sa etanolom (dva puta), dC-završna cDNA je otopljena u 40 µlTE otopine koja sadrži 100 mM NaCl The single-stranded cDNA obtained in (1) was dissolved in distilled water. 60 µ1 dC-chain was added to the solution by adding a buffer (400 mM potassium cacodylate, 50 mM Tris-HCl (pH 6.9), 4 mM dithiotrietol, 1 mM CoCl2 1 mM dCTP) and the mixture was heated at 37°C for 5 minutes. . 3 µl of terminal transferase (27 units/µl; P-1 Biochem.) was added to the reaction solution and the mixture was heated at 37°C for 2.5 minutes. After further treatment with phenol/chloroform (once) and precipitation with ethanol (twice), the dC-terminated cDNA was dissolved in 40 µl TE solution containing 100 mM NaCl
(3) Sinteza dvostruke cDNA (3) Synthesis of double-stranded cDNA
U 40 µl DNA otopine dobivene u 2), dodano je 4 µl oligo(dG)12-18 (200 µg/ml; P-L Biochem.) i smjesa je prvo grijana 5 minuta na 65°C a zatim 30 minuta na 42°C. Dok je reakcijska otopina održavana na 0°C dodano je 80 µl pufera (100 mM Tris-HCl (pH 7,5), 20 mM MgCl2, 50 mM (NH4)2S04, i 500 mM KCl/, 4 µl 4MM dNTP (koji sadrži po 4 mM dATP, dCTP, dGTP i dTTP), 60 µl 1 mM β-NAD, 210 µl destilirane vode, 20 µ1 E. coli DNA polimeraze I (Takara Shuzo Co., Ltd.), 15 µl E. coli DNA ligaze (Takara Shuzo Co., Ltd.) i 15 µl E. coli RNase H (Takara Shuzo Co., Ltd.) i smjesa je podvrgnuta reakciji na 1°C tijekom 1 sata. Nakon dodatka 4 mM dNTP (4 µl), reakcija je vršena 1 sat na 25°C. Dalje, tretiranjem sa fenol/klorformom i taloženjem sa etanolom (jednom) dobiveno je oko 8 µg dvostruke cDNA. Ova dvostruka cDNA je otopljena u TE otopini i podvrgnuta 1,2 agaroza gel elektroforezi. Fragmenti koji odgovaraju veličini od oko 560 bp do oko 2 kbp adsorbirani su na Whatman DE81 odakle je eluiranjem dobiveno oko 0,2 µg dvostruke cDNA. In 40 µl of the DNA solution obtained in 2), 4 µl of oligo(dG)12-18 (200 µg/ml; P-L Biochem.) was added and the mixture was first heated for 5 minutes at 65°C and then at 42°C for 30 minutes. . While the reaction solution was maintained at 0°C, 80 µl buffer (100 mM Tris-HCl (pH 7.5), 20 mM MgCl2, 50 mM (NH4)2SO4, and 500 mM KCl/, 4 µl 4MM dNTP (which containing 4 mM dATP, dCTP, dGTP and dTTP each), 60 µl 1 mM β-NAD, 210 µl distilled water, 20 µl E. coli DNA polymerase I (Takara Shuzo Co., Ltd.), 15 µl E. coli DNA ligase (Takara Shuzo Co., Ltd.) and 15 µl of E. coli RNase H (Takara Shuzo Co., Ltd.) and the mixture was subjected to reaction at 1°C for 1 hour.After the addition of 4 mM dNTP (4 µl), the reaction was carried out for 1 hour at 25° C. Further, treatment with phenol/chloroform and precipitation with ethanol (once) yielded about 8 µg of double-stranded cDNA. This double-stranded cDNA was dissolved in TE solution and subjected to 1,2 agarose gel electrophoresis. Fragments corresponding to a size of about 560 bp to about 2 kbp were adsorbed on Whatman DE81 from which about 0.2 µg of double-stranded cDNA was obtained by elution.
(4) Pripajanje dC-lanca dvostrukoj cDNA (4) Attachment of the dC-strand to the double-stranded cDNA
Dvostruka cDNA dobivena u 3) otopljena je u 40 µl TE otopine. Zatim je dodano 8 µl dC-završnog pufera tipa navedenog u 2) i smjesa je 2 minute grijana na 37°C. Nakon dodatka 1 µl terminalne transferaze (27 jedinica/µl), smjesa je podvrgnuta reakciji na 37°C tijekom 3 minute. Nakon ovoga, reakcijska otopina je odmah ohlađena na 0°C i reakcija je zaustavljena dodatkom 1 µl 0,5M EDTA. Dalje, nakon tretiranja sa fenol/kloroformom i taloženjem sa etanolom, dobiveni talog je supendiran u 10 µl TE otopine. The double cDNA obtained in 3) was dissolved in 40 µl of TE solution. Then 8 µl of dC-terminating buffer of the type specified in 2) was added and the mixture was heated at 37°C for 2 minutes. After the addition of 1 µl of terminal transferase (27 units/µl), the mixture was subjected to a reaction at 37°C for 3 minutes. After this, the reaction solution was immediately cooled to 0°C and the reaction was stopped by the addition of 1 µl of 0.5M EDTA. Further, after treatment with phenol/chloroform and precipitation with ethanol, the obtained precipitate was suspended in 10 µl of TE solution.
(5) Stvaranje pBR-linije cDNA biblioteke (5) Creation of pBR-line cDNA library
4 mikrolitra komercijalnog oligo(dG)-završnog pBR322 vektora (Bethesda Research Laboratories; 10 µg/µl) i 2 µl dC-završne dvostruke cDNA dobivene u 4) je kaljeno u TE otopini koja sadrži 75 µg 0,1 m NaCl. Kaljenje obuhvata tri stupnja: zagrijavanje na 65°C tijekom 5 minuta; zatim zagrijavanje na 40°C tijekom 2 sata i hlađenje na sobnu temperaturu. 4 microliters of commercial oligo(dG)-terminated pBR322 vector (Bethesda Research Laboratories; 10 µg/µl) and 2 µl of dC-terminated duplex cDNA obtained in 4) were quenched in TE solution containing 75 µg of 0.1 m NaCl. Tempering includes three stages: heating to 65°C for 5 minutes; then heating to 40°C for 2 hours and cooling to room temperature.
Prema metodi opisanoj u laboratorijskom priručniku Maniatis-a et al. /Molecular Cloning, Cold Spring Harbor, str. 249 ff. (1982)/ (mogu se također koristiti i druge rutinske tehnike), odgovarajuće stanice dobivene iz E. coli vrste X1776, i transformirane sa kaljenim plazmidom tako da se dobivaju transformanti. According to the method described in the laboratory manual of Maniatis et al. /Molecular Cloning, Cold Spring Harbor, p. 249 ff. (1982)/ (other routine techniques may also be used), appropriate cells obtained from E. coli strain X1776, and transformed with the hardened plasmid to obtain transformants.
Primjer 7: Example 7:
Sinteza cDNA (Stvaranje biblioteke λ fage) Synthesis of cDNA (Creation of λ phage library)
1) Sinteza jednostruke cDNA 1) Synthesis of single-stranded cDNA
Prema postupcima opisanim u primjeru 5, 3,8 g zamrznutih CHU-2 stanica pročišćenih dva puta na oligo (dT)-celuloznoj koloni prerađeno je tako da se dobiva 400 µg poli(A+)RNA. According to the procedures described in example 5, 3.8 g of frozen CHU-2 cells purified twice on an oligo (dT)-cellulose column were processed to obtain 400 µg of poly(A+)RNA.
TE otopina (10 µl), koji ima 12 µg poli(A+)RNA otopljene u sebi je stavljena u reakcijsku epruvetu, koja sadrži 10 µg aktinomicina D (Sigma). Zatim je epruveta dopunjena sa reagensima po sljedećem redosljedu: 20 µl pufera reversne transkriptaze /250 mM Tris-HCl (pH 8,3); 40 mM MgCl2 250 mM KCl/; 20 µl 5 mM dNTP (koji sadrži po 5 mM dATP, dGTP, dCTP i dTTP); 20 µl oligo (dT)12-18 (0,2 µg/ml; P-L Biokemikalije); 1 µl 1 M ditiotrietiola; 2 µl RNasin-a (30 jedinica/µl; Promega Biotech); 10 µl reverzne transkriptaze (10 jedinica/µl; Seikagaku Kogyo Co.,Ltd.): 1 µl α-32P-dATP (10 µCi; Amerscham); i 16 µ1 vode. The TE solution (10 µl), which has 12 µg of poly(A+)RNA dissolved in it, was placed in a reaction tube containing 10 µg of actinomycin D (Sigma). Then the test tube was supplemented with reagents in the following order: 20 µl reverse transcriptase buffer / 250 mM Tris-HCl (pH 8.3); 40 mM MgCl2 250 mM KCl/; 20 µl of 5 mM dNTP (containing 5 mM each of dATP, dGTP, dCTP and dTTP); 20 µl oligo (dT)12-18 (0.2 µg/ml; P-L Biochemicals); 1 µl 1 M dithiotrietiol; 2 µl of RNasin (30 units/µl; Promega Biotech); 10 µl reverse transcriptase (10 units/µl; Seikagaku Kogyo Co.,Ltd.): 1 µl α-32P-dATP (10 µCi; Amerscham); and 16 µl of water.
Reakcijska otopina je bila ukupno 100 µl i držana je 2 sata na 42°C, a zatim je reakcija zaustavljena dodavanjem 0,5 M EDTA (5 µl) i 20% SDS (1 µl). Tretiranjem sa fenolkloroformom (100 µl) i taloženjem sa etanolom (dva puta), dobiveno je oko 4 µg jednostruke cDNA. The reaction solution was a total of 100 µl and was kept for 2 hours at 42°C, then the reaction was stopped by adding 0.5 M EDTA (5 µl) and 20% SDS (1 µl). By treatment with phenolchloroform (100 µl) and precipitation with ethanol (twice), about 4 µg of single-stranded cDNA was obtained.
2) Sinteza dvostruke cDNA 2) Synthesis of double cDNA
cDNA dobivena u 1 je otopljena u 29 µl TE otopine i reakcijska otopina je dobivena dodavanjem sljedećih reagensa po navedenom redoslijedu: 25 µl pufera polimeraze (400 mM Hepes (pH 7,6); 16 mM MgCl2, 63 mM p-merkaptoetanola, i 270 mM KCl/; 10 µl 5 mM dNTP; 1,0 µl 15 mM β-NAD; 1,0 µl a-32P-dATP (10 µCi/µl); 0,2 µl E. coli DNA polimeraze I (New England Biolabs; 10 jedinica/ µ1); 0,1 µl RNase H (60 jedinica/µl; Takara Shuzo Co., Ltd.); 28,7 µl destilirane vode. The cDNA obtained in 1 was dissolved in 29 µl TE solution and the reaction solution was obtained by adding the following reagents in the order indicated: 25 µl polymerase buffer (400 mM Hepes (pH 7.6); 16 mM MgCl2, 63 mM p-mercaptoethanol, and 270 mM KCl/; 10 µl 5 mM dNTP; 1.0 µl 15 mM β-NAD; 1.0 µl α-32P-dATP (10 µCi/µl); 0.2 µl E. coli DNA polymerase I (New England Biolabs ; 10 units/µ1); 0.1 µl RNase H (60 units/µl; Takara Shuzo Co., Ltd.); 28.7 µl distilled water.
Reakcijska otopina inkubirana je na 14°C 1 sat, a onda ostavljen na sobnoj temperaturi još jedan sat. Tada je reakcija zaustavljena dodatkom 0,5 M EDTA (5 µl) i 20% SDS (1 µl) i zatim tretirana sa fenol/kloroformom i taloženje je izvršeno sa etanolom. Dobivena RNA je otopljena u 20 µ1 0,5 M EDTA i pripremljena je reakcijska otopina dodatkom 3 µl Klenow pufera /500 mM Trias-HCl (pH 8,0) i 50 mM MgCl2/, 3 µl 5 mM dNTP, i 4 µl vode. Po dodatku 1 µl DNA polimeraze (Klenow fragmwnt; Takar Shuzo Co., Ltd.) reakcijska otopina je inkubirana 15 minuta na 30°C. The reaction solution was incubated at 14°C for 1 hour, and then left at room temperature for another hour. Then the reaction was stopped by the addition of 0.5 M EDTA (5 µl) and 20% SDS (1 µl) and then treated with phenol/chloroform and precipitated with ethanol. The obtained RNA was dissolved in 20 µl of 0.5 M EDTA and the reaction solution was prepared by adding 3 µl of Klenow buffer /500 mM Trias-HCl (pH 8.0) and 50 mM MgCl2/, 3 µl of 5 mM dNTP, and 4 µl of water. . After adding 1 µl of DNA polymerase (Klenow fragment; Takar Shuzo Co., Ltd.), the reaction solution was incubated for 15 minutes at 30°C.
Inkubirana reakcijska otopina je razrijeđena sa 70 µl TE otopine i reakcija je za-ustavljena dodatkom 0,5 M EDTA (5 µl) i 20% SDS (1 µl). zatim je tretiranjem sa fenol/kloroformom i taloženjem sa etanolom dobiveno oko 8 µl dvostruke cDNA. The incubated reaction solution was diluted with 70 µl of TE solution and the reaction was stopped by adding 0.5 M EDTA (5 µl) and 20% SDS (1 µl). then by treatment with phenol/chloroform and precipitation with ethanol, about 8 µl of double-stranded cDNA was obtained.
3) Metiliranje dvostruke cDNA 3) Methylation of double cDNA
Vodena otopina (30 µl) dvostruke cDNA sintetizirane u 2) je izmješano sa 40 µ1 metilacijskog pufera /500 mM Tris-HCl (pH 8,0); 50mM EDTA/, 20 µl SAM otopine /800 nm S-adenozil-L-metilmetionina (SAM); 50 mM β-merkaptoetanola/, i 100 µl vode. Smjesi je dodano 15 µl EcoRI metilaze (New England Biolabs; 20 jedinica/µl), tako da se dobiva reakcijska otopina ukupnog volumena od 200 µl. Poslije inkubacije na 37°C tijekom 2 sata, tretmana sa fenolom i eterom i taloženja sa etanolom izdvojena je DNA. An aqueous solution (30 µl) of the double cDNA synthesized in 2) was mixed with 40 µl of methylation buffer/500 mM Tris-HCl (pH 8.0); 50 mM EDTA/, 20 µl SAM solution /800 nm S-adenosyl-L-methylmethionine (SAM); 50 mM β-mercaptoethanol/, and 100 µl water. 15 µl of EcoRI methylase (New England Biolabs; 20 units/µl) was added to the mixture, so that a reaction solution with a total volume of 200 µl was obtained. After incubation at 37°C for 2 hours, treatment with phenol and ether, and precipitation with ethanol, DNA was isolated.
4) Dodatak EcoRI veziva 4) Addition of EcoRI binder
U oko 1,2 µg metilirane dvostruke cDNA, 1,5 µl pufera liagaze /250 mM Tris-HCl (pH 7,5) i 100 mM MgCl2/, 0,5 µl prethodno fosforiliziranog EcoRI veziva (10 mer; Takara Shuzo Co.,Ltd.), 1,5 µl 10 mM ATP, 1,5 l 100 mM ditiotrietiola i 2 µl vode, dodano je tako da je ukupni volumen otopine 15 µ1. Nakon dodatka 0,7 µ1 T4DNA ligaze (3,4 jedinica/µl; Takara Shuzo Co.,Ltd.), reakcija se odvijala tijekom noći na 4°C. Zatim je ligaza deaktivirana zagrijavanjem na 65°C tijekom 10 minuta. Reakcijska otopina je dovedena na ukupni volumen od 50 µl dodatkom 100 mM Tris-HCl (pH 7,5) i 5 mM MgCl2 50 mM Nad i 100 µg/ml želatine. Poslije dodavanja EcoRI (3,5 µl; 10 jedinica/µl), reakcija je vršena 2 sata na 37°C. Zatim je dodano 2,5 µl 0,5 M EDTA i 0,5 µl 20% SDS i otopina je tretirana sa fenol/kloroformom i taložena sa etanolom, tako da se izdvaja DNA. Zatim je uklonjen neizreagirani EcoRI filtriranjem gela na Ultragel AcA34 (LKB) ili agaroza-gel elektroforezom, tako da se izdvoji oko 0,5-0,7 µg dvostruke cDNA, kojoj je dodano vezivo. In about 1.2 µg of methylated duplex cDNA, 1.5 µl of lyase buffer /250 mM Tris-HCl (pH 7.5) and 100 mM MgCl2/, 0.5 µl of pre-phosphorylated EcoRI binder (10 mer; Takara Shuzo Co. ,Ltd.), 1.5 µl of 10 mM ATP, 1.5 l of 100 mM dithiotriethylol, and 2 µl of water were added so that the total volume of the solution was 15 µl. After addition of 0.7 µl of T4DNA ligase (3.4 units/µl; Takara Shuzo Co.,Ltd.), the reaction proceeded overnight at 4°C. The ligase was then deactivated by heating at 65°C for 10 minutes. The reaction solution was brought to a total volume of 50 µl by the addition of 100 mM Tris-HCl (pH 7.5) and 5 mM MgCl2, 50 mM Nad and 100 µg/ml gelatin. After addition of EcoRI (3.5 µl; 10 units/µl), the reaction was performed for 2 hours at 37°C. Then 2.5 µl of 0.5 M EDTA and 0.5 µl of 20% SDS were added and the solution was treated with phenol/chloroform and precipitated with ethanol, so that the DNA was isolated. Unreacted EcoRI was then removed by gel filtration on Ultragel AcA34 (LKB) or agarose-gel electrophoresis, so that about 0.5-0.7 µg of double-stranded cDNA was isolated, to which the binder was added.
5) Pripajanje dvostruke cDNA na λgt10 vektor 5) Ligation of the double cDNA to the λgt10 vector
Dvostruka cDNA kojoj je dodano vezivo izmješana je sa 2,4 λg prethodno EcoRI-tretiranog vektora λgt10 vektora (Vektor Cloning System), 1,4 λl pufera ligaze (250 mM Tris-HCl i 100 mM MgCl2) i 6,5 λl destilirane vode, i smjesa je 15 minuta grijana na 42°C. Zatim je dodano, 1 λ1 10 mM ATP, 1 λl 0,1 M ditiotrietiola i 0,5 ditiotriedola i 0,5 λl T4DNA ligaze tako da nastane 15 λl ukupnog volumena reakcijske otopine koja je na 12°C ostavljena preko noći. Double-stranded cDNA to which the binder was added was mixed with 2.4 λg of previously EcoRI-treated λgt10 vector (Vektor Cloning System), 1.4 λl of ligase buffer (250 mM Tris-HCl and 100 mM MgCl2) and 6.5 λl of distilled water. , and the mixture was heated to 42°C for 15 minutes. Then, 1 λ1 of 10 mM ATP, 1 λl of 0.1 M dithiotrietiol and 0.5 dithiotriedol and 0.5 λl of T4DNA ligase were added so that 15 λl of the total volume of the reaction solution was created, which was left at 12°C overnight.
6) Pakiranje in vitro 6) In vitro packaging
Oko trideset rekombinanntih DNA dobivenih u 5) su pakirane sa in vitro pakirajućim priborom (Promega Biotech) tako da se dobije faga zaraza. About thirty recombinant DNAs obtained in 5) were packaged with an in vitro packaging kit (Promega Biotech) so that phage infection was obtained.
Primjer 8: Example 8:
Testiranje biblioteke pBR-linije sa probom (IWQ) Testing the pBR-line library with probe (IWQ)
Whatman 541 papir stavljen je na agar podlogu za rast kolonije i ostavljen da stoji 2 sata na 37°C. Zatim je filtar papir tretiran na sljedeći način, Taub i Thomson /Anal. Biochem., 126, 222(1982)/. Whatman 541 paper was placed on a colony growth agar medium and allowed to stand for 2 hours at 37°C. The filter paper was then treated as follows, Taub and Thomson /Anal. Biochem., 126, 222(1982)/.
Kolonije prenesene na 541 papir su dalje rasle na agarnom mediju koji sadrži kloramfenicil (250µg/µl) preko noći na 37°C. Colonies transferred to 541 paper were further grown on agar medium containing chloramphenicil (250 µg/µl) overnight at 37°C.
541 papir je izvađen i ostavljen na sobnoj temperaturi tijekom 3 minute na drugi list filtar papira koji je bio impregniran sa 0,5 N otopinom NaOH. Ovaj postupak je ponovljen dva puta. Sličan postupak je ponovljen dva puta po 3 minute koristeći 0,5 M Tris-HCl (pH 8). Na 4°C, tretiranja su vršena sa otopinom 0,05 M Tris-HCl (pH 8) tijekom 3 minute, a sa 1,5 mg/ml otopinom lizozima /koji sadrži 0,05 M tris-HCl (pH 8) i 25% saharoze/ tijekom 10 minuta; tada na 37°C tretmani su vršeni otopinom lxSSC (0,15 M NaCl i 0,015 M natrij-citrat) tijekom 2 minute i sa 1xSSC otopinom koja sadrži 200 µg/ml proteinaze K tijekom 30 minuta; najzad na sobnoj temperaturi tretmani su vršeni sa 1xSSC otopinom tijekom 2 minute, i sa 95% otopinom etanola tijekom 2 minute. Posljednji stupanj je ponovljen dva puta. Zatim je osušen 541 papir. Osušeni 541 papir je uronjen u 25:24:1 smjesu fenol/kloroform/izoamilni alkohol /uravnotežen sa 100 mM Tris-HCl (pH 8,5), 100 mM NaCl i 10 mM EDTA/ tijekom 30 minuta na sobnoj temperaturi. Zatim, slični postupci su ponovljeni tri puta sa 95% otopinom etanola 3 minute. Zatim je filtar papir osušen. 541 paper was taken out and left at room temperature for 3 minutes on another sheet of filter paper that was impregnated with 0.5 N NaOH solution. This procedure was repeated twice. A similar procedure was repeated twice for 3 minutes each using 0.5 M Tris-HCl (pH 8). At 4°C, treatments were performed with a 0.05 M Tris-HCl (pH 8) solution for 3 minutes, and with a 1.5 mg/ml lysozyme solution/containing 0.05 M Tris-HCl (pH 8) and 25% sucrose/ during 10 minutes; then at 37°C treatments were performed with 1xSSC solution (0.15 M NaCl and 0.015 M sodium citrate) for 2 minutes and with 1xSSC solution containing 200 µg/ml proteinase K for 30 minutes; finally, at room temperature, treatments were performed with 1xSSC solution for 2 minutes, and with 95% ethanol solution for 2 minutes. The last step was repeated twice. Then the 541 paper was dried. The dried 541 paper was immersed in a 25:24:1 mixture of phenol/chloroform/isoamyl alcohol /equilibrated with 100 mM Tris-HCl (pH 8.5), 100 mM NaCl and 10 mM EDTA/ for 30 minutes at room temperature. Then, similar procedures were repeated three times with 95% ethanol solution for 3 minutes. Then the filter paper is dried.
Proba (IWQ) obilježena sa 32P prema rutinskom postupku (vidi Molecular Cloning) i hibridizacija kolonije su izvedeni prema metodi Wallace-a et al. /Nucleic Acids Res., 9, 879(1981)/. Probe (IWQ) labeled with 32P according to the routine procedure (see Molecular Cloning) and colony hybridization were performed according to the method of Wallace et al. /Nucleic Acids Res., 9, 879(1981)/.
Prehibridizacija je vršena 4 sata na 65°C u hibridizacijskom puferu koji sadrži 6xNET /0,9 M NaCl; 0,09 M Tris-Hd (pH 7,5); i 6 mM EDTA/, 5xDenhardt-ova otopina, 0,1% SDS i 0,1 mg/ml denaturirane DNA (timus teleta). Zatim, hibridizacija je vršena preko noći na 56°C u hibridizacijskom puferu (za njegovo spravljanje pogledaj naprijed) koji sadrži 1x106 cpm/ml radioobilježene probe (IWQ). Po završetku reakcije, 541 papir je ispran dva puta sa 6xSSC otopinom (koja sadrži 0,1% SDS) tijekom 30 minuta na sobnoj temperaturi, i na 56°C je ispiran tijekom 1,5 minute. Isprani 541 papir je onda podvrgnut autoradiografiji. Prehybridization was performed for 4 hours at 65°C in a hybridization buffer containing 6xNET /0.9 M NaCl; 0.09 M Tris-Hd (pH 7.5); and 6 mM EDTA/, 5xDenhardt's solution, 0.1% SDS and 0.1 mg/ml denatured DNA (calf thymus). Then, hybridization was performed overnight at 56°C in hybridization buffer (for its preparation see ahead) containing 1x106 cpm/ml radiolabeled probe (IWQ). Upon completion of the reaction, the 541 paper was washed twice with 6xSSC solution (containing 0.1% SDS) for 30 minutes at room temperature, and washed at 56°C for 1.5 minutes. The washed 541 paper was then subjected to autoradiography.
Plazmid je odvojen od pozitivnih klonova i podvrgnut Southern upijanju s probom (IWQ). Hibridizacija i autoradiografija su vršeni u istim uvjetima kao što je opisano naprijed. Plasmid was isolated from positive clones and subjected to Southern blotting with probe (IWQ). Hybridization and autoradiography were performed under the same conditions as described above.
Slično, Southern upijanje je izvršeno sa probom (A). Koristeći hibridizacijski pufer koji je načinjen kao što je dano naprijed, hibridizacija je vršena prvo na 49°C tijekom 1 sata. Nakon stajanja na 39°C hibridizacija je dalje nastavljena na ovoj temperaturi tijekom 1 sata. Po završetku reakcije, nitrocelulozm filtar je dva puta ispran sa 0,1% SDS koji sadrži 6xSSC tijekom 30 minuta na sobnoj temperaturi, tada je 3 minute ispiran na 39°C. Isprani papir je podvrgnut autoradiografiji. Similarly, Southern blotting was performed with sample (A). Using hybridization buffer prepared as above, hybridization was performed first at 49°C for 1 hour. After standing at 39°C, the hybridization was further continued at this temperature for 1 hour. Upon completion of the reaction, the nitrocellulose filter was washed twice with 0.1% SDS containing 6xSSC for 30 minutes at room temperature, then washed at 39°C for 3 minutes. The washed paper was subjected to autoradiography.
Nađeno je da je uočen jedan pozitivan klon. Ispitivanje nukleotidnog niza dideoksi metodom pokazuje da ovaj klon ima DNA sastavljenu od 308 baznih parova koji obuhvaćaju dijelove proba (IWQ) i (A). pBR322 izvedeni plazmid koji sadrži ovaj dio ne nazvan pHCS-1. It was found that one positive clone was observed. Nucleotide sequence analysis by the dideoxy method shows that this clone has DNA composed of 308 base pairs spanning parts of probes (IWQ) and (A). pBR322 derived plasmid containing this part not called pHCS-1.
Primjer 9: Example 9:
Testiranje biblioteke λfaga linije sa pHCS-1 izvedenom DNA probom Testing the λphage line library with a pHCS-1 derived DNA probe
Hibridiziranje zaraze je vršeno prema metodi Benton i Davis /Science, 196, 180(1977)/. pHCS-1 dobiven u primjeru 8 je tretiran sa Sau3A i EcoRI tako da se dobije DNA fragment od oko 600 bp. Ovaj DNA fragment je radioobilježen translacijskim zasijecanjem prema rutinskim postupcima. Nitrocelulozni filtar (S&S) je stavljen na faga agarnu podlogu za kultiviranje zaraze radi prenošenja faga na filtar. Nakon denaturiranja faga sa 0,5 M NaOH, filter papir je tretiran na sljedeći način: tretman sa 0,1 M NaOH i 1,5 M NaCl tijekom 20 sekundi; dva tretmana sa 0,5 M Tris-HCl (pH 7,5) i 1,5 M NaCl tijekom 20 sekundi; najzad tretman sa 120 mM NaCl, 15 mM natrij-citrata, 13 mM KH2PO4 i 1 mM EDTA (pH 7,3) tijekom 20 sekundi. Infection hybridization was performed according to the method of Benton and Davis /Science, 196, 180 (1977)/. pHCS-1 obtained in example 8 was treated with Sau3A and EcoRI so that a DNA fragment of about 600 bp was obtained. This DNA fragment was radiolabeled by translational cleavage according to routine procedures. A nitrocellulose filter (S&S) was placed on a phage agar medium for culturing the infection to transfer phage to the filter. After denaturing the phage with 0.5 M NaOH, the filter paper was treated as follows: treatment with 0.1 M NaOH and 1.5 M NaCl for 20 seconds; two treatments with 0.5 M Tris-HCl (pH 7.5) and 1.5 M NaCl for 20 seconds; finally treatment with 120 mM NaCl, 15 mM sodium citrate, 13 mM KH2PO4 and 1 mM EDTA (pH 7.3) for 20 seconds.
Filtar je zatim osušen i grijan 2 sata na 80°C radi imobiliziranja DNA. Prehibridizacija je vršena preko noći na 42°C u prehibridizacijskom puferu koji sadrži 5xSSC, 5xDenhardtovu otopinu, 50 mM fosfatnog pufera, 50% formamida, 0,25 mg/ml denaturirane DNA (DNA sperme lososa) i 0,1% SDS. Zatim, hidbridizacija je vršena na 42°C tijekom 20 sati u hibridizacijskom puferu koji sadrži 4x105 cpm/ml pHCS-1 probe koja je bila radioobilježena pomoću translacijskog zasijecanja. Ovaj hibridizacijski pufer je bio smjesa 5xDenhardtove otopine, 20 mM fosfatnog pufera (pH 6,0), 50% formamida, 0,1% SDS, 10% dekstran-sulfata i 0,1 mg/ml denaturirane DNA (DNA sperme lososa). The filter was then dried and heated for 2 hours at 80°C to immobilize the DNA. Prehybridization was performed overnight at 42°C in prehybridization buffer containing 5xSSC, 5xDenhardt's solution, 50 mM phosphate buffer, 50% formamide, 0.25 mg/ml denatured DNA (salmon sperm DNA) and 0.1% SDS. Next, hybridization was performed at 42°C for 20 hours in hybridization buffer containing 4x105 cpm/ml pHCS-1 probe that was radiolabeled using translational cleavage. This hybridization buffer was a mixture of 5xDenhardt's solution, 20 mM phosphate buffer (pH 6.0), 50% formamide, 0.1% SDS, 10% dextran sulfate, and 0.1 mg/ml denatured DNA (salmon sperm DNA).
Hibridizirani nitrocelulozni filtar je 20 minuta ispiran sa 2xSSC koji sadrži 0,1% SDS na sobnoj temperaturi, i tada 30 minuta sa 0,1xSSC koji sadrži 0,1% SDS na 44°C i najzad 10 minuta sa 0,1xSSC na sobnoj temperaturi. Tada je izvršena autoradiografska detekcija. The hybridized nitrocellulose filter was washed for 20 minutes with 2xSSC containing 0.1% SDS at room temperature, then for 30 minutes with 0.1xSSC containing 0.1% SDS at 44°C and finally for 10 minutes with 0.1xSSC at room temperature. . Autoradiographic detection was then performed.
Dobiveno je 5 pozitivnih klonova (G1-G5). Klon koji je sadržavao punu dužinu niza cDNA je ispitivan radi određivanja DNA nukleotidnog niza dideoksi metodom i identificiran je nukleotidni niz prikazan na slici 3(A). Ova cDNA je poslije isjecanja λgt10 vektora pripojena na pBR327 /Sorberon et aL, Gene, 9, 287(1980)/ na EcoRI mjestu tako da nastaje plazmid koji se može dobiti u velikim količinama. Ovaj plazmid je nazvan pBRG4. 5 positive clones (G1-G5) were obtained. The clone containing the full length cDNA sequence was tested for DNA nucleotide sequence determination by the dideoxy method and the nucleotide sequence shown in Figure 3(A) was identified. After cutting the λgt10 vector, this cDNA was joined to pBR327 /Sorberon et al, Gene, 9, 287(1980)/ at the EcoRI site, so that a plasmid is produced that can be obtained in large quantities. This plasmid was named pBRG4.
Primjer 10: Example 10:
Testiranje biblioteke λfaga linije sa pBRG4-izvedenom probom i (LC) probom Screening of the λphage line library with the pBRG4-derived probe and the (LC) probe
Hibridizacija zaraze je vršena prema metodi Benton i Davis (vidi takoder Science) korištenoj u primjeru 9. Nitrocelulozni filtar (S&S) je stavljen na faga agarni medij za kultiviranje zaraze radi prenošenja faga na filtar. Nakon denaturiranja fage DNA sa 0,5 M NaOH, filtar je tretiran sljedećim postupcima: tretman sa 0,1 M NaOH i 1,5 M NaCl tijekom 20 sekundi; zatim dva puta tretmani sa 0,5 M Tris-Hd (pH 7,5) i 1,5 M NaCl tijekom 20 sekundi; najzad tretman sa 120 mM NaCl, 15 mM natrij-citrata, 13 mM KH2P04 i 1 mM EDTA (pH 7,2) tijekom 20 sekundi. Filtar je zatim osušen i grijan 2 sata na 80°C radi imobiliziranja DNA. Dva lista istog filtra su pripremljena na način kako je opisano naprijed i podvrgnuta su testiranju sa pBRG4-izvedenom DNA probom i probom (LC). Infection hybridization was performed according to the method of Benton and Davis (see also Science) used in Example 9. A nitrocellulose filter (S&S) was placed on phage agar infection culture medium to transfer phage to the filter. After denaturing phage DNA with 0.5 M NaOH, the filter was treated with the following procedures: treatment with 0.1 M NaOH and 1.5 M NaCl for 20 seconds; then two treatments with 0.5 M Tris-Hd (pH 7.5) and 1.5 M NaCl for 20 seconds; finally treatment with 120 mM NaCl, 15 mM sodium citrate, 13 mM KH2PO4 and 1 mM EDTA (pH 7.2) for 20 seconds. The filter was then dried and heated for 2 hours at 80°C to immobilize the DNA. Two sheets of the same filter were prepared as described above and tested with the pBRG4-derived DNA probe and the probe (LC).
Testiranje sa pBRG4-izvedenom DNA probom je vršeno na sljedeći način. pBRG4 je tretiran sa EcoRI tako da se dobije DNA fragment sa oko 1500 bp. Ovaj fragment je radioobilježen translacijskim zasijecanjem prema rutinskim postupcima. Jedan od dva nitrocelulozna filtra je podvrgnut prehibridizaciji preko noći na 42° C u prehibridizacijskom puferu koji sadrži 5xSSC, 5xDenhardtovu otopinu, 50 mM fosfatnog pufera, 50% formamida, 0,25 mg/ml denaturirane DNA (DNA sperme lososa) i 0,1% SDS. Zatim, filtar je podvrgnut hibiridizaciji na 42°C tijekom 20 sati u hibridizacijskom puferu koji sadrži radioobilježenu probu DNA (oko 1x106 cpm/ml) od oko 1500 bp. Ovaj hibridizacijski pufer je smjesa 5xSSC, 5xDenhard-tovu otopinu, 20 mM fosfatnog pufera (pH 6,0), 50% formamida, 0,1% SDS, 10% dekstran-sulfata i 0,1 mg/ml denaturirane DNA (DNA sperme lososa). Hibridiziram nitrocelulozom filtar je 20 minuta ispiran sa 2xSSC koji sadrži 0,1% SDS na sobnoj temperaturi., i tada 30 minuta sa 0,1xSSC koji sadrži 0,1% SDS na 44°C, i konačno, 10 minuta sa 0,1% SSC na sobnoj temperaturi. Izvršena je detekcija pomoću autoradiografije. Testing with the pBRG4-derived DNA probe was performed as follows. pBRG4 was treated with EcoRI to obtain a DNA fragment with about 1500 bp. This fragment was radiolabeled by translational cleavage according to routine procedures. One of the two nitrocellulose filters was subjected to prehybridization overnight at 42°C in prehybridization buffer containing 5xSSC, 5xDenhardt's solution, 50 mM phosphate buffer, 50% formamide, 0.25 mg/ml denatured DNA (salmon sperm DNA) and 0.1 % SDS. Then, the filter was subjected to hybridization at 42°C for 20 hours in a hybridization buffer containing a radiolabeled DNA probe (about 1x106 cpm/ml) of about 1500 bp. This hybridization buffer is a mixture of 5xSSC, 5xDenhard's solution, 20 mM phosphate buffer (pH 6.0), 50% formamide, 0.1% SDS, 10% dextran sulfate, and 0.1 mg/ml denatured DNA (sperm DNA salmon). I hybridize with nitrocellulose, the filter was washed for 20 minutes with 2xSSC containing 0.1% SDS at room temperature, and then for 30 minutes with 0.1xSSC containing 0.1% SDS at 44°C, and finally, for 10 minutes with 0.1 % SSC at room temperature. Detection was performed using autoradiography.
Testiranje sa probom (LC) je izvršeno pomoću sljedećih postupaka. Drugi filtar je prethodno tretiran sa 5xSSC koji sadrži 0,1% SDS 2 sata na 65°C. Tada je vršena prehibridizacija 2 sata na 65°C u otopini koja sadrži 6xNET, 1xDenhardt-ove otopine i 100 µg/ml denaturirane DNA (DNA sperma lososa). Hibridizacija je zatim izvedena preko noći na 63°C u hibridizacijskom puferu koji sadrži radioobilježenu probu (LC) (2x106 cpm/ml). Ovaj hibridizacijski pufer također je smjesa 6xNET, 1xDenhardt-ova otopina i 100 µg/ml denaturirane DNA (DNA sperme lososa). Hibridizirani nitrocelulozni filter je tri puta ispran (svaki puta po 20 minuta) sa 3xSSC koji sadrži 0,1% SDS na sobnoj temperaturi, tada je ispiran 2 minute na 63°C sa 6xSSC koji sadrži 0,1% SDS. Trial testing (LC) was performed using the following procedures. The second filter was pretreated with 5xSSC containing 0.1% SDS for 2 hours at 65°C. Then prehybridization was performed for 2 hours at 65°C in a solution containing 6xNET, 1xDenhardt's solution and 100 µg/ml denatured DNA (salmon sperm DNA). Hybridization was then performed overnight at 63°C in hybridization buffer containing radiolabeled probe (LC) (2x106 cpm/ml). This hybridization buffer is also a mixture of 6xNET, 1xDenhardt's solution and 100 µg/ml denatured DNA (salmon sperm DNA). The hybridized nitrocellulose filter was washed three times (20 minutes each time) with 3xSSC containing 0.1% SDS at room temperature, then washed for 2 minutes at 63°C with 6xSSC containing 0.1% SDS.
Filtar je osušen i detektiran autoradiografski. The filter was dried and detected by autoradiography.
U testiranju naprijed opisanom, klonovi koji su bili pozitivni u obje probe su odvojeni i klon koji je imao punu dužinu lanca cDNA je ispitivan dideoksi metodom radi utvrđivanja nukleotidnog niza. Nađeno je da ima nukleotidni niz prikazan na slici 4(A). Ova dna je odsječena od λt10 vektora i pridružena pBR327 na EcoRI mjestu tako da stvara plazmid pBRV2. In the assay described above, clones that were positive in both assays were separated and the clone that had the full-length cDNA chain was tested by the dideoxy method to determine the nucleotide sequence. It was found to have the nucleotide sequence shown in Figure 4(A). This DNA was excised from the λt10 vector and ligated into pBR327 at the EcoRI site to generate plasmid pBRV2.
Primjer 11: Example 11:
Testiranje biblioteke humanog kromosomskog gena Human chromosomal gene library testing
1) Stvaranje biblioteke humanog kromosomskog gena 1) Creating a human chromosomal gene library
Biblioteka humanog kromosomskog gena dobivena zahvaljujući ljubaznosti Dr. Maniatis-a sa Harvard University je pripremljena sljedećim postupcima: Cijela DNA kromosoma je ekstrahirana iz jetre ljudskog embrija sa fenolom ili drugim odgovarajućim kemikalijama i djelomično je razorena sa restrikcijskim enzimima, HaeIII i AluI; dobiveni DNA fragmenti su tretirani pomoću centrifugalnog gradijenta gustoće saharoze tako da koncentrat fragmenata koji ima lance dužine od oko 18-25 kb; koncentrirani fragmenti su pripojeni na arm DNA E. coli fagu λ Charon 4A, sa sintetskim nukleotidima kratkih lanaca koju su ubačeni na mjestima kidanja restrikcijskog enzima EcoRI, tako da se stvara infekcijska faka DNA rekombinanti; u cilju smanjenja infektivnosti, dobivene su pakovanjem čestice čistije λ fage. Tako dobivena biblioteka humanog gena je teorijski razmatrana kao set rekombinanata koji sadrže humane DNA s lancima dužine od 18-25 kb koji sadrže praktično svi humani geni. Human chromosomal gene library obtained thanks to the kindness of Dr. Maniatis from Harvard University was prepared by the following procedures: Whole chromosome DNA was extracted from human embryo liver with phenol or other appropriate chemicals and partially digested with restriction enzymes, HaeIII and AluI; the obtained DNA fragments were treated using a centrifugal sucrose density gradient so that a concentrate of fragments with chains of about 18-25 kb in length; the concentrated fragments were attached to the arm DNA of E. coli phage λ Charon 4A, with synthetic nucleotides of short chains that were inserted at the cleavage sites of the restriction enzyme EcoRI, so that infectious phage DNA recombinants were created; in order to reduce infectivity, purer λ phage particles were obtained by packaging. The thus obtained human gene library was theoretically considered as a set of recombinants containing human DNA with chains of 18-25 kb in length that contain practically all human genes.
2) Testiranje biblioteke humanog kromosomskog gena sa pHCS-1 izvedenom DNA probom 2) Testing of the human chromosomal gene library with pHCS-1 derived DNA probe
Hibridizacija zaraze je izvedena prema metodi Benton i Davis /Science, 196, 180(1977)/. pHCS-1 dobiven u primjeru 8 tretiran je sa Sau3A i EcoRI tako da se dobije fragment od oko 600 bp. Ovaj DNA fragment je radioobilježen pomoću translacijskog zasijecanja prema rutinskim postupcima. Nitrocelulozni filtar (S&S) je stavljen na faga agarni medij za kultiviranje zaraze radi prenošenja faga na filtar. Nakon denaturiranja faga DNA sa 0,5 M NaOH, filter papir je tretiran sljedećim postupcima; tretman sa 1,5 M NaCl tijekom 20 sekundi; dva tretmana sa 0,5 M Tris-HCl (pH 7,5) i 1,5 M NaCl tijekom 20 sekundi; konačno, tretman sa 120 mM NaCl, 15 mM natrij-citrata, 13 mM KH2PO4 i 1 mM EDTA (pH 7,2) tijekom 20 sekundi. Infection hybridization was performed according to the method of Benton and Davis /Science, 196, 180(1977)/. pHCS-1 obtained in Example 8 was treated with Sau3A and EcoRI to obtain a fragment of about 600 bp. This DNA fragment was radiolabeled using translational cleavage according to routine procedures. A nitrocellulose filter (S&S) was placed on the phage agar medium for culturing the infection to transfer phage to the filter. After denaturing the phage DNA with 0.5 M NaOH, the filter paper was treated with the following procedures; treatment with 1.5 M NaCl for 20 seconds; two treatments with 0.5 M Tris-HCl (pH 7.5) and 1.5 M NaCl for 20 seconds; finally, treatment with 120 mM NaCl, 15 mM sodium citrate, 13 mM KH2PO4 and 1 mM EDTA (pH 7.2) for 20 seconds.
Filtar je zatim osušen i zagrijavan na 80°C tijekom 2 sata radi imobiliziranja DNA. Prehibridizacija je vršena preko noći na 42°C u prehibridizacijskom puferu koji sadrži 5xSSC, 5xDenhardtovu otopinu, 50 mM fosfatnog pufera, 50% formamida, 0,25 mg/ml denaturirane DNA (DNA sperme lososa) i 0,1% SDS. Zatim je hibridizacija vršena 20 sati na 42°C u hibridizacijskom puferu koji sadrži 4x105 cpm/ml pHCS-1 probe koja je bila radioobilježena translacijskim zasijecanjem. Ovaj hibridizacijski pufer je bio smjesa 5xSSC, 5xDenhardova otopina, 20 mM fosfatnog pufera (pH 6,0), 50% formamida, 0,1% SDS, 10% dekstran-sulfat i 0,1 mg/ml denaturirane DNA (DNA sperme lososa). The filter was then dried and heated at 80°C for 2 hours to immobilize the DNA. Prehybridization was performed overnight at 42°C in prehybridization buffer containing 5xSSC, 5xDenhardt's solution, 50 mM phosphate buffer, 50% formamide, 0.25 mg/ml denatured DNA (salmon sperm DNA) and 0.1% SDS. Then hybridization was performed for 20 hours at 42°C in a hybridization buffer containing 4x105 cpm/ml pHCS-1 probe that was radiolabeled by translational cleavage. This hybridization buffer was a mixture of 5xSSC, 5xDenhard's solution, 20 mM phosphate buffer (pH 6.0), 50% formamide, 0.1% SDS, 10% dextran sulfate, and 0.1 mg/ml denatured DNA (salmon sperm DNA ).
Hibridizirani nitrocelulozni filtar je 20 minuta ispiran sa 2xSSC koji sadrži 0,1% SDS na sobnoj temperaturi, i tada 30 minuta sa 0,1xSSC koji sadrži 0,1% SDS na 44°C i konačno 10 minuta sa 0,1xSSC na sobnoj temperaturi. Detekcija je dalje vršena autoradiografski. The hybridized nitrocellulose filter was washed for 20 minutes with 2xSSC containing 0.1% SDS at room temperature, and then for 30 minutes with 0.1xSSC containing 0.1% SDS at 44°C and finally for 10 minutes with 0.1xSSC at room temperature. . Detection was further performed autoradiographically.
Dobiveno je 10 rasparenih pozitivnih klonova. Rekombinanti DNA su dobiveni od ovih klonova metodom Maniatis /Cell, 15, 687(1978)/. Dobivene DNA su tretirane sa restrikcijskim enzimima takvim kao EcoRI, BemHI i BglII, analizirane agaroza gel elektroforezom i mapa njihovoga rekstrikcijskog enzima je dobivena prema metodi Fritsh-a et al. (vidi također Cell). 10 split positive clones were obtained. Recombinant DNA was obtained from these clones by the Maniatis method /Cell, 15, 687 (1978)/. The obtained DNAs were treated with restriction enzymes such as EcoRI, BemHI and BglII, analyzed by agarose gel electrophoresis and a map of their restriction enzyme was obtained according to the method of Fritsh et al. (see also Cell).
Southern hibridizacija je vršena sa probom koja je radioobilježena pHCS-1 izvedenim DNA fragmentom koji je isti kao onaj korišten u gore ispitivanom testiranju. DNA fragment od oko 8 kbp koje je isječen sa EcoRI je izabran od klonova koji su hibridizirani s probom. Fragment je podkloniran u EcoRI mjesto pBR327. Podklonirana DNA je podvrgnuta drugom tretmanu sa restrikcijskim enzimima i Southern hibridizacija je vršena više puta. DNA fragment od oko 4 kbp koji je isječen sa EcoRI i Xhol je nađeno da sadrži gen koji kodira humani G-CSF polipeptid. Ovaj DNA fragment je ispitan radi određivanja niza njegovog 3-kbp dideoksi metodom i identificiran je nukleotidni niz prikazan na slici 5. Ovaj fragment ima mjesto cijepanja restrikcijskog enzima prikazan na slici 7. Southern hybridization was performed with a probe radiolabeled with a pHCS-1 derived DNA fragment identical to that used in the above assay. A DNA fragment of about 8 kbp cut with EcoRI was selected from clones that hybridized with the probe. The fragment was subcloned into the EcoRI site of pBR327. The subcloned DNA was subjected to a second treatment with restriction enzymes and Southern hybridization was performed several times. A DNA fragment of about 4 kbp cut with EcoRI and XhoI was found to contain the gene encoding the human G-CSF polypeptide. This DNA fragment was tested to determine the sequence of its 3-kbp dideoxy method and the nucleotide sequence shown in Figure 5 was identified. This fragment has a restriction enzyme cleavage site shown in Figure 7.
Testiranje humanih kromosomskih gena je također vršeno korištenjem pBRG4-izvedene DNA i pBRV2-izvedene DNA kao proba. U oba slučaja DNA fragment od 1500 bp koji je tretiran sa EcoRI je izravno radioobilježen translatomim usijecanjem na način opisan gore, ili alternativno, DNA fragment od oko 700 bp koji je dobiven naizmjeničnim tretmanima sa EcoRI i DraI radioobilježenim translacijskim usijecanjem. Tako dobivena proba je korištena u hibridizaciji zaraze koja je izvršena u istim uvjetima kao što je opisano naprijed. Odabrani klonovi su analizirani Southern hibridizacijom tako da se dobiva DNA fragment koji ima nukleotidni niz prikazan na slici 5. Tako dobiven plazmid je nazvan pBRCE3. Human chromosomal gene testing was also performed using pBRG4-derived DNA and pBRV2-derived DNA as probes. In both cases, a DNA fragment of 1500 bp treated with EcoRI was directly radiolabeled by translatome cleavage in the manner described above, or alternatively, a DNA fragment of about 700 bp obtained by alternating treatments with EcoRI and DraI was radiolabeled with translational cleavage. The sample thus obtained was used in the infection hybridization, which was carried out under the same conditions as described above. The selected clones were analyzed by Southern hybridization to obtain a DNA fragment with the nucleotide sequence shown in Figure 5. The resulting plasmid was named pBRCE3.
Primjer 12: Example 12:
Stvaranje E.coli rekombinantnog vektora (+VSE) i transformacija (korištenjem tac promotora koji sadrži vektor) Creation of E.coli recombinant vector (+VSE) and transformation (using tac promoter containing vector)
1) Stvaranje rekombinantnog vektora 1) Creation of recombinant vector
(i) Pripremanje vektora (i) Preparation of vectors
5 mikrograma tac promotora koji sadrži vektor pKK223-3 (Pharmacia) je tretiranio sa 8 jedinica EcoRI (Takara Shuzo Co., Ltd.) tijekom 2 sata na 37°C u 30 (µl reakcijske otopine (40 mM Tris-HCl, 7 mM MgCl2, 100 mM NaCl, i 7 mM 2-merkaptoetanola). 5 micrograms of tac promoter containing vector pKK223-3 (Pharmacia) was treated with 8 units of EcoRI (Takara Shuzo Co., Ltd.) for 2 hours at 37°C in 30 (µl reaction solution (40 mM Tris-HCl, 7 mM MgCl2, 100 mM NaCl, and 7 mM 2-mercaptoethanol).
Zatim, 3 µl alkalne fosfataze (Takara Shuzo Co., Ltd.) je dodano i tretman je vršen na 60°C tijekom 30 minuta. DNA fragment je izdvojen trostrukim tretmanom s fenolom i jednostrukim tretmanom s eterom i taložen s etanolom, sve je vršeno prema rutinskim postupcima. Then, 3 µl of alkaline phosphatase (Takara Shuzo Co., Ltd.) was added and the treatment was carried out at 60°C for 30 minutes. The DNA fragment was isolated by triple treatment with phenol and single treatment with ether and precipitated with ethanol, everything was done according to routine procedures.
Izdvojeni fragment DNA je otopljen u 50 mikrolitara smjese koja je sastavljena od 50 mM Tris-HCl, 5 mM MgCl2, 10 mM DTT i po 1 mM sATP, dCTP, dGTP i dTTP. Nakon dodatka 3 µl E. coli DNA polimeraze I -Klenow fragment (Takara Shuzo Co., Ltd.), reakcija je vršena na 14°C tijekom 2 sata tako da se stvore otvoreni krajevi. The isolated DNA fragment was dissolved in 50 microliters of a mixture composed of 50 mM Tris-HCl, 5 mM MgCl2, 10 mM DTT and 1 mM each of sATP, dCTP, dGTP and dTTP. After adding 3 µl of E. coli DNA polymerase I -Klenow fragment (Takara Shuzo Co., Ltd.), the reaction was performed at 14°C for 2 hours to create open ends.
(ii) Dobivanje sintetskog veziva (ii) Obtaining a synthetic binder
Tri mikrograma oligonukleotida koji ima nizove sintetskih veziva, CGAATGACCCCCCTGGGCC i CAGGGGGGTCATTGG, je fosforiliziran vršenjem reakcije u 40 µl reakcijske otopine (sastavljene od 50 mM Tris-HCl, 10 mM MgCl2, 10 mM 2-merkaptoetanola i 1 mM ATP) na 37°C tijekom 60 minuta u prisutnosti 4 jedinice T4 polinukleotidne kinaze. Svaki od fosforiliziranih oligonukleotida (0,2 µg) je otopljen u 20 µl 100 mM NaCl-koji sadrži TE otopinu /10 mM Tris-Hd (pH 8,0) i 1 mM EDTA). Nakon tretmana na 65°C tijekom 10 minuta, oligonukleotidi su kaljeni sporim hlađenjem do sobne temperature. Three micrograms of oligonucleotide having the sequences of synthetic linkers, CGAATGACCCCCCTGGGCC and CAGGGGGGTCATTGG, was phosphorylated by carrying out the reaction in 40 µl reaction solution (composed of 50 mM Tris-HCl, 10 mM MgCl2, 10 mM 2-mercaptoethanol and 1 mM ATP) at 37°C for 60 minutes in the presence of 4 units of T4 polynucleotide kinase. Each of the phosphorylated oligonucleotides (0.2 µg) was dissolved in 20 µl of 100 mM NaCl-containing TE solution (10 mM Tris-Hd (pH 8.0) and 1 mM EDTA). After treatment at 65°C for 10 minutes, the oligonucleotides were quenched by slow cooling to room temperature.
(iii) Dobivanje G-CSF cDNA fragmenta (iii) Obtaining the G-CSF cDNA fragment
60 mikrograma pBRG4 dobivenog u primjeru 9 koji sadrži cDNA prikazan na slici 3(A) je tretirano sa 100 jedinica restrikcijskog enzima Apal (New England Biolabs) i 50 jedinica Dral (Takara Shuzo Co., Ltd.) na 37°C tijekom 3 sata u 200 µl reakcijske otopine sastavljene od 6 mM Tris-HCl, 6 mM MgCl2 i 6 mM 2-merkaptoetanola. Oko 2 µg Apal -Dral fragmenta (oko 590 bP) je izdvojeno pomoću 1,2% agaroza gel elektroforeze. 60 micrograms of pBRG4 obtained in Example 9 containing the cDNA shown in Figure 3(A) was treated with 100 units of restriction enzyme ApaI (New England Biolabs) and 50 units of Dral (Takara Shuzo Co., Ltd.) at 37°C for 3 hours. in 200 µl reaction solution composed of 6 mM Tris-HCl, 6 mM MgCl2 and 6 mM 2-mercaptoethanol. About 2 µg of the Apal-Dral fragment (about 590 bp) was separated by 1.2% agarose gel electrophoresis.
(iv) Vezanje fragmenta (iv) Binding of fragments
Po oko 0,1 mikrograma svakog od fragmenata dobivenih u (i) do (iii) je otopljeno u 20 µl otopine za vezanje (66 mM Tris-HCl, 6,6 mM MgCl2, 10 mM DTT i 1 mM ATP). Nakon dodatka 175 jedinica T4 ligaze, otopina je preko noći držana na 4°C tako da se dobije rekombinantni vektor (slika 8). About 0.1 microgram of each of the fragments obtained in (i) to (iii) was dissolved in 20 µl of binding solution (66 mM Tris-HCl, 6.6 mM MgCl2, 10 mM DTT and 1 mM ATP). After the addition of 175 units of T4 ligase, the solution was kept overnight at 4°C so that the recombinant vector was obtained (Figure 8).
2) Transformiranje 2) Transforming
Koristeći 20 µl reakcijske otopine koja sadrži rekombinantni vektor dobiven u (iv), E. coli vrsta JMI05 je transformirana postupkom sa rubidij-kloridom (vidi Maniatis et al., Molecular Cloning, str. 252 (1982)/. Plazmid je odvojen od amplicilin-rezistentne kolonije kulture transformata i tretiran je s restrikcijskim enzimima, BamHI, AccII i Apal radi potvrđivanja da su transformati oni koji su željeni. Using 20 µl of the reaction solution containing the recombinant vector obtained in (iv), E. coli strain JMI05 was transformed by the rubidium chloride procedure (see Maniatis et al., Molecular Cloning, p. 252 (1982)). The plasmid was separated from amplicillin -resistant colonies of the transformant culture and treated with the restriction enzymes, BamHI, AccII and ApaI to confirm that the transformants are the desired ones.
Primjer 13: Example 13:
Stvaranje E. coli rekombinantnog vektora (+VSE) i transformacija (korištenjem PL promotora koji sadrži vektor) Creation of an E. coli recombinant vector (+VSE) and transformation (using the PL promoter containing the vector)
1) Stvaranje rekombinantnog vektora 1) Creation of recombinant vector
(i) Dobivanje vektora (i) Obtaining a vector
100 mikrograma PL promotora koji sadrži vektor pPL-lambda (Pharmacia) je preko noći tretirano na 37°C sa 50 jedinica restrikcijskog enzima BamHI u 100 µ1 reakcijske otopine /10 mM Tris-HCl (pH 7,6), 7 mM MgCl2 100 mM NaCl i 10 mM DTT/. 100 micrograms of the PL promoter containing the vector pPL-lambda (Pharmacia) was treated overnight at 37°C with 50 units of the restriction enzyme BamHI in 100 µl reaction solution /10 mM Tris-HCl (pH 7.6), 7 mM MgCl2 100 mM NaCl and 10 mM DTT/.
Podvrgavanjem reakcijske otopine 1% agaroza gel elektroforezi, izdvojeno je oko 49 µg približno 4-kb fragmenta i oko 11 µg približno 1,2-kb fragmenta. By subjecting the reaction solution to 1% agarose gel electrophoresis, about 49 µg of the approximately 4-kb fragment and about 11 µg of the approximately 1.2-kb fragment were isolated.
4-kb fragment je otopljen u 100 µl TE pufera (za njegov sastav pogledaj naprijed) i defosforiliziran je reakcijom s alkalnom fosfatazom (Takara Shuzo Co., Ltd.) na 60°C tijekom 60 minuta. The 4-kb fragment was dissolved in 100 µl of TE buffer (see ahead for its composition) and dephosphorylated by reaction with alkaline phosphatase (Takara Shuzo Co., Ltd.) at 60°C for 60 minutes.
Drugi fragment od oko 1,2 kb dužine otopljen je u 20 µl pufera (10 mM Tris-HCl, 10 mM MgCl2 i 1 mM DTT) i tretirano preko noći sa 20 jedinica restrikcijskog enzima MboII (New England Biolabs) na 37°C. The second fragment of about 1.2 kb in length was dissolved in 20 µl of buffer (10 mM Tris-HCl, 10 mM MgCl2 and 1 mM DTT) and treated overnight with 20 units of restriction enzyme MboII (New England Biolabs) at 37°C.
4% poliakrilamid gel elektroforezom izdvojeno je oko 0,9 µg MboII-BamHI fragmenata. About 0.9 µg of MboII-BamHI fragments were isolated by 4% polyacrylamide gel electrophoresis.
(ii) Dobivanje sintetskog veziva (ii) Obtaining a synthetic binder
Oligonukleotidi koji imaju nizove sintetičkih veziva TAAGGAGAATTCATCGAT i TCGATGAATTCTCCTTAG, fosforilizirani su i kaljeni kao u (ii) u Primjeru 12, tako da se dobiva sintetsko S/D vezivo. Oligonucleotides having the synthetic linker sequences TAAGGAGAATTCATCGAT and TCGATGAATTCTCCTTAG are phosphorylated and annealed as in (ii) in Example 12, so that a synthetic S/D linker is obtained.
(iii) Dobivanje vektora izražavanja (iii) Obtaining expression vectors
0,1 µg oko 4-kb fragmenta, po 0,05 µg BamHI fragmenta koji ima OLPL područje i MbOII-BamHI fragmenta koji ima tL1 područje /tri fragmenta dobivena u (i)/ i 0,1 ukapljenog sintetskog S/D veziva dobivenog u (ii) je podvrgnuto reakciji preko noći na 12°C u 40 µl reakcijske otopine (66 mM Tris-HCl, 6,6 mM MgCl2, 10 mM DTT i 1 mM ATP) u prisutnosti 175 jedinica T4DNA ligaze (Takara Shuzo Co., Ltd.). 20 µl reakcijske otopine upotrijebljeno za transformiranje E. coli vrste N99CI+ (Pharmacia) postupkom sa kalcij-kloridom (vidi također Molecular Cloning). 0.1 µg of about 4-kb fragment, 0.05 µg each of BamHI fragment having OLPL region and MbOII-BamHI fragment having tL1 region /three fragments obtained in (i)/ and 0.1 liquefied synthetic S/D binder obtained in (ii) was subjected to overnight reaction at 12°C in 40 µl reaction solution (66 mM Tris-HCl, 6.6 mM MgCl2, 10 mM DTT and 1 mM ATP) in the presence of 175 units of T4DNA ligase (Takara Shuzo Co. , Ltd.). 20 µl of the reaction solution was used to transform E. coli strain N99CI+ (Pharmacia) by the calcium chloride method (see also Molecular Cloning).
Transformanti su kultivirani i izdvojen je plazmid iz kulture njihovih ampicilin-rezistentnih kolonija. Tretman plazmida sa restrikcijskim enzimima EcoRI, BamHI i Smal pokazuju da je to bio željeni plazmid. The transformants were cultured and the plasmid was isolated from the culture of their ampicillin-resistant colonies. Treatment of the plasmid with the restriction enzymes EcoRI, BamHI and SmaI showed that it was the desired plasmid.
Dva mikrograma ovog plazmida reagiralo je sa restrikcijskim enzimom ClaI (New England Biolabs) na 37°C tijekom 2 sata u 20 µl pufera (10 mM Tris-HCl, 6 mM MgCl2 i 50 mM NaCl). Potom je enzim deaktiviran grijanjem 10 minuta na 65°C. Two micrograms of this plasmid was reacted with the restriction enzyme ClaI (New England Biolabs) at 37°C for 2 hours in 20 µl buffer (10 mM Tris-HCl, 6 mM MgCl2 and 50 mM NaCl). The enzyme was then deactivated by heating for 10 minutes at 65°C.
Jedan mikrolitar reakcijske otopine je preko noći reagirao na 120C sa 175 jedinica T4DNA ligaze (Takara Shuzo Co., Ltd.) u otopini za vezanje, koja ima gore opisani sastav. One microliter of the reaction solution was reacted overnight at 120C with 175 units of T4DNA ligase (Takara Shuzo Co., Ltd.) in a binding solution having the composition described above.
Reakcijska otopina je tada korištena za transformiranje E. coli vrste N99cI+ (Pharmacia). Plazmid je izdvojen iz kulture ampicilin-rezistentnih kolonija transformanata i tretiran sa EcoRI i BamHI, tako da se potvrdi da je spomenuti plazmid onaj željeni. The reaction solution was then used to transform E. coli strain N99cI+ (Pharmacia). The plasmid was isolated from the culture of ampicillin-resistant transformant colonies and treated with EcoRI and BamHI, so as to confirm that the mentioned plasmid is the desired one.
(iv) Dobivanje G-CSF izražavajućeg rekombinantnog vektora i transformanata (iv) Obtaining G-CSF expressing recombinant vector and transformants
Plazmid ekspresije dobiven u (iii) je tretiran sa restrikcijskim enzimom ClaI. Poslije stvaranja otvorenih krajeva, plazmid je obrađen kao u primjeru 12, tako da se dobiva rekombinantni vektor ubačenog cDNA fragmenta G-CSF-a. Ovaj vektor je korišten za transformiranje E. coli vrste N4830 (Pharmacia Fine Chemicals) postupkom sa kalcij-kloridom opisanom u Molecular Cloning, (isto). Identificiranje željenih transformanata izvršeno je kao u primjeru 12 (slika 9). The expression plasmid obtained in (iii) was treated with the restriction enzyme ClaI. After creating the open ends, the plasmid was processed as in example 12, so that a recombinant vector of the inserted cDNA fragment of G-CSF was obtained. This vector was used to transform E. coli strain N4830 (Pharmacia Fine Chemicals) by the calcium chloride procedure described in Molecular Cloning, (ibid.). The desired transformants were identified as in example 12 (Figure 9).
Primjer 14: Example 14:
Stvaranje E. coli rekombinantnog vektora (+VSE) i transformacija (koristeći trp promotor koji ima vektor) Generation of E. coli recombinant vector (+VSE) and transformation (using the trp promoter that the vector has)
1) Građenje rekombinantnog vektora 1) Construction of recombinant vector
(i) Pripremanje vektora (i) Preparation of vectors
Plazmid pOYl, dobiven je ubacivanjem triptofan promotora koji sadrži HpaII-TagI fragmenta (oko 330 bp) u pBR322 na ClaI mjesto. Deset mikrograma ovog plazmida tretirano je sa 7 jedinica restrikcijskog enzima ClaI i 8 jedinica PvuII na 37°C, tijekom 3 sata u 30 µl reakcijske otopine sastavljene od 10 mM Tris-HCl, 6 mM MgCl2 i 50 mM NaCl. Plasmid pOY1 was obtained by inserting the tryptophan promoter containing the HpaII-TagI fragment (about 330 bp) into pBR322 at the ClaI site. Ten micrograms of this plasmid were treated with 7 units of restriction enzyme ClaI and 8 units of PvuII at 37°C for 3 hours in 30 µl reaction solution composed of 10 mM Tris-HCl, 6 mM MgCl2 and 50 mM NaCl.
Zatim je dodano 2 µl alkalne fosfataze (Takara Shuzo Co., Ltd.) i reakcija je vršena na 60°C tijekom 1 sata. Then, 2 µl of alkaline phosphatase (Takara Shuzo Co., Ltd.) was added and the reaction was performed at 60°C for 1 hour.
DNA fragment (oko 2,5 µg) od oko 2,6 kb dužine izdvojen je iz reakcijske otopine 1% agaroza gel elektroforezom. A DNA fragment (about 2.5 µg) of about 2.6 kb in length was separated from the reaction solution by 1% agarose gel electrophoresis.
(ii) Dobivanje sintetskog veziva (ii) Obtaining a synthetic binder
Oligonukleotidi koji imaju nizove sintetskih veziva CGCGAATGACCCCCCTGGGCC i CAGGGGGGTCATTCG su fosforilizirani i kaljeni kao što je opisano u (ii) u primjeru 12, tako da se dobiva sintetsko vezivo. Oligonucleotides having the synthetic linker sequences CGCGAATGACCCCCCTGGGCC and CAGGGGGGTCATTCG were phosphorylated and annealed as described in (ii) in Example 12, so that a synthetic linker was obtained.
(iii) Dobivanje rekombinantnog vektora (iii) Obtaining a recombinant vector
Oko 1 µg vektor fragmenta dobivenog u (i), oko 1 µg sintetskog veziva dobivenog u (ii) i oko 1 µg G-CSF fragmenta dobivenog u (iii) u primjeru 12, reagiralo je sa 175 jedinica T4DNA ligaze preko noći na 12°C u 20 µl otopine za vezanje, koja ima sastav opisan u primjeru 12, 1) (iv), tako da se dobije rekombinantni vektor (slika 10). About 1 µg of the vector fragment obtained in (i), about 1 µg of the synthetic linker obtained in (ii) and about 1 µg of the G-CSF fragment obtained in (iii) in Example 12 were reacted with 175 units of T4DNA ligase overnight at 12° C in 20 µl of the ligation solution, which has the composition described in example 12, 1) (iv), so that the recombinant vector is obtained (Figure 10).
2) Transformiranje 2) Transforming
Dvadeset mikrolitara reakcione otopine dobivene u (iii) korišteno je za transformiranje E. coli DH1 postupkom sa rubidij-kloridom opisanim u Molecular Cloning, isto. Twenty microliters of the reaction solution obtained in (iii) was used to transform E. coli DH1 by the rubidium chloride procedure described in Molecular Cloning, ibid.
Kao i u primjeru 12 plazmid je izdvojen iz ampicilin-rezistentnih kolonija transformanata, i tretiranje ovog plazmida sa reakcijskim enzimima Apal, Dral, Nrul i PstI pokazuje da su dobiveni željeni transformanti. As in Example 12, the plasmid was isolated from ampicillin-resistant transformant colonies, and treatment of this plasmid with the reaction enzymes ApaI, Dral, Nrul and PstI showed that the desired transformants were obtained.
Primjer 15: Example 15:
Kultiviranje transformanata Cultivation of transformants
1) Kultiviranje transformanata (sa tac) dobivenih u primjeru 12 1) Cultivation of transformants (with tac) obtained in example 12
Transformanti su kultivirani preko noći na 37°C i 1 ml kulture je dodan u 100 ml Luria medija, koji sadrži 25 µg/ml ili 50 µg/ml ampicilina. Kultiviranje je vršeno 2-3 sata na 37°C. Transformants were cultured overnight at 37°C and 1 ml of culture was added to 100 ml of Luria medium, containing 25 µg/ml or 50 µg/ml ampicillin. Cultivation was carried out for 2-3 hours at 37°C.
Kultiviranje je nastavljeno na 37°C tijekom 2-4 sata poslije dodavanja izopropil-β-D-tiogalactoze, tako da se postigne krajnja koncentracija od 2 mM. Cultivation was continued at 37°C for 2-4 hours after the addition of isopropyl-β-D-thiogalactose, so as to reach a final concentration of 2 mM.
2) Kultiviranje transformanata (sa PL) dobivenim u primjeru 13 2) Cultivation of transformants (with PL) obtained in example 13
Transformanti su kultivirani preko noći na 28°C, i 1 ml kulture je dodan u 100 ml Luria medija koja sadrži 25 ili 50 µg/ml ampicilina. Kultiviranje je vršeno oko 4 sata na 28°C. Transformants were cultured overnight at 28°C, and 1 ml of culture was added to 100 ml of Luria medium containing 25 or 50 µg/ml ampicillin. Cultivation was carried out for about 4 hours at 28°C.
Kultiviranje je nastavljeno 2-4 sata na 42°C. Cultivation was continued for 2-4 hours at 42°C.
3) Kultiviranje transformanata (sa trP) dobivenim u primjeni 14 3) Cultivation of transformants (with trP) obtained in application 14
Transformanti su kultivirani preko noći na 37°C, i 1 ml kulture je dodan u 100 ml M9 medija, koja sadrži 0,5% glukoze, 0,5% Casamino kiselina (Difco) i 25 ili 50 µg/ml ampicilina. Kultiviranje je vršeno 4-6 sati na 37°C. Poslije dodatka 50 µg/ml 3-β-indolakrilne kiseline (IAA), kultiviranje je nastavljeno 4-8 sati na 37°C. Transformants were cultured overnight at 37°C, and 1 ml of culture was added to 100 ml of M9 medium, containing 0.5% glucose, 0.5% Casamino acid (Difco), and 25 or 50 µg/ml ampicillin. Cultivation was carried out for 4-6 hours at 37°C. After the addition of 50 µg/ml 3-β-indolacrylic acid (IAA), cultivation was continued for 4-8 hours at 37°C.
Primjer 16: Example 16:
Izdvajanje i pročišćavanje G-CSF polipeptida iz E. coli Isolation and purification of G-CSF polypeptide from E. coli
1) Izdvajanje 1) Extraction
Tri vrste transformanata kultiviranih u primjeru 15 je podvrgnuto sljedećim postupcima izdvajanja. Three types of transformants cultured in Example 15 were subjected to the following isolation procedures.
Kultura (100 ml) je centrifugirana tako da se dobije tableta stanica, koja je zatim suspendirana u 5 ml smjese 20 mM Tris-HCl (pH 7,5) i 30 mM NaCl. The culture (100 ml) was centrifuged to obtain a pellet of cells, which was then suspended in 5 ml of a mixture of 20 mM Tris-HCl (pH 7.5) and 30 mM NaCl.
Tada su dodani 0,2 M fenilmetilsulfonil fluorid, 0,2 M EDTA i lizozim u odgovarajućim koncentracijama od 1 mM, 10 mM i 0,2 mg/ml i suspenzija je stavljena 30 minuta na 0°C. Then 0.2 M phenylmethylsulfonyl fluoride, 0.2 M EDTA and lysozyme were added in the respective concentrations of 1 mM, 10 mM and 0.2 mg/ml and the suspension was placed for 30 minutes at 0°C.
Stanice su lizirane pomoću tri ciklusa zamrzavanja/taljenja. Lizat je centrifugiran, tako da se dobije supernatant. Alternativno, lizat je tretiran sa 8 M gunidin hidrokloridom, tako da njegova krajnja koncentracija bude 6 M guanidin hidroklorida, a zatim centrifugiran na 30000 obr/min tijekom 5 sati, i tada je izdvojen supernantant. Cells were lysed using three freeze/thaw cycles. The lysate was centrifuged, so that the supernatant was obtained. Alternatively, the lysate was treated with 8 M guanidine hydrochloride, so that its final concentration was 6 M guanidine hydrochloride, and then centrifuged at 30,000 rpm for 5 hours, and then the supernatant was collected.
2) Pročišćavanje 2) Purification
(i) Supernatant dobiven u 1) podvrgnut je gel filtriranju na Ultrogel AcA54 koloni (4,6 cm promjerax90 cm dužine) pri protoku od oko 50 ml/sat sa 0,01 M Tris-HCl puferom (pH 7,4), koji sadrži 0,15 M NaCl i 0,01% Tween 20 (Nakai Kagaku Co., Ltd.). (i) The supernatant obtained in 1) was subjected to gel filtration on an Ultrogel AcA54 column (4.6 cm diameter x 90 cm length) at a flow rate of about 50 ml/hour with 0.01 M Tris-HCl buffer (pH 7.4), which containing 0.15 M NaCl and 0.01% Tween 20 (Nakai Kagaku Co., Ltd.).
Frakcije koje pokazuju aktivnost pri analizi metodom CSA ispitivanja (b) (opisanoj ranije u ovoj specifikaciji) su odabrane i koncentrirane do volumena od oko 5 ml sa ultrafiltracionom aparaturom pM-10 (Amicon). Fractions showing activity when analyzed by CSA assay method (b) (described earlier in this specification) were selected and concentrated to a volume of about 5 ml with an ultrafiltration apparatus pM-10 (Amicon).
(ii) Koncentriranim frakcijama je dodan n-propanol (čistoće pogodne za ispitivanje aminokiselinskog niza; Tokyo Kasei C, Ltd.) i trifluoroctena kiselina, i smjesa je obrađena tako da krajnja koncentracija n-propanola i trifluoroctene kiseline budu 30% i 0,1%, respektivno. Obrađena smjesa je ostavljena u ledu tijekom oko 15 minuta i centrifugirana na 15000 obr/mm tijekom 10 minuta, radi izdvajanja taloga. Supernatant je adsorbiran na µ-Bondapak C18 koloni (polupreparatorske kvalitete; Waters; 8 mmx30 cm), koja je bila uravnotežena sa vodenom otopinom koja sadrži n-propanol (vidi gore) i trifluoroctenom kiselinom. Kolona je neprekidno eluirana sa vodenom otopinom 0,1% tnfluoroctene kiseline, koja sadrži n-propanol, sa linearnim gradijentom gustoće od 30-60%. Sa Hitachi Model 685-50 (HPLC-aparaturom Hitachi, Ltd.) i Hitachi Model 638-41 (detektorom Hitachi, Ltd.), koji su korišteni, istovremeno je mjerena adsorpcija na 220 nm i 280 nm. Poslije eluiranja, po 10 µl alikvota svake frakcije je razrijeđeno 100 puta i otopine su testirane na aktivne frakcije metodom CSA ispitivanja (b). Opažena je aktivnost u pikovima koji su eluirani 40% n-propanolom. Ovi pikovi su kombinirani i ponovno kromatografirani pod istim uvjetima kao gore i frakcije su ispitivane na svoju aktivnost metodom (b). Ponovno, aktivnost je nađena u pikovima za 40% n-propanol. Ovi aktivni pikovi su sakupljeni (četiri frakcije=4ml) i suho zamrznuti. (ii) n-propanol (purity suitable for amino acid sequence testing; Tokyo Kasei C, Ltd.) and trifluoroacetic acid were added to the concentrated fractions, and the mixture was worked up so that the final concentrations of n-propanol and trifluoroacetic acid were 30% and 0.1 %, respectively. The treated mixture was left in ice for about 15 minutes and centrifuged at 15,000 rev/mm for 10 minutes to separate the precipitate. The supernatant was adsorbed onto a µ-Bondapak C18 column (semi-prep grade; Waters; 8 mmx30 cm), which was equilibrated with an aqueous solution containing n-propanol (see above) and trifluoroacetic acid. The column was continuously eluted with an aqueous solution of 0.1% trifluoroacetic acid, containing n-propanol, with a linear density gradient of 30-60%. With the Hitachi Model 685-50 (HPLC apparatus Hitachi, Ltd.) and Hitachi Model 638-41 (detector Hitachi, Ltd.), which were used, the adsorption at 220 nm and 280 nm was measured simultaneously. After elution, a 10 µl aliquot of each fraction was diluted 100 times and the solutions were tested for active fractions by the CSA test method (b). Activity was observed in peaks eluted with 40% n-propanol. These peaks were combined and rechromatographed under the same conditions as above and the fractions were assayed for activity by method (b). Again, activity was found in peaks for 40% n-propanol. These active peaks were collected (four fractions=4ml) and freeze-dried.
(iii) Suho smrznuti prah je otopljen u 200 µl vodene otopine 0,1% trifluoroctene kiseline koja sadrži 40% n-propanol, i otopina je podvrgnuta HPLC na TSK-G3000SW koloni (7,5 mmx60 cm; Toyo Soda Manufacturing Co., Ltd.). Eluiranje je vršeno pri protoku od 0,4 ml/mm s vodenom otopinom 0,1% trifluoroctene ki-seline koja sadrži 40% n-propanola, i frakcije od 0,4 ml su sakupljane sakupljačem frakcija, RRAC-100 (Pharmacia Fine Chemicals). Frakcije su ispitivane na CSA kao što je opisano naprijed i aktivne frakcije su izdvojene. Dalje su pročišćavane na µ-Bondapak C18 koloni (4,6 mmx30 cm), glavni pik je izdvojen i suho smrznut. (iii) The freeze-dried powder was dissolved in 200 µl of an aqueous solution of 0.1% trifluoroacetic acid containing 40% n-propanol, and the solution was subjected to HPLC on a TSK-G3000SW column (7.5 mmx60 cm; Toyo Soda Manufacturing Co., Ltd.). Elution was performed at a flow rate of 0.4 ml/mm with an aqueous solution of 0.1% trifluoroacetic acid containing 40% n-propanol, and fractions of 0.4 ml were collected with a fraction collector, RRAC-100 (Pharmacia Fine Chemicals ). Fractions were assayed for CSA as described above and active fractions were separated. They were further purified on a µ-Bondapak C18 column (4.6 mmx30 cm), the main peak was separated and freeze-dried.
Tako dobiveni protein je tretiran sa 2-merkaptoetanolom i podvrgnut SDS-poliakril-amid gel (15,0%) elektroforezi (15 mV, 6 sati). Nakon bojanja sa Coomassie Blue, željeni G-CSF polipeptid se može identificirati kao jednostruka vrpca. The thus obtained protein was treated with 2-mercaptoethanol and subjected to SDS-polyacrylamide gel (15.0%) electrophoresis (15 mV, 6 hours). After staining with Coomassie Blue, the desired G-CSF polypeptide can be identified as a single band.
Primjer 17: Example 17:
Ispitivanje G-CSF aktivnosti (+VSE) Test of G-CSF activity (+VSE)
CSF uzorak dobiven u primjeru 16 je ispitivan prema metodi CSF ispitivanja (a) opisanoj ranije u ovoj specifikaciji. Ovi rezultati su prikazani u tablici 1. The CSF sample obtained in Example 16 was tested according to the CSF test method (a) described earlier in this specification. These results are shown in Table 1.
Tablica 1 Table 1
[image] [image]
Primjer 18: Example 18:
Analiza aminokiselina (+VSE) Amino acid analysis (+VSE)
1) Analiza sastava aminokiselina 1) Analysis of amino acid composition
CSF pročišćen u primjeru 16 je hidroliziran rutinskim postupcima. Sastav aminokiselina proteinskog dijela hidrolizata je analiziran metodom analize aminokiselina sa automatskim analizatorom aminokiselina, Hitachi 835 (Hitachi Ltd.). Rezultati su prikazani u tablici 2. Hidroliza je vršena u sljedećim uvjetima: The CSF purified in Example 16 was hydrolyzed by routine procedures. The amino acid composition of the protein part of the hydrolyzate was analyzed using the amino acid analysis method with an automatic amino acid analyzer, Hitachi 835 (Hitachi Ltd.). The results are shown in table 2. Hydrolysis was performed under the following conditions:
(i) 6 N HCl, 110°C, 24 sata u vakuumu (i) 6 N HCl, 110°C, 24 hours in vacuo
(ii) 4 N metansulfonska kiselina + 0,2% 3-(2-aminoetil)indol, 110°C, 24 sata, 48 sati, 72 sata, u vakuumu. (ii) 4 N methanesulfonic acid + 0.2% 3-(2-aminoethyl)indole, 110°C, 24 hours, 48 hours, 72 hours, in vacuum.
Uzorak je otopljen u otopini (1,5 ml) koja sadrži 40% n-propanola i 0,1% tri-fluoroctene kiseline. Alikvoti, svaki mase 0,1 ml su osušeni sa suhim plinovitim dušikom i nakon dodatka reagenasa navedenih u (i) ili (ii), posude su u vakuumu zataljene, a zatim je vršena hidroliza. The sample was dissolved in a solution (1.5 ml) containing 40% n-propanol and 0.1% trifluoroacetic acid. Aliquots, each with a mass of 0.1 ml, were dried with dry nitrogen gas and after the addition of the reagents specified in (i) or (ii), the vessels were sealed in a vacuum, and then hydrolysis was performed.
Svaka od vrijednosti prikazane u tablici 2 je srednja vrijednost 4 mjerenja, vrijednosti za 24 sata (i) i vrijednosti za 24, 48 i 72 sata (ii), osim što su sadržaji Thr, Ser, 1/2Cys, Met, Val, Ile i Trp izračunate sljedećim metodama (vidi "Tampaku Kagaku (Protein Chemistry) II" Course m Biochemical Experiments, Tokyo Kagaku Dohjin); Each of the values shown in Table 2 is the mean of 4 measurements, 24 hour values (i) and 24, 48 and 72 hour values (ii), except that the contents of Thr, Ser, 1/2Cys, Met, Val, Ile and Trp calculated by the following methods (see "Tampaku Kagaku (Protein Chemistry) II" Course m Biochemical Experiments, Tokyo Kagaku Dohjin);
Za Thr, Ser, 1/2Cys i Met, vremenska zavisnost profila od 24, 48 i 72 sata vrijednosti za (ii) je ekstrapolirana na 0 sati. For Thr, Ser, 1/2Cys and Met, the time dependence of the profile of the 24, 48 and 72 hour values for (ii) was extrapolated to 0 hours.
Za Val i Ile, korištena je 72 satna vrijednost za (ii). Za Trp, korištena je srednja vrijednost od 24, 48 i 72 satne vrijednosti za (ii). For Val and Ile, the 72 hour value for (ii) was used. For Trp, the mean of the 24, 48 and 72 hourly values for (ii) was used.
Tablica 2 Table 2
Podaci analize aminokiselina Amino acid analysis data
[image] [image]
2) Analiza N-terminalnih aminokiselina 2) Analysis of N-terminal amino acids
Uzorak je podvrgnut Edman razlaganju sa plin-faznim ispitivačem niza (Appplied Biosystems) i dobivena PTH aminokiselina je analizirana rutinskim postupcima sa HPLC aparaturom (Beckman Instruments) i Ultrasphere-ODS kolonom (Beckman Instruments). The sample was subjected to Edman decomposition with a gas-phase array analyzer (Applied Biosystems) and the obtained PTH amino acid was analyzed by routine procedures with an HPLC apparatus (Beckman Instruments) and an Ultrasphere-ODS column (Beckman Instruments).
Kolona (5 µm; promjera 4,6 mm i dužine 250 mm) uravnotežena je polaznim puferom /vodena otopina koja sadrži 15 mM natrij-acetatnog pufera (pH 4,5) i 40% acetonitrila/, uzorak (otopljen u 20 µl polaznog pufera) je ubačen i izvršeno je jednokratno eluiranje s polaznim puferom. Tijekom ove operacije, protok je održavan na 1,4 ml/min a temperatura kolone na 40°C. The column (5 µm; diameter 4.6 mm and length 250 mm) is equilibrated with the starting buffer /aqueous solution containing 15 mM sodium acetate buffer (pH 4.5) and 40% acetonitrile/, the sample (dissolved in 20 µl of the starting buffer ) was inserted and a single elution was performed with the starting buffer. During this operation, the flow rate was maintained at 1.4 ml/min and the column temperature at 40°C.
Detekcija PTH amino-kiseline je izvršena korištenjem apsorpcija u ultraljubičastom području na 269 nm i 320 nm. Standardni uzorci (svaki mase 2 nmol) PTH aminokiseline (Sigma) su odvojeni na istoj liniji radi određivanja njihovih retencijskih vremena, koja su uspoređivana s onima za uzorak u cilju identifikacije N-terminalnih aminokiselina. Na ovaj način detektirani su PTH-metionin i PTH-treonin. Detection of PTH amino acid was performed using absorption in the ultraviolet region at 269 nm and 320 nm. Standard samples (2 nmol each) of PTH amino acid (Sigma) were separated on the same line to determine their retention times, which were compared with those of the sample in order to identify the N-terminal amino acids. PTH-methionine and PTH-threonine were detected in this way.
Primjer 19: Example 19:
Stvaranje E. coli rekombinantnog vektora (-VSE) i transformacija Creation of E. coli recombinant vector (-VSE) and transformation
1) Koristeći tac promotor koji sadrži vektor 1) Using the tac promoter containing vector
Postupci iz primjera 12 su ponovljeni osim što "pBRG4 dobiven u primjeru 9 koji sadrži cDNA prikazanu na slici 3(A)" /vidi (iii) u primjeru 12/ je zamijenjen sa "pBRV2 dobivenim u primjeru 10 koji sadrži cDNA prikazanu na slici 4(A)". Kao u primjeru 12, dobiveni transformanti su potvrđeni kao poželjni (slika 11). The procedures of Example 12 were repeated except that "pBRG4 obtained in Example 9 containing the cDNA shown in Figure 3(A)" /see (iii) in Example 12/ was replaced with "pBRV2 obtained in Example 10 containing the cDNA shown in Figure 4 (AND)". As in Example 12, the obtained transformants were confirmed as desirable (Figure 11).
2) Koristeći PL promotor koji sadrži vektor 2) Using a PL promoter containing vector
Postupci iz primjera 13 su ponovljeni koristeći cDNA (-VSE) i dobiveni transformanti su potvrđeni kao poželjni (slika 12). The procedures of Example 13 were repeated using cDNA (-VSE) and the resulting transformants were confirmed as desirable (Figure 12).
3) Korištenje trp promotora koji sadrži vektor 3) Using the trp promoter containing vector
Ponovljeni su postupci iz primjera 14 koristeći cDNA (-VSE) i transformanti su potvrđeni kao poželjni (slika 13). The procedures of Example 14 were repeated using cDNA (-VSE) and the transformants were confirmed as desirable (Figure 13).
Primjer 20: Example 20:
Ispitivanje G-CSF aktivnosti (-VSE) G-CSF activity test (-VSE)
Tri vrste tranformanata dobivenih u primjeru 19 su kultivirane metodom opisanom u primjeru 15. Iz kultiviranih E. coli, G-CSF polipeptidi su izdvojeni i pročišćeni metodom opisanom u primjeru 16, tako da se dobiva humani G-CSF polipeptid kao jednostruka vrpca. Three types of transformants obtained in example 19 were cultivated by the method described in example 15. From cultured E. coli, G-CSF polypeptides were isolated and purified by the method described in example 16, so that human G-CSF polypeptide was obtained as a single band.
Tako dobiveni G-CSF uzorak je ispitan metodom ispitivanja CSF aktivnosti (a) opisanom ranije u ovoj specifikaciji. Rezultati su prikazani u tablici 3. The G-CSF sample thus obtained was tested by the CSF activity test method (a) described earlier in this specification. The results are shown in Table 3.
Tablica 3 Table 3
[image] [image]
Primjer 21: Example 21:
Analiza aminokiselina (-VSE) Amino acid analysis (-VSE)
1) Analiza sastava aminokiselina 1) Analysis of amino acid composition
Sastav aminokiselina CSF uzorka pročišćenog u primjeru 20 je analiziran metodom opisanom u 1) u primjeru 18. Rezultati su prikazani u tablici 4. The amino acid composition of the CSF sample purified in Example 20 was analyzed by the method described in 1) in Example 18. The results are shown in Table 4.
Tablica 4 Table 4
Podaci analize aminokiselina Amino acid analysis data
[image] [image]
2) Analiza N-terminalnih aminokisleina 2) Analysis of N-terminal amino acids
Uzorak je podvrgnut analizi N-terminalnih aminokiselina prema metodi opisanoj u 2) primjeru 18. Na ovaj način detektirani su PTH-metionin i PTH-treonin. The sample was subjected to N-terminal amino acid analysis according to the method described in 2) example 18. In this way, PTH-methionine and PTH-threonine were detected.
Primjer 22: Example 22:
Dobivanje pHGA410 vektora (za primjenu sa životinjskim stanicama +VSE linije) Obtaining the pHGA410 vector (for use with animal cells +VSE lines)
EcoRI fragment dobiven u primjeru 9 čija je DNA prikazana na slici 3(A) je tretiran sa restrikcijskim enzimom Dral na 37°C tijekom 2 sata, a zatim je tretiran sa Klenow fragmentom DNA polimeraze 1 (Takara Shuzo Co., Ltd.) tako da nastaju otvoreni krajevi. Jedan mikrogram BglII veziva (8 mer, Takara Shuzo Co., Ltd.) je fosforilizirano sa ATP i pripojeno je na 1 µg posebno dobivene smjese DNA fragmenata. Pripojeni fragmenti su tretirani sa restrikcijskim enzimom, BglII, i podvrgnuti su agaroza gel elektroforezi. Zatim je izdvojen samo najveći DNA fragment. The EcoRI fragment obtained in Example 9 whose DNA is shown in Figure 3(A) was treated with the restriction enzyme Dral at 37°C for 2 hours, and then it was treated with the Klenow fragment of DNA polymerase 1 (Takara Shuzo Co., Ltd.) as follows to create open ends. One microgram of BglII binder (8 mer, Takara Shuzo Co., Ltd.) was phosphorylated with ATP and attached to 1 µg of a specially prepared mixture of DNA fragments. The joined fragments were treated with the restriction enzyme, BglII, and subjected to agarose gel electrophoresis. Then only the largest DNA fragment was isolated.
Ovaj DNA fragment bio je ekvivalentan sa oko 170 baznih parova koji sadrže humani G-CSF polipeptid kodiranog dijela (vidi sliku 6). Vektor pDKCR /Fukunga et al., Proc. Natl. Acad. Sci., USA, 81, 5086(1984)/ je tretiran sa restrikcijskim enzimom BamHI i zatim je defosforiliziran s alkalnom fosfatazom (Takara Shuzo Co., Ltd.). Vektor DNA dobiven je pripajanjem na 710 bp cDNA fragmenta u prisutnosti T4 DNA ligaze (Takara Shuzo Co., Ltd.), tako da se dobiva pHGA410 (slika 14). Kao što je prikazano na slici 14, ovaj plazmid sadrži promotor SV40 ranijeg gena, replikacija replikanta začetka SV40, dio zečjeg β-globin gen, replikaciju injicirajućeg područja pBR322 i pBR322-izvedeni β-laktamaza gen (Amp1), s humanim genom pripojenim ispod promotora SV40 ranijeg gena. This DNA fragment was equivalent to about 170 base pairs containing the human G-CSF polypeptide coding portion (see Figure 6). Vector pDKCR /Fukunga et al., Proc. Natl. Acad. Sci., USA, 81, 5086(1984)/ was treated with restriction enzyme BamHI and then dephosphorylated with alkaline phosphatase (Takara Shuzo Co., Ltd.). Vector DNA was obtained by ligating the 710 bp cDNA fragment in the presence of T4 DNA ligase (Takara Shuzo Co., Ltd.), thus obtaining pHGA410 (Figure 14). As shown in Figure 14, this plasmid contains the SV40 early gene promoter, replication of the SV40 origin replicator, part of the rabbit β-globin gene, replication of the pBR322 injection region, and the pBR322-derived β-lactamase gene (Amp1), with the human gene inserted below the promoter SV40 of the earlier gene.
Primjer 23: Example 23:
Stvaranje rekombinantnog vektora (+VSE) za upotrebu u transformiranju C127 stanica Generation of a recombinant vector (+VSE) for use in transforming C127 cells
1) Stvaranje pHGA410 (H) 1) Creation of pHGA410 (H)
Dvadeset mikrograma plazmida pHGA410 (slika 14) dobivenog u primjeru 22 je otopljeno u reakcijskoj otopini sastavljenoj od 50 mM Tris-HCl (pH 7,5); 7 mM MgCl2, 100 mM NaCl, 7 mM 2-merkaptoetanola i 0,01% albumin seruma teleta (BSA). Twenty micrograms of plasmid pHGA410 (Figure 14) obtained in example 22 was dissolved in a reaction solution composed of 50 mM Tris-HCl (pH 7.5); 7 mM MgCl2, 100 mM NaCl, 7 mM 2-mercaptoethanol and 0.01% calf serum albumin (BSA).
Restrikcijski enzim, EcoRI (10-15 jedinica; Takara Shuzo Co., Ltd.) je dodan u reakcijsku otopinu i držan na 37°C tijekom oko 30 minuta radi djelomičnog raspada sa EcoRI. Zatim je DNA fragment podvrgnut dvama tretmanima sa 1:1 smjesom fenol/kloroform, jednim tretmanom s eterom i taloženjem s etanolom. The restriction enzyme, EcoRI (10-15 units; Takara Shuzo Co., Ltd.) was added to the reaction solution and kept at 37°C for about 30 minutes for partial digestion with EcoRI. The DNA fragment was then subjected to two treatments with a 1:1 mixture of phenol/chloroform, one treatment with ether and precipitation with ethanol.
Dobiveni DNA fragment je otopljen u 50 µl otopine sastavljene od 50 mM Tris-Hd, 5 mM MgCl2, 10 mM DTT, i po jednim mM dATP, dCTP, dGTP i DTTP. Zatim je dodano 5 µ1 Klenow fragmenta i otopina je inkubirana na 14°C tijekom 2 sata tako da nastanu otvoreni krajevi. The obtained DNA fragment was dissolved in 50 µl of a solution composed of 50 mM Tris-Hd, 5 mM MgCl2, 10 mM DTT, and one mM each of dATP, dCTP, dGTP and DTTP. Then 5 µl of Klenow fragment was added and the solution was incubated at 14°C for 2 hours to form open ends.
Zatim je pomoću 0,8% agaroza gel elektroforeze izdvojeno 6 µg DNA fragmenta od oko 5,8 kb dužine. Then, using 0.8% agarose gel electrophoresis, 6 µg of a DNA fragment of about 5.8 kb in length was isolated.
Pet mikrograma izdvojenog DNA fragmenta je otopljeno u 50 µl reakcijske otopi-ne sastavljene od 50 mM Tris-HCl (pH 7,6), 10 mM MgCl2 10 mM DTT i 1 mM ATP. Nakon dodatka 2 µg HindIII veziva (Takara Shuzo Co., Ltd.) reakcija je vršena preko noći na 40C. Five micrograms of the isolated DNA fragment were dissolved in 50 µl of reaction solution composed of 50 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 10 mM DTT and 1 mM ATP. After adding 2 µg of HindIII binder (Takara Shuzo Co., Ltd.), the reaction was carried out overnight at 40C.
Dalje su vršeni tretmani sa fenolom i eterom i taloženje sa etanolom. Talog je otopljen u 30 µ1 otopine sastavljene od 10 mM Tris-HCl (pH 7,5), 7 mM MgCl2 i 60 mM NaCl, i otopina je inkubirana 3 sata na 37°C u prisutnosti 10 jedinica HindIII. Nakon ponovnog tretmana sa T4 ligazom, dobivena DNA je upotrebljena za transformiranje E. coli vrste DH1 postupkom sa rubidij-kloridom (vidi također Molecular Cloning). Iz ampicilin-rezistentnog (Ampr) odabrana je kolonija transformanata stanica koje uzgajaju plazmid koji je identičan sa pHGA410 osim što je HindIII ubačen na EcoRI mjesto. Tako dobiveni plazmid je nazvan pHGA410 (H) (slika 15). Further, treatments with phenol and ether and precipitation with ethanol were carried out. The pellet was dissolved in 30 µl of a solution composed of 10 mM Tris-HCl (pH 7.5), 7 mM MgCl 2 and 60 mM NaCl, and the solution was incubated for 3 hours at 37°C in the presence of 10 units of HindIII. After retreatment with T4 ligase, the obtained DNA was used to transform E. coli strain DH1 by the rubidium chloride method (see also Molecular Cloning). A colony of transformant cells was selected from ampicillin-resistant (Ampr) cells growing a plasmid identical to pHGA410 except that HindIII was inserted into the EcoRI site. The resulting plasmid was named pHGA410 (H) (Figure 15).
2) Stvaranje rekombinantnog vektora ekspresije pTN-G4 2) Creation of recombinant pTN-G4 expression vector
Dvadeset mikrograma pHGA410 (H) tako dobivenog je otopljeno u 50 µ1 reakcijske otopine koja je sastavljena od 10 mM Tris-HCl (pH 7,5) i 7 mM MgCl2, 175 mM NaCl, 0,2 mM EDTA, 7 mM 2-merkaptoetanola i 0,01% seruma albumina goveda. Poslije dodatka 20 jedinica SalI (Takara Shuzo Co., Ltd.), reakcijska otopina je inkubirana na 37°C tijekom 5 sati. Poslije tretmana sa fenolom i taloženja sa etanolom, inkubacija je vršena kao u 1) tijekom 2 sata na 14°C u prisustvu Klenow fragmenta DNA polimeraze (Takara Shuzo Co.,Ltd), tako da se grade otvoreni krajevi. Bez prethodnog izdvajanja DNA pomoću agaroza gel elektroforeze, reakcijska otopina je odmah podvrgnuta taloženju sa etanolom. Dobiveni DNA fragment je tretiran sa HindIII i 5 µg HindIII-SalI fragmenta (oko 2,7 kbp) je izdvojeno pomoću 1% agaroza gel elektroforeze. U posebnom stupnju, plazmid pdBPV-1, koji ima papilloma virus goveda (BPV) (ovaj plazmid je dobiven zahvaljujući dobroti Dr. Howley i opisan je u Sarver, N., Sbyrne, J.C. & Howley, P.M. Proc. Natl. Acad. Sci., USA, 79, 7147-7151(1982)/ je tretiran sa HindIII i PvuII, kao što je opisao Nagata et al., /Fukunga, Sokawa i Nagata, Proc. Natl. Acad. Sci., USA, 81, 5086-5090(1984), tako da se dobije 8,4 kb DNA fragment. Ovaj 8,4 kb DNA fragment i posebni dobiveni HindIII-SalI DNA fragment (oko 2,7 kbp) je povezan sa T4DNA ligazom. Produkt povezivanja je ko-rišten za transformiranje E. coli vrste DH1 pomoću postupka sa rubidij-kloridom opisanom u Molecular Cloning. Izdvojene su E. coli kolonije koje gaje plazmid koji ima pHGA410-izvedenu G-CSF cDNA. Ovaj plazmid je nazvan pTN-G4 (slika 15). Twenty micrograms of pHGA410 (H) thus obtained was dissolved in 50 µl of a reaction solution composed of 10 mM Tris-HCl (pH 7.5) and 7 mM MgCl2, 175 mM NaCl, 0.2 mM EDTA, 7 mM 2-mercaptoethanol. and 0.01% bovine serum albumin. After addition of 20 units of SalI (Takara Shuzo Co., Ltd.), the reaction solution was incubated at 37°C for 5 hours. After treatment with phenol and precipitation with ethanol, incubation was performed as in 1) for 2 hours at 14°C in the presence of Klenow fragment of DNA polymerase (Takara Shuzo Co., Ltd), so that open ends are built. Without prior separation of DNA using agarose gel electrophoresis, the reaction solution was immediately subjected to ethanol precipitation. The resulting DNA fragment was treated with HindIII and 5 µg of the HindIII-SalI fragment (about 2.7 kbp) was separated by 1% agarose gel electrophoresis. In particular, plasmid pdBPV-1, which has bovine papilloma virus (BPV) (this plasmid was obtained through the kindness of Dr. Howley and is described in Sarver, N., Sbyrne, J.C. & Howley, P.M. Proc. Natl. Acad. Sci ., USA, 79, 7147-7151(1982)/ was treated with HindIII and PvuII, as described by Nagata et al., /Fukunga, Sokawa and Nagata, Proc. Natl. Acad. Sci., USA, 81, 5086 -5090(1984), so that an 8.4 kb DNA fragment is obtained. This 8.4 kb DNA fragment and a separate HindIII-SalI DNA fragment obtained (about 2.7 kbp) are ligated with T4DNA ligase. The ligation product is co- was prepared to transform E. coli strain DH1 using the rubidium chloride procedure described in Molecular Cloning. E. coli colonies growing a plasmid having a pHGA410-derived G-CSF cDNA were isolated. This plasmid was named pTN-G4 (Figure 15).
Adenovirus tipa II /Tanpakushitsu, Kakusan, Koso (Proteini, Nucleic Acids i Enzy-mes), 27, Decembar, 1982, Kyoritsu Shuppan/ slično je tretiran tako da se dobije plazmid. pVA koji sadrži oko 1700-bp SalI-HindIII fragmenta VAI i VAII i fragment koji sadrži VAI i VAII su izdvojeni iz ovog plazmida. Ovaj fragment je ubačen u pTNG4 na HindIII mjesto, tako da se dobiva pTNG4VAα i pTNG4VAγ (slika 15). Zbog toga VA gen adenovirusa, ovih plazmida je sposoban da istakne ekspresiju transkripcijskog produkta iz ranijeg promotora SV40. Adenovirus type II /Tanpakushitsu, Kakusan, Koso (Proteini, Nucleic Acids i Enzymes), 27, December, 1982, Kyoritsu Shuppan/ was similarly treated to obtain a plasmid. pVA containing about 1700-bp SalI-HindIII fragments VAI and VAII and a fragment containing VAI and VAII were isolated from this plasmid. This fragment was inserted into pTNG4 at the HindIII site, resulting in pTNG4VAα and pTNG4VAγ (Figure 15). Because of this, the VA gene of adenoviruses of these plasmids is able to highlight the expression of the transcription product from the early promoter of SV40.
Primjer 24: Example 24:
Transformiranje C127 stanica i G-CSF izražavanje (+VSE) Transforming C127 cells and G-CSF expression (+VSE)
Prije nego što je upotrijebljen za transformiranje C127 stanica miša, pTN-G4 dobiven u primjeru 23 je tretiran sa restrikcijskim enzimom BamHI. Dvadeset mikrograma plazmida pTN-G4 je otopljeno u 100 µl reakcijske otopine /10 mM Tris-HCl (pH 8,0), 7 mM MgCl2 100 mM NaCl, 2 mM 2-merkaptoetanola i 0,01% BSA/ i tretirano je sa 20 jedinica BamHI (Takara Shuzo Co.,Ltd.), a zatim je tretirano sa fenolom i eterom i taloženo sa etanolom. Before being used to transform mouse C127 cells, pTN-G4 obtained in Example 23 was treated with the restriction enzyme BamHI. Twenty micrograms of plasmid pTN-G4 was dissolved in 100 µl of reaction solution /10 mM Tris-HCl (pH 8.0), 7 mM MgCl2, 100 mM NaCl, 2 mM 2-mercaptoethanol and 0.01% BSA/ and treated with 20 unit of BamHI (Takara Shuzo Co., Ltd.), and then treated with phenol and ether and precipitated with ethanol.
C127 stanice miša uzgajane u Dulbecco-ovoj minimalnoj osnovnoj sredini, koja sadrži 10% seruma zametka goveda (Gibco). C127 stanice uzgajane na plitici promjera 5 cm su transformirane sa 10 µg, po plititci, posebno dobivene DNA postupkom sa kalcij-fosfatom /vidi Haynes, J. & Weissmann, C., Nucleic Acids Res., 11, 687-706 (1983)/. Poslije tretiranja sa glicerolom stanice su inkubirane na 37°C tijekom 12 sati. Mouse C127 cells grown in Dulbecco's minimal essential medium, containing 10% fetal bovine serum (Gibco). C127 cells grown in 5 cm diameter plates were transformed with 10 µg, per plate, of DNA specifically obtained by the calcium phosphate method / see Haynes, J. & Weissmann, C., Nucleic Acids Res., 11, 687-706 (1983) /. After treatment with glycerol, the cells were incubated at 37°C for 12 hours.
Inkubirane stanice su transformirane na tri svježe plitice promjera 5 cm i medij je mijenjan dvaput tjedno. 1 dana foke su bile prenesene na svježe plitice i podvrgnute serijskom kultiviranju na Dulbecco-ovoj minimalnoj osnovnoj sredini, koja sadrži 10% seruma zametka goveda (Gibco), tako da se odvoje klonovi koji imaju visoku brzinu produkcije G-CSF. Ovi klonovi su proizveli G-CSF, od oko 1 mg/ml. Dalje kloniranje daje porast u klonovima koji su sposobni producirati G-CSF na razini 10 mg/ml ili više. Dodatno u C127I stanicama, NIH3T3 također mogu biti korištene stanice domaćina. Incubated cells were transformed into three fresh 5 cm diameter plates and the medium was changed twice a week. At 1 day, seals were transferred to fresh plates and serially cultured on Dulbecco's minimal essential medium, containing 10% fetal bovine serum (Gibco), to isolate clones having a high rate of G-CSF production. These clones produced G-CSF of about 1 mg/ml. Further cloning gives rise to clones capable of producing G-CSF at a level of 10 mg/ml or more. In addition to C127I cells, NIH3T3 can also be used host cells.
Primjer 25: Example 25:
Izražavanje G-CSF u CHO stanicama (+VSE) Expression of G-CSF in CHO cells (+VSE)
1) Stvaranje pHGG4-dhfr 1) Creation of pHGG4-dhfr
Dvadeset mikrograma plazmida pHGA410 dobivenog u primjeru 22 je otopljeno u 100 µl reakcijske otopine koja sadrži 10 mM Tris-HCl (pH 7,5), 7 mM MgCl2, 175 mM NaCl, 0,2 mM EDTA, 0,7 mM 2-merkaptoetanola i 0,01% BSA. Reakcija je vršena preko noći na 37°C u prisustvu 20 jedinica restrikcijskog enzima Sall (Takara Shuzo Co., Ltd.), a zatim je izvršeno tretiranje sa fenolom i eterom i taloženje sa etanolom. Twenty micrograms of plasmid pHGA410 obtained in example 22 was dissolved in 100 µl of reaction solution containing 10 mM Tris-HCl (pH 7.5), 7 mM MgCl2, 175 mM NaCl, 0.2 mM EDTA, 0.7 mM 2-mercaptoethanol. and 0.01% BSA. The reaction was carried out overnight at 37°C in the presence of 20 units of restriction enzyme SalI (Takara Shuzo Co., Ltd.), followed by treatment with phenol and ether and precipitation with ethanol.
Talog DNA je otopljen u 100 µl reakcijske otopine, koja sadrži 50 mM Tris-HCl, 5mM MgCl2, 10 mM DTT i po 1 mM dATP, dCTP, dGTP i dTTP i reakcija je izvršena na 14°C tijekom 2 sata u prisustvu Klenow fragmenta E. coli i DNA polimeraze (10 µl; Takara Shuzo Co., Ltd.), a zatim je tretiranje vršeno sa fenolom i eterom i taloženje sa etanolom. The DNA precipitate was dissolved in 100 µl of the reaction solution, which contains 50 mM Tris-HCl, 5 mM MgCl2, 10 mM DTT and 1 mM each of dATP, dCTP, dGTP and dTTP and the reaction was carried out at 14°C for 2 hours in the presence of the Klenow fragment E. coli and DNA polymerase (10 µl; Takara Shuzo Co., Ltd.), followed by treatment with phenol and ether and precipitation with ethanol.
EcoRI vezivo je pripojeno na DNA u talogu sljedećim postupcima: The EcoRI binder was attached to the DNA in the precipitate by the following procedures:
DNA otopljena u 50 µl reakcijske otopine koja sadrži 50 mM Tris-HCl (pH 7,4), 10 mM DTT, 0,05 mM spermidma, 2 mM ATP, 2 mM heksamin-kobalt klorida i 20 µg/ml BSA. Reakcija je vršena na 4°C tijekom 12-16 sati u prisustvu EcoRI veziva (Takara Shuzo Co., Ltd.) i 200 jedinica T4DNA ligaze (Takara Shuzo Co., Ltd.). Poslije tretiranja sa fenolom, ispiranja sa eterom i taloženja sa etanolom, sve je vršeno prema rutinskim postupcima. DNA talog je djelomično razoren sa EcoRI i 3 µg fragmenata od oko 2,7 kpb dužine je izdvojeno pomoću 1% agar gel elektroforeze. DNA dissolved in 50 µl reaction solution containing 50 mM Tris-HCl (pH 7.4), 10 mM DTT, 0.05 mM spermide, 2 mM ATP, 2 mM hexamine-cobalt chloride and 20 µg/ml BSA. The reaction was performed at 4°C for 12-16 hours in the presence of EcoRI binder (Takara Shuzo Co., Ltd.) and 200 units of T4DNA ligase (Takara Shuzo Co., Ltd.). After treatment with phenol, washing with ether and precipitation with ethanol, everything was done according to routine procedures. The DNA pellet was partially digested with EcoRI and 3 µg fragments of about 2.7 kbp in length were separated by 1% agar gel electrophoresis.
Plazmid pAdD26SVpA /Kaufman, R.G. & Sharp, P.A., Mol. Cell Biol., 2, 1304-1319(1982)/ tretirano je sa E. coli i defosforilizirano tretiranjem sa bakterijskom alkalnom fosfatazom (BAP). Određenije, 20 µg pAdD26SVpA i 20 jedinica EcoRI je dodano reakcijskoj otopini /50 mM Tris-HCl (pH 7,5), 7 mM MgCl2, 100 mM NaCl, 7 mM 2-merkaptoetanola i 0,01% BSA/ i reakcija je vršena na 37°C tijekom 10 sati. Zatim je reakcijskoj otopini dodano 5 jedinica BAP i reakcija je vršena na 68°C tijekom 30 minuta. Poslije tretmana sa fenolom, EcoRI fragment pAdD26SVpA je izdvojen elektroforezom u prinosu od oko 5 µg. Plasmid pAdD26SVpA /Kaufman, R.G. & Sharp, P.A., Mol. Cell Biol., 2, 1304-1319(1982)/ was treated with E. coli and dephosphorylated by treatment with bacterial alkaline phosphatase (BAP). More specifically, 20 µg of pAdD26SVpA and 20 units of EcoRI were added to the reaction solution (50 mM Tris-HCl (pH 7.5), 7 mM MgCl2, 100 mM NaCl, 7 mM 2-mercaptoethanol and 0.01% BSA) and the reaction was carried out. at 37°C for 10 hours. Then 5 units of BAP were added to the reaction solution and the reaction was performed at 68°C for 30 minutes. After treatment with phenol, the EcoRI fragment of pAdD26SVpA was isolated by electrophoresis in a yield of about 5 µg.
Fragment od oko 2,7 kbp dužine i pAdD26SVpA, svaki mase 0,5 µg je kaljeno. Dobiveni plazmid je upotrijebljen radi transformiranja E. coli vrste DH1, postupkom sa rubidij-kloridom, i odvojene su kolonije odgajivača plazmida pHGG4-dhfr. Dobiveni plazmid je nazvan pHGG4-dhfr (slika 16a). A fragment of about 2.7 kbp in length and pAdD26SVpA, each weighing 0.5 µg, was annealed. The resulting plasmid was used to transform E. coli strain DH1, using the rubidium chloride method, and colonies of the pHGG4-dhfr plasmid grower were separated. The resulting plasmid was named pHGG4-dhfr (Figure 16a).
Alternativni postupak je sljedeći: Plazmid pHGA410 je tretiran sa Sall i djelomično razoren sa RcoRI, bez dodatka EcoRI veziva. DNA fragment od oko 2,7 kbp dužine je izdvojen i tretiran sa Klenow fragmentom E. coli DNA polimeraze, tako da se stvore otvoreni krajevi. EcoRI fragment koji ima otvorene krajeve je dobiven iz pAdD26SVpA, kao što je opisano gore. Ovaj EcoRI fragment i posebno pripremljen fragment (oko 2,7 kbp) je tretiran sa T4DNA ligazom, tako da se dobiva pHGG4-dhfr. An alternative procedure is as follows: Plasmid pHGA410 was treated with SalI and partially digested with RcoRI, without addition of EcoRI binder. A DNA fragment of about 2.7 kbp in length was isolated and treated with the Klenow fragment of E. coli DNA polymerase to create open ends. An open-ended EcoRI fragment was obtained from pAdD26SVpA, as described above. This EcoRI fragment and a specially prepared fragment (about 2.7 kbp) were treated with T4DNA ligase, so that pHGG4-dhfr was obtained.
pHGA410 (H) dobiven u primjeru 23 je tretiran sa restrikcijskim enzimima HindIII i Sall, kao što je opisano u 2) u primjeru 23 i HindIII-SalI fragment je pripojen na otvoreni kraj EcoRI fragmenta pAdD26SVpA, opisanog gore. Ova metoda može se također koristiti za dobivanje pHGG4-dhfr (Slika 16b). pHGA410 (H) obtained in Example 23 was treated with restriction enzymes HindIII and SalI, as described in 2) in Example 23 and the HindIII-SalI fragment was ligated to the open end of the EcoRI fragment of pAdD26SVpA, described above. This method can also be used to obtain pHGG4-dhfr (Figure 16b).
2) Stvaranje pG4DR1 i pG4DR2 2) Creation of pG4DR1 and pG4DR2
Deset mikrograma plazmida pAdD26SVpA, spomenutog u 1) otopljeno je u 50 ml reakcijske otopine koja sadrži 50 mM Tris-HCl (pH 7,5), 7 mM MgCl2, 100 mM NaCl, 7 mM 2-merkaptoetanola i 0,01% BSA. Poslije dodavanja po 10 jedinica svakog od restrikcijskih enzima EcoRI i BamHI reakcija je vršena na 37°C tijekom 10 sati, a zatim je tretiranje nastavljeno sa fenolom i ispiranje sa eterom. DNA fragment od oko 2 kb je izdvojen elektroforezom kroz 1% agarozu gel niske točke taljenja. Izdvojeni DNA fragment je tretiran sa Klenow fragmentom DNA polimeraze rutinskim postupcima, tako da se grade otvoreni krajevi. DNA fragment otvorenih krajeva je podvrgnut tretmanu sa fenolom, ispran sa eterom i taložen sa etanolom. Ten micrograms of plasmid pAdD26SVpA, mentioned in 1) was dissolved in 50 ml of reaction solution containing 50 mM Tris-HCl (pH 7.5), 7 mM MgCl2, 100 mM NaCl, 7 mM 2-mercaptoethanol and 0.01% BSA. After adding 10 units of each of the restriction enzymes EcoRI and BamHI, the reaction was carried out at 37°C for 10 hours, and then the treatment was continued with phenol and washing with ether. A DNA fragment of about 2 kb was separated by electrophoresis through a 1% low melting point agarose gel. The separated DNA fragment was treated with Klenow fragment DNA polymerase by routine procedures, so that open ends are built. The open-ended DNA fragment was treated with phenol, washed with ether and precipitated with ethanol.
Deset mikrograma plazmida pHGA410, dobivenog u 1) primjera 23 otopljeno je u 50 µl reakcijske otopine koja sadrži 10 mM Tris-HCl (pH 7,5), 7 mM MgCl2 60 mM NaCl. Reakcija je vršena tijekom 6 sati na 37°C, u prisutnosti 10 jedinica HindIII. DNA fragment je izdvojen elektroforezom iz 1% agaroza gela niske točke taljenja, izvršenom rutinskim postupcima. Izdvojeni DNA fragment je zatim tretiran sa BAP i stvoreni su otvoreni krajevi, tretiranjem sa Klenow fragmentom. Tada je ̧tretiranjem sa fenolom i pranjem sa eterom DNA fragment pripojen na otvorene krajeve prethodno dobivenog 2-kb DNA fragmenta sa T4DNA ligazom prema sljedećim postupcima: po 1 µg svakog DNA fragmenta je otopljen u 30 µl reakcijske otopine koja sadrži 66 mM Tris-HCl (pH 7,5), 6,6 mM MgCl2, 5 mM DTT i 1 mM ATP i reakcija je izvedena na 6°C tijekom 12 sati, u prisustvu 50 jedinica T4DNA ligaze. Produkt povezivanja je upotrijebljen radi transformiranja E. coli vrste DH1. Na ovaj način dobiveni su pG4DR1 i pG4DR2, prikazani na slici 16c. Ten micrograms of plasmid pHGA410, obtained in 1) of example 23 were dissolved in 50 µl of reaction solution containing 10 mM Tris-HCl (pH 7.5), 7 mM MgCl2, 60 mM NaCl. The reaction was carried out for 6 hours at 37°C, in the presence of 10 units of HindIII. The DNA fragment was separated by electrophoresis from a 1% low-melting point agarose gel, performed by routine procedures. The isolated DNA fragment was then treated with BAP and open ends were created by treatment with the Klenow fragment. Then, by treating with phenol and washing with ether, the DNA fragment was attached to the open ends of the previously obtained 2-kb DNA fragment with T4DNA ligase according to the following procedures: 1 µg of each DNA fragment was dissolved in 30 µl of a reaction solution containing 66 mM Tris-HCl ( pH 7.5), 6.6 mM MgCl2, 5 mM DTT and 1 mM ATP and the reaction was performed at 6°C for 12 hours, in the presence of 50 units of T4DNA ligase. The ligation product was used to transform E. coli strain DH1. In this way, pG4DR1 and pG4DR2 were obtained, shown in Figure 16c.
3) Transformiranje i izražavanje 3) Transforming and expressing
CHO stanice (dhfr-vrsta; dobivena ljubaznošću Dr. L. Chasin-a sa Kolumbia Univerziteta) su kultivirane radi odgajanja u alfa-minimalnom osnovnom mediju, koji sadrži 10% osnovnog seruma goveda (α-MEN dopunjenom sa adenozinom, dezoksiadenozinom i timidinom) u plitici promjera 9 cm (Nune). Kultivirane stanice su transformirane postupkom sa kalcij-fosfatom (Wigler et al., Cell, 14, 725(1978)/ na sljedeći način: CHO cells (dhfr-type; courtesy of Dr. L. Chasin, Columbia University) were cultured for growth in alpha-minimal essential medium, containing 10% basic bovine serum (α-MEN supplemented with adenosine, deoxyadenosine, and thymidine). in a plate with a diameter of 9 cm (Nune). Cultured cells were transformed by the calcium-phosphate method (Wigler et al., Cell, 14, 725(1978)) as follows:
Nosač DNA (timus DNA teleta) je dodan u odgovarajućoj količini u 1 µg plaz-mida pHGG4-dhfr, dobivenog u 1), i smjesa je otopljena u 375 µl TE otopine, što je dalje praćeno dodatkom 125 µl 1M CaCl2. Otopina je ohlađena na ledu tijekom 3-5 minuta, i otopini je dodano 500 µl 2xHBS (50 mM Hepes, 280 mM NaCl i 1,5 mM fosfatnog pufera). Poslije ponovnog hlađenja na ledu, otopina je izmiješana sa 1 ml kulture CHO stanica, prenesenih na plitice i inkubirana je 9 sati u CO2 inkubatoru. Sredina je izdvojena iz plitice i isprana je sa TBS (Tris-puferni salin), dodano joj je 20% glicerola koji sadrži TBS, i medij je ponovno ispran, poslije čega je dodan neselektivni medij (α-MEN medij, opisan gore, osim što je dopunjen sa nukleotidima). Nakon drugog dana inkubacije, 10-puta razrijeđena kultura je prenesena u selektivni medij (koji nije dopunjen nukleotidima). Kultiviranje je nastavljeno, tako što je medij zamjenjivan selektivnim svježim medijem svaka 2 dana i dobivene kolonije su odabrane i prenesene na svježe plitice, gdje stanice odrastaju u prisutnosti 0,02 µM metotreksata (MTX), što je praćeno kloniranjem kroz rast u prisutnosti 0,05 µM MTX, što je dalje povećano na 0,1 µM MTX. Carrier DNA (calf thymus DNA) was added in an appropriate amount to 1 µg of plasmid pHGG4-dhfr, obtained in 1), and the mixture was dissolved in 375 µl of TE solution, which was further followed by the addition of 125 µl of 1M CaCl2. The solution was cooled on ice for 3-5 min, and 500 µl of 2xHBS (50 mM Hepes, 280 mM NaCl, and 1.5 mM phosphate buffer) was added to the solution. After re-cooling on ice, the solution was mixed with 1 ml of CHO cell culture, transferred to plates and incubated for 9 hours in a CO2 incubator. The medium was removed from the plate and washed with TBS (Tris-buffered saline), 20% glycerol containing TBS was added, and the medium was washed again, after which non-selective medium (α-MEN medium, described above, except is supplemented with nucleotides). After the second day of incubation, the 10-fold diluted culture was transferred to selective medium (not supplemented with nucleotides). Cultivation was continued by replacing the selective medium with fresh medium every 2 days and the resulting colonies were picked and transferred to fresh plates, where the cells were grown in the presence of 0.02 µM methotrexate (MTX), followed by cloning through growth in the presence of 0, 05 µM MTX, which was further increased to 0.1 µM MTX.
Transformacija CHO stanica može biti također izvršena pomoću kontratransformacije sa pHGG4 i pAdD26SVpA /vidi Shaill et al., Proc. Natl. Acad. ScL, USA, 80, 4654-4658(1983)/. Transformation of CHO cells can also be performed by counter-transformation with pHGG4 and pAdD26SVpA /see Shaill et al., Proc. Natl. Acad. ScL, USA, 80, 4654-4658(1983)/.
CHO stanice su također transformirane sljedećim postupcima: CHO cells were also transformed by the following procedures:
pG4DR1 ili pG4DR2, koji su dobiveni u 2) su prethodno tretirani sa Sall i KpnI, radi dobivanja DNA fragmenata i 10 µg ovih fragmenata je upotrijebljeno radi transformiranja CHO stanica, kao gore; transformirane stanice su podvrgnute kontinuiranom kultiviranju u seriji selektivnih medija na način opisan gore; oko 7 dana kasnije ne manje od 100 razdvojenih kolonija pojavljuje se u svakoj plitici; ove kolonije su prenesene u masi na svježu pliticu i podvrgnute nastavljenom kultiviranju u seriji selektivnih medija, u prisutnost 0,01 mM MTX, dok se ne pojavi deset neparnih kolonija; isti postupci su ponovljeni i sa MTX koncentracijama od 0,02, 0,05, i 2,01 µM, i kolonije koje su preživjele su odabrane; selekcija kolonija se može izvršiti na sličan način, čak i kada se dobivenih 10 neparnih kolonija podvrgne kultiviranju sa rastućim koncentracijama MTX-a. pG4DR1 or pG4DR2, which were obtained in 2) were pretreated with SalI and KpnI, to obtain DNA fragments and 10 µg of these fragments were used to transform CHO cells, as above; transformed cells were subjected to continuous cultivation in a series of selective media in the manner described above; about 7 days later no less than 100 separated colonies appear in each plate; these colonies were transferred en masse to a fresh plate and subjected to continued cultivation in a series of selective media, in the presence of 0.01 mM MTX, until ten odd colonies appeared; the same procedures were repeated with MTX concentrations of 0.02, 0.05, and 2.01 µM, and the colonies that survived were selected; colony selection can be performed in a similar manner, even when the resulting 10 odd colonies are subjected to cultivation with increasing concentrations of MTX.
Rekombinantni vektor koji gaji "policitronski gen", može se također koristiti za transformiranje CHO stanica. Primjer ove alternativne metode je sljedeći: A recombinant vector harboring the "polycitron gene" can also be used to transform CHO cells. An example of this alternative method is as follows:
pAdD26SVpA je tretiran sa PstI i izdvojena dva fragmenta su pripojena pBRG4 -izvedenom CSF cDNA fragmentu, tako da se gradi rekombinantni vektor, gdje su adenovirus promotor, CSF cDNA, DHFR i poli (A), mjesto SV40 ubačeni u napisanom poretku. Ovaj rekombinantni vektor je upotrijebljen za transformiranje CHO stanica. pAdD26SVpA was treated with PstI and the separated two fragments were joined to the pBRG4-derived CSF cDNA fragment, so that a recombinant vector was built, where the adenovirus promoter, CSF cDNA, DHFR and poly(A), SV40 site were inserted in the written order. This recombinant vector was used to transform CHO cells.
Primjer 26: Example 26:
Ispitivanje G-CSF aktivnosti (+VSE) Test of G-CSF activity (+VSE)
Supernatanti kultura C127 stanica i CHO stanica koje su dobivene u primjerima 24, odnosno 25, podvrgnute su podešenom pH od 4 sa 1N octenom kiselinom. Culture supernatants of C127 cells and CHO cells obtained in examples 24 and 25, respectively, were subjected to an adjusted pH of 4 with 1N acetic acid.
Nakon dodatka jednakog volumena n-propanola, dobiveni talog je uklonjen centrifugiranjem. Supernatant je propušten kroz otvorenu kolonu (promjera 1 cm i dužine 2 cm) ispunjenu sa C8 reverzno-faznim nosačem (Vamamura Kagaku K.K.) i eluiranje je izvršeno sa 50% n-propanola. After the addition of an equal volume of n-propanol, the resulting precipitate was removed by centrifugation. The supernatant was passed through an open column (diameter 1 cm and length 2 cm) filled with C8 reverse-phase carrier (Vamamura Kagaku K.K.) and eluted with 50% n-propanol.
Eluat je razrijeđen dva puta s vodom i podvrgnut je reverzno-faznoj HPLC na YMC-C8 koloni (Yamamura Kagaku K.K.), a zatim je eluiranje vršeno sa n-propanolom (30-60% linearni gradijent gustoće) koji sadrži 0,1% TFA. Frakcije koje su eluirane sa n-propanolom koncentracija od oko 40% su izdvojene, suho zamrznute i otopljene u 0,1 M glicinskog pufera (pH 9). Zbog ovih postupaka, humani G-CSF u C127 i CHO stanicama je koncentriran oko 20 puta. The eluate was diluted twice with water and subjected to reverse-phase HPLC on a YMC-C8 column (Yamamura Kagaku K.K.), then eluting with n-propanol (30-60% linear density gradient) containing 0.1% TFA . Fractions eluted with n-propanol concentration of about 40% were separated, freeze-dried and dissolved in 0.1 M glycine buffer (pH 9). Due to these procedures, human G-CSF in C127 and CHO cells is concentrated about 20 times.
Kao kontrola, stanice su transformirane s humanim G-CSF cDNA-slobodnim plazmidima i supernatanti njihovih kultura su koncentrirani prema postupcima opisanim naprijed. Aktivnosti humanih G-CSF uzoraka ispitivane su metodom ispitiva-nja aktivnosti humanog G-CSF (a) opisanom ranije u ovoj specifikaciji. Ako je djelotovornost ekspresije dovoljno visoka, supernatanti kultura mogu biti izravno ispitivani bez prethodnog koncentriranja. Rezultati su dani u tablici 5, gdje su podaci bazirani na koncentriranim uzorcima. As a control, cells were transformed with human G-CSF cDNA-free plasmids and their culture supernatants were concentrated according to the procedures described above. The activities of the human G-CSF samples were tested by the method for testing the activity of human G-CSF (a) described earlier in this specification. If the expression efficiency is high enough, culture supernatants can be directly assayed without prior concentration. The results are given in Table 5, where the data are based on concentrated samples.
Tablica 5 Table 5
Ispitivanje aktivnosti humane G-CSF Assay of the activity of human G-CSF
[image] [image]
Primjer 27: Example 27:
Analiza aminokiselina i analiza šećera (+VSE) Amino acid analysis and sugar analysis (+VSE)
1) Analiza sastava aminokiselina 1) Analysis of amino acid composition
Sirovi CSF uzorak dobiven u primjeru 26 pročišćen je prema postupcima opisanim u primjeru (iii). Pročišćeni uzorak CSF je hidroliziran rutinskim postupcima, protein dijela hidrolizata je ispitivan na aminokiselinski sastav specijalnom metodom analize aminokiseline sa Hitachi 835 automatskim analizatorom aminokiselina (Hitachi, Ltd.). Rezultati su prikazani u tablici 6. Hidroliza je vršena u sljedećim uvjetima: The crude CSF sample obtained in Example 26 was purified according to the procedures described in Example (iii). The purified CSF sample was hydrolyzed by routine procedures, the protein part of the hydrolyzate was examined for amino acid composition using a special method of amino acid analysis with a Hitachi 835 automatic amino acid analyzer (Hitachi, Ltd.). The results are shown in table 6. Hydrolysis was performed under the following conditions:
(i) 6 N HCl, 110°C, 24 sata, u vakuumu (i) 6 N HCl, 110°C, 24 hours, in vacuo
(ii) 4 N metansulfonska kiselina +0,2% 3-(2-aminoetil)indol, 110°C, 24 sata, 48 sati, 72 sata, u vakuumu. (ii) 4 N methanesulfonic acid +0.2% 3-(2-aminoethyl)indole, 110°C, 24 hours, 48 hours, 72 hours, in vacuo.
Uzorak je otopljen u otopini (1,5 ml) koja sadrži 40% n-propanola i 0,1% tri-fluoroctene kiseline. Alikvoti otopine, svaki od 0,1 ml, su osušeni sa suhim plinovitim dušikom, nakon dodatka reagensa navedenih u (i) ili (ii), posude sa otopinom su zataljene u vakuumu, što je dalje praćeno hidrolizom sadržaja. The sample was dissolved in a solution (1.5 ml) containing 40% n-propanol and 0.1% trifluoroacetic acid. Aliquots of the solution, each of 0.1 ml, were dried with dry nitrogen gas, after the addition of the reagents specified in (i) or (ii), the vessels with the solution were sealed in vacuum, which was further followed by hydrolysis of the contents.
Svaka od vrijednosti prikazanih u tablici 6 je srednja vrijednost 4 mjerenja, 24 vrijednosti za (i) i 24, 48 i 72 satne vrijednosti za (ii), izuzevši što sadržaju Thr, Ser, 1/2Cys, Met, Val, Ile i Trp su izračunati sljedećim metodama (vidi "Tampaku Kagaku (Protein Chemistry) II" A Course in Biochemical Experiments. Tokyo Ka-gaku Dohjin): Each of the values shown in table 6 is the mean value of 4 measurements, 24 values for (i) and 24, 48 and 72 hour values for (ii), except for the content of Thr, Ser, 1/2Cys, Met, Val, Ile and Trp were calculated by the following methods (see "Tampaku Kagaku (Protein Chemistry) II" A Course in Biochemical Experiments. Tokyo Ka-gaku Dohjin):
Za thr, Ser, 1/2Cys i Met, vremenska zavisnost profila od 24, 48 i 72 satnih vrijednosti za (ii) je ekstrapolirana na 0 sati. For thr, Ser, 1/2Cys and Met, the time dependence of the profile of 24, 48 and 72 hour values for (ii) was extrapolated to 0 hours.
Za Val i Ile, korištena je 72 satna vrijednost za (ii). Za Trp, korištena je srednja vrijednost 24, 48 i 72 satnih vrijednosti za (ii). For Val and Ile, the 72 hour value for (ii) was used. For Trp, the mean of the 24, 48 and 72 hourly values for (ii) was used.
Tablica 6 Table 6
Podaci analiza aminokiselina Amino acid analysis data
[image] [image]
2) Analiza sadržaja šećera 2) Analysis of sugar content
Unutarnji standard (25 nmol inozitola) je dodano u 200 ng pročišćenog CSF uzorka korištenog za analizu sastava aminokiselina 1). Nakon dodatka otopine metanola (µ1) koji sadrži 1,5 N HCl, reakcija je vršena na 90°C tijekom 4 sata u pročišćenom N2 u zatvorenoj epruveti. Epruveta je zatim otvorena i dodan je srebro-karbonat (Ag2CO3) radi neutraliziranja sadržaja. Zatim je dodano 50 µl acetan-hidrida i epruveta je neko vrijeme mućkana. Epruveta je ostavljena preko noći u tamnom na sobnoj temperaturi. Gornji sloj je stavljen u epruvetu uzorka i osušen plinovitim dušikom. Talogu je dodan metanol i smjesa je isprana i centrifugirana na svjetlu. Gornji sloj je stavljen u istu epruvetu uzorka i osušen. Nakon dodatka 50 µl TMS reagensa (5:1:1 smjesa piridina:heksametil disilazana i trimetilkloro-silana), reakcija je vršena na 40°C tijekom 20 minuta i reakcijski produkt je stavljen u zamrzivač za duboko zamrzavanje. Standard je dobiven kombiniranjem 25 nmol inozitola sa po 50 nmol galaktoze (Gal), N-acetil galaktozamina (Gal NAc), sialinske kiseline i nekog drugog odgovarajućeg reagensa. An internal standard (25 nmol of inositol) was added to 200 ng of the purified CSF sample used for amino acid composition analysis 1). After the addition of a methanol solution (µ1) containing 1.5 N HCl, the reaction was carried out at 90°C for 4 hours in purified N2 in a closed test tube. The tube was then opened and silver carbonate (Ag2CO3) was added to neutralize the contents. Then 50 µl of acetic hydride was added and the test tube was shaken for some time. The tube was left overnight in the dark at room temperature. The upper layer was placed in a sample tube and dried with nitrogen gas. Methanol was added to the precipitate and the mixture was washed and centrifuged in the light. The top layer was placed in the same sample tube and dried. After adding 50 µl of TMS reagent (5:1:1 mixture of pyridine:hexamethyldisilazane and trimethylchloro-silane), the reaction was carried out at 40°C for 20 minutes and the reaction product was placed in a freezer for deep freezing. The standard was obtained by combining 25 nmol of inositol with 50 nmol each of galactose (Gal), N-acetyl galactosamine (Gal NAc), sialic acid and some other appropriate reagent.
Tako dobiveni uzorci su podvrgnuti plinskoj kromatografskoj analizi u sljedećim uvjetima: The samples thus obtained were subjected to gas chromatographic analysis under the following conditions:
Uvjeti analize Analysis conditions
Kolona: 2% OV-17 VINport HP, 0,25-0,18 mm, 3 m, staklo Column: 2% OV-17 VINport HP, 0.25-0.18 mm, 3 m, glass
Temperatura: povišena od 110 do 250°C na 4°C/min. Temperature: increased from 110 to 250°C at 4°C/min.
Tlak nosećeg plina (N2): početni 0,12-0,16 Pa Carrier gas pressure (N2): initial 0.12-0.16 Pa
krajnji 0,2-0,25 Pa ultimate 0.2-0.25 Pa
Osjetljivost: 103 M područje, 0,1-0,4 volta Sensitivity: 103 M range, 0.1-0.4 volts
Tlak: H2, 0,08 Pa Pressure: H2, 0.08 Pa
zrak, 0,08 Pa air, 0.08 Pa
Uzorak 2,5-3,0 µl Sample 2.5-3.0 µl
Na ovaj način galktozam N-acetil galktozamin i sialinska kiselina su identificirani u CSF uzorku ovoga izuma. In this way galctosome N-acetyl galctosamine and sialic acid were identified in the CSF sample of the present invention.
Primjer 28: Example 28:
Dobivanje pHGV2 vektora (za upotrebu sa životinjskim stanicama, -VSE linije) Obtaining the pHGV2 vector (for use with animal cells, -VSE lines)
EcoRI fragment dobiven u primjeru 10 koji ima cDNA prikazanu na slici 4(A) je tretiran sa restrikcijskim enzimom DraI tijekom 2 sata na temperaturi 37°C, a zatim sa Klenow fragmentom DNA polimeraze I (Takara Shuzo Co., Ltd.) tako da nastaju otvoreni krajevi. Jedan mikrogram BglII veziva (8 mer, Takara Shuzo Co., Ltd.) je fosforilizirano sa ATP i pripojeno na oko 1 µg posebno dobivene smjese DNA fragmenata. Pripojeni fragmenti su tretirani sa restrikcijskim enzimom, BglII, i podvrgnuti agaroza gel elektroforezi. Zatim je izdvojen samo najveći DNA fragment. The EcoRI fragment obtained in Example 10 having the cDNA shown in Figure 4(A) was treated with the restriction enzyme DraI for 2 hours at a temperature of 37°C, and then with the Klenow fragment of DNA polymerase I (Takara Shuzo Co., Ltd.) so that open ends are formed. One microgram of BglII binder (8 mer, Takara Shuzo Co., Ltd.) was phosphorylated with ATP and attached to about 1 µg of a specially obtained mixture of DNA fragments. The joined fragments were treated with the restriction enzyme, BglII, and subjected to agarose gel electrophoresis. Then only the largest DNA fragment was isolated.
Ovaj DNA fragment je ekvivalentan sa oko 700 bp koji sadrži kodirajući dio humanog G-CSF. Vektor pdKCR /fukunaga et al., Proc. Natl. Acad. Sci., USA 81, 5086(1984)/ je tretiran sa restrikcijskim enzimom, MaMHI, i zatim je defosforiliziran s alkalnom fosfatazom (Takara Shuzo Co., Ltd.). Dobiveni vektor DNA je pripojen na oko 700 cDNA fragment u prisutnosti T4 DNA ligaze (Takara Shuzo Co., Ltd.), tako da se dobiva pHGV2 (slika 17). Kao što je prikazano na slici 17, ovaj plazmid sadrži promotor SV40 ranog gena, replikaciju početne regije SC40 dijela γ-globin zeca, replikaciju početne regije pBR322 i pBR322-izvedenog β-laktamaza gena (Ampr), s humanim G-CSF genom spojenim ispod promotora za SV40 rani gen. This DNA fragment is equivalent to about 700 bp containing the coding part of human G-CSF. Vector pdKCR /fukunaga et al., Proc. Natl. Acad. Sci., USA 81, 5086(1984)/ was treated with the restriction enzyme, MaMHI, and then dephosphorylated with alkaline phosphatase (Takara Shuzo Co., Ltd.). The obtained vector DNA was joined to about 700 cDNA fragment in the presence of T4 DNA ligase (Takara Shuzo Co., Ltd.), so that pHGV2 was obtained (Figure 17). As shown in Figure 17, this plasmid contains the SV40 early gene promoter, replication of the SC40 start region of the rabbit γ-globin portion, replication of the start region of pBR322 and the pBR322-derived β-lactamase gene (Ampr), with the human G-CSF gene fused below promoter for SV40 early gene.
Primjer 29: Example 29:
Stvaranje rekombinantnog vektora (-VSE) za korištenje u transformiranju C127 stanica Generation of a recombinant vector (-VSE) for use in transforming C127 cells
1) Stvaranje pHGV2(H) 1) Creation of pHGV2(H)
Dvadeset mikrograma plazmida pHGV2 (slika 17) dobivenog u primjeru 28 je tretirano postupcima opisanim u 1) u primjeru 23, tako da nastane plazmid nazvan pHGV2(H) (slika 18). Twenty micrograms of plasmid pHGV2 (Figure 17) obtained in Example 28 was treated with the procedures described in 1) in Example 23, so that a plasmid named pHGV2(H) (Figure 18) was formed.
2) Stvaranje rekombinantnih vektora ekspresije pTN-V2, pTNVAα i pTNVAβ 2) Creation of recombinant expression vectors pTN-V2, pTNVAα and pTNVAβ
Korištenjem 20 µg pHGV2(H), ponovljeni su postupci opisani u 2) u primjeru 2 radi odabiranja E. coli uzgojenog plazmida koji ima pHGV2-izvedenu G-CSF cDNA. Ovaj plazmid je nazvan pTN-V2 (slika 18). Using 20 µg of pHGV2(H), the procedures described in 2) in Example 2 were repeated to select an E. coli cultured plasmid having a pHGV2-derived G-CSF cDNA. This plasmid was named pTN-V2 (Figure 18).
Adenovirus tip II /Tampakushitsu, Kakusan Koso (Proteins, Nucleic Acids and Enzymes), 27, December, 1982, Kyoritsu Shuppan/ su slično tretirani da se dobije plazmid ∆pVA, koji sadrži oko 1700 bp SaII-HindIII dio koji uzgaja VAI i VAII i fragment koji sadrži VAI i VAII je izdvojen iz ovog plazmida. Ovaj fragment je ubačen u PTN-2 na HindIII mjesto tako da se dobivaju pTNVAα i pTNVAβ (slika 18). Zbog VA gena adenoviroze, ovaj plazmid je sposoban pojačati ekspresiju transkripcije produkta iz ranijeg promotora SV40. Adenovirus type II /Tampakushitsu, Kakusan Koso (Proteins, Nucleic Acids and Enzymes), 27, December, 1982, Kyoritsu Shuppan/ were similarly treated to obtain plasmid ∆pVA, which contains about 1700 bp SaII-HindIII portion that grows VAI and VAII and a fragment containing VAI and VAII was isolated from this plasmid. This fragment was inserted into PTN-2 at the HindIII site to obtain pTNVAα and pTNVAβ (Figure 18). Due to the VA gene of adenovirus, this plasmid is able to enhance the transcriptional expression of the product from the earlier SV40 promoter.
Primjer 30: Example 30:
Transformacija C127 stanica i G-CSF ekspresija u (-VSE) Transformation of C127 cells and G-CSF expression in (-VSE)
pTN-V2 dobiven u primjeru 29 je tretiran sa restrikcijskim enzimom BamHI prije nego što je iskorišten za transformiranje C127 stanica miša. pTN-V2 obtained in Example 29 was treated with the restriction enzyme BamHI before being used to transform mouse C127 cells.
Stanice C127 miša su transfornmirane s tako dobivenom DNA radi ekspresije G-CSF (vidi primjer 24) i odabrani su klonovi koji posjeduju visoku G-CSF brzinu produkcije. Ovi klonovi produciraju G-CSF od oko 1 mg/1. Mouse C127 cells were transformed with the DNA thus obtained to express G-CSF (see Example 24) and clones possessing a high G-CSF production rate were selected. These clones produce G-CSF of about 1 mg/l.
Daljnjim kloniranjem mogu biti izdvojeni klonovi sposobni da produciraju G-CSF od oko 10 mg/1. Na sličan način C127 stanice su transformirane sa pTNVAα i pTNVAβ dobivene u primjeru 29, i odabrani su transformati za klonove koji imaju visoku sposobnost G-CSF produkcije. Tako se mogu dobiti klonovi sa pTNVAα koji su sposobni producirati G-CSF u prinosu 20 mg/1 ili više, dok se sa pTNVAβ dobivaju klonovi koji posjeduju nižu produktivnost (nekoliko mg/1). By further cloning, clones capable of producing G-CSF of about 10 mg/1 can be isolated. Similarly, C127 cells were transformed with pTNVAα and pTNVAβ obtained in Example 29, and transformants were selected for clones having high G-CSF production capacity. Thus, clones with pTNVAα that are capable of producing G-CSF in a yield of 20 mg/1 or more can be obtained, while clones with lower productivity (several mg/1) are obtained with pTNVAβ.
Osim C127 stanica također se mogu rabiti i NIH3T3 stanice. In addition to C127 cells, NIH3T3 cells can also be used.
Primjer 31: Example 31:
Ekspresija G-CSF u CHO stanicama (-VSE) Expression of G-CSF in CHO cells (-VSE)
1) Građenje pHGV2-dhfr 1) Construction of pHGV2-dhfr
DNA fragment od oko 2,7 kbp dužine je dobiven iz 20 µg plazmida pHGV2 (Primjer 28) postupcima opisanim u 1) u primjeru 25. Ovaj fragment (0,5 µg) i EcoRI fragment pAdD26SVpA (0,5 µg) su prekaljeni. Dobiveni plazmid je upotrijebljen za transformiranje E. coli vrste DH1 postupkom sa rubidij-kloridom i odabrane su kolonije koje odgajaju pHGV2-dhfr plazmid. Dobiveni plazmid je nazvan pHGV2-dhfr (slika 19a). A DNA fragment of about 2.7 kbp in length was obtained from 20 µg of plasmid pHGV2 (Example 28) by the procedures described in 1) in Example 25. This fragment (0.5 µg) and the EcoRI fragment of pAdD26SVpA (0.5 µg) were annealed. The resulting plasmid was used to transform E. coli DH1 by the rubidium chloride method and colonies growing the pHGV2-dhfr plasmid were selected. The resulting plasmid was named pHGV2-dhfr (Figure 19a).
Alternativni postupak je sljedeći: plazmid pHGV2 je tretiran sa Sa1L i djelomično je razoren sa EcoRI bez pripajanja nekog veziva EcoRI. DNA fragment od oko 2,7 kbp dužine je izdvojen i tretiran sa Klenow fragmentom E. coli DNA polimeraze, tako da se stvaraju otvoreni krajevi. Otvoreni krajevi EcoRI fragmenta se dobivaju iz pAdD26SVpA, kao što je opisano gore. Ovaj EcoRI fragment i odvojeno pripremljen fragment (oko 2,7 kbp) su tretirani sa T4DNA ligazom, tako da nastane pHGV2-dhfr. An alternative procedure is as follows: plasmid pHGV2 was treated with Sa1L and partially digested with EcoRI without attaching any EcoRI linker. A DNA fragment of about 2.7 kbp in length was isolated and treated with the Klenow fragment of E. coli DNA polymerase, so that open ends are created. The exposed ends of the EcoRI fragment were obtained from pAdD26SVpA, as described above. This EcoRI fragment and a separately prepared fragment (about 2.7 kbp) were treated with T4DNA ligase to generate pHGV2-dhfr.
pHGV2 (H) dobiven u 1) Primjera 29 je tretiran sa restrikconim enzimom HindIII i SandII, kao što je opisano u 2) u primjeru 29, i HindIII-SandII fragment je pripojen na otvorene krajeve EcoRI fragmenta pAdD26SVpA, opisanog gore. Ova metoda se može također primijeniti za pripremanje pHGG4-dhfr (slika 19b). pHGV2 (H) obtained in 1) of Example 29 was treated with the restriction enzyme HindIII and SandII, as described in 2) of Example 29, and the HindIII-SandII fragment was ligated to the open ends of the EcoRI fragment of pAdD26SVpA, described above. This method can also be applied to prepare pHGG4-dhfr (Figure 19b).
2) Nastajanje pV2DR1 i pV2DR2 2) Formation of pV2DR1 and pV2DR2
Deset mikrograma plazmida pAdD26SVpA, spomenutog u 1) je otopljeno u 50 ml reakcijske otopine koja sadrži 50 mM Tris-HCl (pH 7,5), 7 mM MgCl2, 100 mM NaCl, 7 mM 2-merkapto-etanola i 0,01% BSA. Reakcija je izvedena na 37°C tijekom 10 sati u prisustvu 10 jedinica svakog od sljedećih enzima, EcoRI i BamHI. Zatim je izvršeno tretiranje sa fenolom i ispiranje sa eterom, rutinskim postupcima. DNA fragment od oko 2 kb je izdvojen elektroforezom, kroz 1% agaroza gel niske (točke taljenja, rutinskim postupcima). Ten micrograms of plasmid pAdD26SVpA, mentioned in 1) was dissolved in 50 ml of reaction solution containing 50 mM Tris-HCl (pH 7.5), 7 mM MgCl2, 100 mM NaCl, 7 mM 2-mercapto-ethanol and 0.01% BSA. The reaction was performed at 37°C for 10 hours in the presence of 10 units of each of the following enzymes, EcoRI and BamHI. Then treatment with phenol and washing with ether was carried out, routine procedures. A DNA fragment of about 2 kb was separated by electrophoresis, through a 1% agarose gel of low (melting point, routine procedures).
Izdvojeni DNA fragment je tretiran sa Klenow fragmentom DNA polimeraze ru-tinskim postupcima, tako da se grade otvoreni krajevi. DNA fragment otvorenih krajeva je podvrgnut tretmanu sa fenolom, ispran je sa eterom i taložen sa etanolom. The separated DNA fragment was treated with the Klenow fragment of DNA polymerase by routine procedures, so that open ends are built. The open-ended DNA fragment was treated with phenol, washed with ether and precipitated with ethanol.
Deset mikrograma plazmida pHGV2(H) dobivenog u 1) primjera 29 je otopljeno u 50 µl reakcijske otopine, koja sadrži 10 mM Tris-HCl (pH 7,5), 7 mM MgCl2, i 60 mM NaCl. Reakcija je izvedena na 37°C tijekom 6 sati u prisutnosti 10 jedinica HindIII. DNA fragment je izdvojen elektroforezom kroz 1% agaroza gel niske točke taljenja, rutinskim postupcima. Izdvojeni fragment je zatim tretiran sa BAP i otvoreni krajevi su dobiveni tretiranjem sa Klenow fragmentom. Koristeći tretiranje fenolom i ispiranje sa eterom, DNA fragment je pripojen na otvorene krajeve prethodno dobivenog 2-kb DNA fragmenta sa T4DNA ligazom, sljedećim postupcima: 1 µg svakog fragmenta je otopljen u 30 µ1 reakcijske otopine koja sadrži 66 mM Tris-HCl (pH 7,5), 6.6 mM MgCl2 5 mM DTT i 1 mM ATP, i reakcija je izvedena na 6°C tijekom 12 sati u prisutnosti 50 jedinica T4DNA ligaze. Proizvod vezanja je korišten za transformiranje E. coli vrste DH1. Na ovaj način dobiveni su pV2DR1 i pV2DR2, prikazani na slici 19c. Ten micrograms of plasmid pHGV2(H) obtained in 1) of example 29 were dissolved in 50 µl of reaction solution, which contains 10 mM Tris-HCl (pH 7.5), 7 mM MgCl 2 , and 60 mM NaCl. The reaction was performed at 37°C for 6 hours in the presence of 10 units of HindIII. The DNA fragment was separated by electrophoresis through a 1% low melting point agarose gel, using routine procedures. The isolated fragment was then treated with BAP and open ends were obtained by treatment with the Klenow fragment. Using phenol treatment and washing with ether, the DNA fragment was ligated to the open ends of the previously obtained 2-kb DNA fragment with T4DNA ligase, using the following procedures: 1 µg of each fragment was dissolved in 30 µl of a reaction solution containing 66 mM Tris-HCl (pH 7 ,5), 6.6 mM MgCl2, 5 mM DTT and 1 mM ATP, and the reaction was performed at 6°C for 12 hours in the presence of 50 units of T4DNA ligase. The ligation product was used to transform E. coli strain DH1. In this way, pV2DR1 and pV2DR2 were obtained, shown in Figure 19c.
3) Transformiranje i ekspresija 3) Transformation and expression
CHO stanice su transformirane sa plazmidom pHGV2-dhfr za G-CSF ekspresiju prema postupcima opisanim u 3) u primjeru 25. CHO cells were transformed with plasmid pHGV2-dhfr for G-CSF expression according to the procedures described in 3) in example 25.
Transformiranje CHO stanica može takoder biti izvršeno kotransformacijama sa pHGV2 i pAdD26SVpA. Transformation of CHO cells can also be performed by co-transformation with pHGV2 and pAdD26SVpA.
CHO stanice su transformirane sljedećim postupcima: pV2DR1 ili pV2DR2 koji su dobiveni u 2) prethodno su tretirani sa SaII i KpmI tako da se dobivaju fragmenti DNA i 10 µg ovih fragmenata je upotrebljeno za transformiranje CHO stanica kao prije; transformirane stanice su podvrgnute produženom kultiviranju u serijama selektivnih medija na način opisan naprijed, oko 7 dana kasnije, ne manje od 100 odvojenih kolonija se pojavilo po plitici; ove kolonije su u masi prenesene na svježu pliticu i podvrgnute su produženom kultiviranju u serijama selektivnih medija u prisutnosti 0,01 µm. MTX, dok se ne pojavi deset neparnih kolonija; isti postupci su ponovljeni sa MTX koncentracijama koje su serijski povećavane do 0,02 µM, 0,05 µm i 0,1 µM i odvojene su preživjele kolonije; selekcija kolonija može biti izvršena na isti način čak i kada je 10 nepamih kolonija dobiveno individualnom selekcijom i podvrgnuto kultiviranju sa rastućim MTX koncentracijama. CHO cells were transformed by the following procedures: pV2DR1 or pV2DR2 obtained in 2) were previously treated with SalII and KpmI to obtain DNA fragments and 10 µg of these fragments were used to transform CHO cells as before; the transformed cells were subjected to prolonged cultivation in series of selective media as described above, about 7 days later, not less than 100 separate colonies appeared per plate; these colonies were transferred en masse to a fresh plate and subjected to extended cultivation in batches of selective media in the presence of 0.01 µm. MTX, until ten odd colonies appear; the same procedures were repeated with MTX concentrations that were serially increased to 0.02 µM, 0.05 µM and 0.1 µM and the surviving colonies were separated; colony selection can be performed in the same way even when 10 non-prime colonies are obtained by individual selection and subjected to cultivation with increasing MTX concentrations.
Rekombinantni vektor koji uzgaja "policistronski gen" može također biti upotrebljen za transformiranje CHO stanica. Primjer alternativne metode je sljedeći: A recombinant vector carrying a "polycistronic gene" can also be used to transform CHO cells. An example of an alternative method is as follows:
pAdD26SVpA je tretirana sa PstI i izdvojena dva fragmenta su pripojena na pBRV2-izvedeni CSF cDNA fragment tako da stvaraju rekombinantni vektor gdje je virus promotor, CSF cDNA, DHFR i poli(A) mjesto SV40 ubačeni po napisanom redoslijedu. Ovaj rekombinantni vektor je korišten za transformiranje CHO stanica. pAdD26SVpA was treated with PstI and the separated two fragments were ligated to the pBRV2-derived CSF cDNA fragment to create a recombinant vector where the virus promoter, CSF cDNA, DHFR and poly(A) site of SV40 were inserted in the order written. This recombinant vector was used to transform CHO cells.
Primjer 32: Example 32:
Ispitivanje G-CSF aktivnosti (-VSE) G-CSF activity test (-VSE)
Postupcima opisanim u primjeru 26, humani G-CSF dobiven iz supernatanata kultura C127 stanica i CHO stanica koje su dobivene u primjerima 30 i 31. Humana G-CSF aktivnost svakog od izdvojenih uzoraka ispitana je kao u primjeru 26. Rezultati su prikazani u tablici 7. By the procedures described in example 26, human G-CSF obtained from culture supernatants of C127 cells and CHO cells obtained in examples 30 and 31. Human G-CSF activity of each of the isolated samples was tested as in example 26. The results are shown in table 7 .
Tablica 7. Table 7.
Ispitivanje humane G-CSF aktivnosti Assay of human G-CSF activity
[image] [image]
Primjer 33: Example 33:
Analiza aminokiselina i analiza šećera (-VSE) Amino acid analysis and sugar analysis (-VSE)
1) Analiza sastava aminokiseline 1) Analysis of amino acid composition
Sirovi CSF uzorak dobiven u primjeru 32 je pročišćen prema postupcima opisanim u primjeru 2(iii). Pročišćeni CSF uzorak je podvrgnut analizi sastava aminokiseline postupcima opisanim u 1) u primjeru 27. Rezultati su prikazani u tablici 8. The crude CSF sample obtained in Example 32 was purified according to the procedures described in Example 2(iii). The purified CSF sample was subjected to amino acid composition analysis by the procedures described in 1) in Example 27. The results are shown in Table 8.
Tablica 8 Table 8
Podaci analize aminokiselina Amino acid analysis data
[image] [image]
2) Analiza sastava šećera 2) Analysis of sugar composition
Pročišćeni CSF uzorak korišten je u analizi sastava aminokiseline u 1) također je podvrgnut analizi sastava šećera istim postupcima i u istim uvjetima kao što je opisano u 2) u primjeru 27. Na taj način je potvrđena prisutnost galaktoze, N-acetil galaktozamina i sialinske kiseline u CSF uzorku ovoga izuma. The purified CSF sample used in the analysis of the amino acid composition in 1) was also subjected to the analysis of the sugar composition using the same procedures and under the same conditions as described in 2) in example 27. In this way, the presence of galactose, N-acetyl galactosamine and sialic acid in CSF sample of this invention.
Primjer 34: Example 34:
Građenje rekombinantnog vektora koji sadrži kromozomski gen za ekspresiju u COS stanicama Construction of a recombinant vector containing a chromosomal gene for expression in COS cells
Plazmid pBRCE3β, koji je dobiven u primjeru 11 i koji sadrži kromozomski gen prikazan u slici 5, je tretiran sa EcoRI. pSVH+K+ plazmid, koji je opisan u Baneriji et al., Cell, 27, 299(1981), je tretiran sa KpnI radi uklanjanja globin gena. Plazmid je dalje podvrgnut djelomičnom razlaganju sa HindIII, radi uklanjanja dijela zaostalog gena SV40. Fragmenti su ponovno pripojeni, tako da se dobiva vektor izražavanja pML-E+. Plasmid pBRCE3β, which was obtained in Example 11 and which contains the chromosomal gene shown in Figure 5, was treated with EcoRI. The pSVH+K+ plasmid, described in Banerija et al., Cell, 27, 299(1981), was treated with KpnI to remove the globin gene. The plasmid was further subjected to partial digestion with HindIII, in order to remove part of the residual SV40 gene. The fragments were rejoined, resulting in the pML-E+ expression vector.
Ovaj vektor je tretiran sa restrikcijskim enzimom, EcoRI i defosforiliziran s alkalnom fosfatazom (Takara Shuzo Co., Ltd.), tako da se dobije vektor DNA, koji je vezan na gore spomenutu kromosomsku DNA pomoću T4DNA ligaze (Takara Shuzo Co., Ltd.), tako da se dobiva pMLCE3. Kao što je prikazano u slici 20, ovaj plazmid sadrži pojačivač SV40 gena, repliku početka SV40, repliku početka pBR322 i pBR322-izveden β-laktamaza gen (AMpr), i humani G-CSF kromosomski gen pripojen ispod pojačivača SV40 gena. This vector was treated with a restriction enzyme, EcoRI, and dephosphorylated with alkaline phosphatase (Takara Shuzo Co., Ltd.) to obtain vector DNA, which was linked to the above-mentioned chromosomal DNA by T4DNA ligase (Takara Shuzo Co., Ltd. ), so that pMLCE3 is obtained. As shown in Figure 20, this plasmid contains the SV40 gene enhancer, the SV40 origin replicon, the pBR322 origin replicon and the pBR322-derived β-lactamase gene (AMpr), and the human G-CSF chromosomal gene fused below the SV40 gene enhancer.
Primjer 35: Example 35:
Ekspresija humanog G-CSF kromosomskog gena u COS stanicama Expression of the human G-CSF chromosomal gene in COS cells
COS-1 stanice (dobivene zahvaljujući ljubaznosti Dr. Gluzman-a iz Cold Spring Harbor Laboratory, USA), koje su izrasle do gustoće od oko 70% u Petrijevim zdjelicama (promjera 9 cm, Nunc), koristeći DMEM sredinu (Dulbecco-ova modifikacija Eagle-ove sredine, nabavljene od Nissui Sieyaku K.K., pod trgovačkim imenom "Nissui") koja sadrži 10% seruma teleta, su transformirane postupkom sa kalcij-fosfatom /Wigler et al., Cell, 14, 725 (1978)/ ili DEAE-dekstran: klorihin metodom /vidi, na primjer, Gordon et al., Science, 228, 810(1985)/. COS-1 cells (obtained courtesy of Dr. Gluzman from Cold Spring Harbor Laboratory, USA), grown to a density of about 70% in Petri dishes (9 cm diameter, Nunc), using DMEM medium (Dulbecco's modification Eagle's medium, obtained from Nissui Sieyaku K.K., under the trade name "Nissui") containing 10% calf serum, was transformed by the calcium-phosphate method /Wigler et al., Cell, 14, 725 (1978)/ or DEAE- by the dextran:chloroquine method /see, for example, Gordon et al., Science, 228, 810(1985)/.
Transformiranje postupkom sa kalcij-fosfatom je izvršeno kao što slijedi: 160 µg plazmida pMLCE3, dobivenog u primjeru 34, je otopljeno u 320 µl TE otopine i poslije dodatka destilirane vode (3,2Ml), dodano je 504 µl 2M CaCl2. Transformation with the calcium-phosphate method was performed as follows: 160 µg of plasmid pMLCE3, obtained in example 34, was dissolved in 320 µl of TE solution and after the addition of distilled water (3.2Ml), 504 µl of 2M CaCl2 was added.
U dobivenu otopinu je dodano 4 ml 2xHBS (50 mM Hepes, 280 mM NaCl, 1,5 mM fosfatnog pufera, pH 7,12) i smjesa je hlađena na ledu tijekom 20-30 minuta. Ohlađena smjesa je ukapavanjem dodata mediju u količini od 1 ml po Petrijevoj zdjelici, gdje su uzgojene COS-1 stanice. Poslije kultiviranja 4 sata na 37°C u CO2 inkubatoru, stanice su isprane sa DMEM medijem bez seruma, i ostavljene da stoje oko 3 minute na sobnoj temperaturi u 5 ml DMEM medija koja sadrži 20% glicerola, i ponovno isprane sa DMEM medijem bez seruma. Nakon uklanjanja DMEM medija koji ne sadrži serum, dodano je 10 ml DMEM medija koja sadrži 10% seruma teleta i kultiviranje je vršeno preko noći u CO2 inkubatoru. Medij je zatim zamijenjen svježim medijem istog tipa i kultiviranje je vršeno dodatna 3 dana. 4 ml of 2xHBS (50 mM Hepes, 280 mM NaCl, 1.5 mM phosphate buffer, pH 7.12) was added to the resulting solution and the mixture was cooled on ice for 20-30 minutes. The cooled mixture was added dropwise to the medium in an amount of 1 ml per Petri dish, where COS-1 cells were grown. After culturing for 4 hours at 37°C in a CO2 incubator, the cells were washed with DMEM medium without serum, and left to stand for about 3 minutes at room temperature in 5 ml of DMEM medium containing 20% glycerol, and washed again with DMEM medium without serum . After removing the DMEM medium that does not contain serum, 10 ml of DMEM medium containing 10% calf serum was added and the cultivation was performed overnight in a CO2 incubator. The medium was then replaced with fresh medium of the same type and cultivation was carried out for an additional 3 days.
Transformiranje metodom DEAE-dekstran: klorihin izvršeno je kao što slijedi: Kao u postupku sa kalcij-fosfatom, COS-1 stanice su kultivirane tako da odrastu do gustoće od 70% i isprane su dva puta sa DMEM medijem, koji ne sadrži serum; ispranim stanicama dodan je DMEM medij koji sadrži serum i sadrži 250 µg/ml DEAE-dekstrana i 2 µg/ml plazmida pMLCE3, dobivenog u primjeru 34 i kultiviranje je vršeno na 37°C tijekom 12 sati; zatim su stanice isprane dva puta sa DMEM medijem koja ne sadrži serum i podvrgnute su daljnjoj kultivaciji na 37°C tijekom 2 sata u DMEM mediju koja sadrži 10% seruma teleta i 1 mM klorokina; zatim su stanice dva puta isprane sa DMEM medijem koji ne sadrži serum i kultivirane na 37°C još tri dana u DMEM mediju koji sadrži 10% seruma teleta. Transformation by the DEAE-dextran: chlorquine method was performed as follows: As in the calcium-phosphate procedure, COS-1 cells were grown to 70% density and washed twice with serum-free DMEM medium; DMEM medium containing serum and containing 250 µg/ml DEAE-dextran and 2 µg/ml plasmid pMLCE3, obtained in example 34, was added to the washed cells and the cultivation was carried out at 37°C for 12 hours; then the cells were washed twice with serum-free DMEM medium and subjected to further cultivation at 37°C for 2 hours in DMEM medium containing 10% calf serum and 1 mM chloroquine; then the cells were washed twice with DMEM medium containing no serum and cultured at 37°C for another three days in DMEM medium containing 10% calf serum.
Supernatant tako dobivene kulture COS-1 stanica je podešen na pH 4 sa 1 N octenom kiselinom. Nakon dodatka istog volumena n-propanola, dobiveni talog je uklonjen centrifugiranjem. Supernatant je propušten kroz otvorenu kolonu promjera 1 i dužine 2 cm, ispunjenom sa C8 reverzno-faznim nosačem (Yamamura Kagaku K.K.) i eluiranje je izvršeno sa 50% n-propanolom. Eluat je razrijeđen dva puta s vodom i podvrgnut reverzno-faznoj HPLC na YMC-C8 koloni (Yamamura Kagaku K.K.), uz eluiranje sa n-propanolom (30-60% linearnog gradijenta gustoće) koji sadrži 0,1% TFA. Frakcije koje su eluirane sa n-propanolom koncentracija od oko 40% su izdvojene, suho smrznute i otopljene u 0,1 M glicidnom puferu (pH 9). Kao rezultat ovih postupaka, humani G-CSF u supernatantu kulture COS-1 stanica koncentriran je oko 20 puta. The supernatant of the COS-1 cell culture thus obtained was adjusted to pH 4 with 1 N acetic acid. After the addition of the same volume of n-propanol, the resulting precipitate was removed by centrifugation. The supernatant was passed through an open column of diameter 1 and length 2 cm, filled with C8 reverse-phase support (Yamamura Kagaku K.K.) and elution was performed with 50% n-propanol. The eluate was diluted twice with water and subjected to reverse-phase HPLC on a YMC-C8 column (Yamamura Kagaku K.K.), eluting with n-propanol (30-60% linear density gradient) containing 0.1% TFA. Fractions eluted with n-propanol concentration of about 40% were separated, freeze-dried and dissolved in 0.1 M glycidic buffer (pH 9). As a result of these procedures, human G-CSF in the culture supernatant of COS-1 cells is concentrated about 20-fold.
Kao kontrola COS-1 stanice su transformirane sa G-CSF kromosomskim genom slobodni pML-E+ gore opisanim postupcima i koncentriran je supernatant dobivene kulture. As a control, COS-1 cells were transformed with G-CSF chromosomal gene free pML-E+ by the procedures described above and the culture supernatant was concentrated.
Humana G-CSF aktivnost dobivenih uzoraka ispitivana je pomoću "Method of Human G-CSF Activity Assay(a)" opisanom ranije u ovoj specifikaciji. Rezultati su dani u tablici 9. The human G-CSF activity of the obtained samples was assayed using the "Method of Human G-CSF Activity Assay(s)" described earlier in this specification. The results are given in table 9.
Tablica 9. Table 9.
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Primjer 36: Example 36:
RNA analiza G-CSF (kromosomskog gena) RNA analysis of G-CSF (chromosomal gene)
COS stanice kultivirane do koncentracije stanica od 8x106 stanica/plitica (promjera 9 cm) su transformirane sa 80 µg plazmida pMLCE3α. Nakon 48 sati, ukupna RNA je dobivena prema postupku Chirgwin-a /Biochemistry, 18, 5294-5299(1979)/. COS cells cultured to a cell concentration of 8x106 cells/plate (diameter 9 cm) were transformed with 80 µg of plasmid pMLCE3α. After 48 hours, total RNA was obtained according to the procedure of Chirgwin /Biochemistry, 18, 5294-5299 (1979)/.
Plazmid pBRG4 dobiven u primjeru 9 je prekinut sa restrikcijskim enzimom AhaIII i dobiven pBRG4-izvedeni DNA fragment je radioobilježen sa /γ32-P/ATP koristeći T polinukleotid kinaze tako da se dobije oko 2,8 kb DNA fragment koji sadrži G-CSF cDNA. Fragment je izdvojen i upotrebljen kao DNA proba. Plasmid pBRG4 obtained in Example 9 was cut with the restriction enzyme AhaIII and the resulting pBRG4-derived DNA fragment was radiolabeled with /γ32-P/ATP using T polynucleotide kinase to obtain an approximately 2.8 kb DNA fragment containing G-CSF cDNA. The fragment was isolated and used as a DNA probe.
Nakon denaturiranja DNA probe (1,5x105 s.p.m., 2,8x106 s.p.m./µg DNA), ova je izmiješana sa 20 µg ukupne RNA dobivene iz CMOS stanica. Hibridizacijom na 45°C tijekom 15 sati, smjesa je razorena sa 200 jedinica/ml ili 400 jedinica/ml S1 nuklaze (p.L. Bichemicals) prema postupku Weaver i Weissmann /Nucleic Acid Res., 7, 1175-1193(1979), što je praćeno 4% poliakrilamid gel elektroforezom u prisutnosti 8,3 M uree. Tada je izvršena detekcija pomoću autoradiografije. After denaturing the DNA sample (1.5x105 s.p.m., 2.8x106 s.p.m./µg DNA), it was mixed with 20 µg of total RNA obtained from CMOS cells. After hybridization at 45°C for 15 hours, the mixture was digested with 200 units/ml or 400 units/ml S1 nuclease (p.L. Bichemicals) according to the procedure of Weaver and Weissmann /Nucleic Acid Res., 7, 1175-1193(1979), which is followed by 4% polyacrylamide gel electrophoresis in the presence of 8.3 M urea. Then the detection was carried out using autoradiography.
Pri tome je opažena vrpca koja odgovara 722 bp kao jaka radioobilježena vrpca u COS stanicama, a također je detektirana i odgovarajuća vrpca za 487 bp. A band corresponding to 722 bp was observed as a strong radiolabeled band in COS cells, and a corresponding band for 487 bp was also detected.
Stoga, RNA COS stanica je nađeno da sadrži G-CSF +VSE i -VSE linije. Therefore, COS cell RNA was found to contain G-CSF of the +VSE and -VSE lines.
Primjer 37: Example 37:
Analiza aminokiseline i analiza šećera (kromosomskog gena) Amino acid analysis and sugar analysis (chromosomal gene)
1) Analiza sastava aminokiseline 1) Analysis of amino acid composition
Sirovi CSF uzorak dobiven u primjeru 35 je pročišćen prema postupcima opisanim u primjeru 2(iii). Pročišćeni CSF uzorak je podvrgnut analizi sastava aminokiseline postupcima opisanim u 1) primjeru 27. Rezultati su prikazani u tablici 10. The crude CSF sample obtained in Example 35 was purified according to the procedures described in Example 2(iii). The purified CSF sample was subjected to amino acid composition analysis by the procedures described in 1) example 27. The results are shown in table 10.
Tablica 10 Table 10
Podaci analize aminokiseline Amino acid analysis data
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2) Analiza sastava šećera 2) Analysis of sugar composition
Pročišćeni CSF uzorak korišten u analizi sastava aminokiseline u 1) je također podvrgnut analizi sastava šećera istim postupcima kao što je opisano u 2) u primjeru 27. Kao rezultat ove analize potvrđena je prisutnost galaktoze, N-acetil galaktozamina i sialinske kiseline u CSF uzorku ovoga izuma. The purified CSF sample used in the analysis of amino acid composition in 1) was also subjected to analysis of sugar composition by the same procedures as described in 2) in example 27. As a result of this analysis, the presence of galactose, N-acetyl galactosamine and sialic acid in the CSF sample of this was confirmed invention.
Primjer 38: Example 38:
Ekspresija humanog G-CSF kromosomskog gena u C127 stanicama Expression of the human G-CSF chromosomal gene in C127 cells
Plazmid pMLCEα, dobiven u primjeru 34 je bio tretiran sa EcoRI i fragment od oko 4 kb je izdvojen postupcima opisanim u Molecular Cloning. Izdvojeni fragment je upotrijebljen kao izvor kromosomskog G-CSF gena. Plasmid pMLCEα, obtained in example 34, was treated with EcoRI and a fragment of about 4 kb was isolated by the procedures described in Molecular Cloning. The isolated fragment was used as the source of the chromosomal G-CSF gene.
Fragment je tretiran sa Klenow fragmentom DNA polimeraze I, tako da se grade otvoreni krajevi (A). The fragment is treated with Klenow fragment of DNA polymerase I, so that open ends are built (A).
Promotor SV40 (oko 0,4 kb EcoRI-EcoRI fragment) je isječen od plazmida pHGA410 (dobivenom u primjeru 22) postupcima opisanim u Molecular Cloning, i zatim je tretiran sa Klenow fragmentom DNA polimeraze (B). The SV40 promoter (about 0.4 kb EcoRI-EcoRI fragment) was excised from plasmid pHGA410 (obtained in Example 22) by the procedures described in Molecular Cloning, and then treated with the Klenow fragment of DNA polymerase (B).
U odvojenom stupnju, plazmid pdBPV-1 koji ima papilloma virus goveda (BPV) /ovaj plazmid je dobiven zahvaljujući ljubaznosti Dr. Howley i opisan je u Sarver, N., Sbyrne, J.C. & Howley, P.M. Proc. Natl. Acad. Sci., USA, 79, 7147-7151 (1982)/ je tretiran sa HindIII i PvuII, tako da se dobije DNA fragment od oko 8,4 kb. Ovaj fragment je tretiran sa Klenow fragmentom DNA polimeraze 1 i defosforiliziran sa bakterijskom alkalnom fosfatazom (C). In a separate step, plasmid pdBPV-1 harboring bovine papilloma virus (BPV)/this plasmid was obtained through the courtesy of Dr. Howley and is described in Sarver, N., Sbyrne, J.C. & Howley, P.M. Proc. Natl. Acad. Sci., USA, 79, 7147-7151 (1982)/ was treated with HindIII and PvuII, so that a DNA fragment of about 8.4 kb was obtained. This fragment was treated with Klenow fragment of DNA polymerase 1 and dephosphorylated with bacterial alkaline phosphatase (C).
DNA fragmenti (A), (B) i (C), svaki mase od 0,1 µg je otopljen u 20 µl reakcijske otopine /50 mM Tris-HCl (pH 7,6), 10 mM MgCl2, 10 mM DTT i 1 mM ATP/ i reakcija je izvedena preko noći na 4°C u prisutnosti 180 jedinica T4DNA ligaze. DNA fragments (A), (B) and (C), each weighing 0.1 µg were dissolved in 20 µl reaction solution /50 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 10 mM DTT and 1 mM ATP/ and the reaction was performed overnight at 4°C in the presence of 180 units of T4DNA ligase.
Reakcijska otopina je zatim tretirana sa rubidij-kloridom, postupkom opisanim u Molecular Cloning, tako da se dobiva plazmid pTNCE3α (slika 21). The reaction solution was then treated with rubidium chloride, according to the procedure described in Molecular Cloning, so that the plasmid pTNCE3α was obtained (Figure 21).
DNA fragmenti (A), korišten kao izvor kromosomskog G-CSF gena može biti za-mijenjen DNA fragmentom od oko 1,87 kb, koji je dobiven sljedećim postupcima: DNA fragments (A), used as the source of the chromosomal G-CSF gene can be replaced by a DNA fragment of about 1.87 kb, which was obtained by the following procedures:
20 µg pMLCE3α je otopljeno sa 100 µl smjese 10 mM Tris-HCl (pH 8,0), 7 mM MgCl2 i 100 mM NaCl, 7 mM 2- merkaptoetanola i 0,01% BSA; otopina je inkubirana na 37°C 5 sati u prisutnosti 20 jedinica StuI i podvrgnuta je elektroforezi kroz 1,2% agaroza gel. 20 µg of pMLCE3α was dissolved with 100 µl of a mixture of 10 mM Tris-HCl (pH 8.0), 7 mM MgCl2 and 100 mM NaCl, 7 mM 2-mercaptoethanol and 0.01% BSA; the solution was incubated at 37°C for 5 hours in the presence of 20 units of StuI and electrophoresed through a 1.2% agarose gel.
Tako dobiven plazmid pTNCE3α je upotrijebljen za transformiranje C127 stanica miša, kao u primjeru 24 i klonovi koji izražavaju humani G-CSF kromosomski gen i izdvojeni su oni koji imaju visok kapacitet za produkciju G-CSF. The thus obtained plasmid pTNCE3α was used to transform C127 mouse cells, as in example 24, and clones expressing the human G-CSF chromosomal gene and those with a high capacity for G-CSF production were isolated.
Primjer 39: Example 39:
Izražavanje humanog G-CSF kromosomskog gena u CHO stanicama Expression of the human G-CSF chromosomal gene in CHO cells
Kao u slučaju izražavanja u C127 stanicama, plazmid pMLCE3α je tretiran sa StuI i izdvojen je DNA fragment od oko 1,78 kb; alternativno, isti plazmid je tretiran sa EcoRI i izdvojen je EcoRI fragment od oko 4 kb. Oba fragmenta su pogodna za korištenje, kao izvori kromosomskog G-CSF gena. As in the case of expression in C127 cells, plasmid pMLCE3α was treated with StuI and a DNA fragment of about 1.78 kb was isolated; alternatively, the same plasmid was treated with EcoRI and an EcoRI fragment of about 4 kb was isolated. Both fragments are suitable for use as sources of the chromosomal G-CSF gene.
Izvor fragmenta je tretiran sa Klenow fragmentom DNA polimeraze 1 (a). The source of the fragment was treated with Klenow fragment of DNA polymerase 1 (a).
Kao u primjeru 38, promotor SV40 (EcoRI-EcoRI fragment) je isječen iz pHGA410, tako da se dobiva fragment od oko 0,4 kb, koji je slično tretiran sa Klenow fragmentom DNA polimeraze (b). As in example 38, the SV40 promoter (EcoRI-EcoRI fragment) was excised from pHGA410, so that a fragment of about 0.4 kb was obtained, which was similarly treated with the Klenow fragment of DNA polymerase (b).
U odvojenom stupnju plazmid pAdD26SVpA plazmid /Kaufman, R.G. & Sharp, P.A., Mol. Cell. Biol., 2, 1304-1319(1982)/ je tretiran sa EcoRI, i tada sa Klenow fragmentom DNA polimeraze, i najzad je defosforiliziran tretmanom sa bakterijskom alkalnom fosfatazom (c). In a separate step, the plasmid pAdD26SVpA plasmid /Kaufman, R.G. & Sharp, P.A., Mol. Cell. Biol., 2, 1304-1319(1982)/ was treated with EcoRI, and then with the Klenow fragment of DNA polymerase, and finally dephosphorylated by treatment with bacterial alkaline phosphatase (c).
Fragmenti (a), (b) i (c), svaki mase od 0,1 µg su otopljeni u 20 µl reakcijske otopine /50 mM Tris-HCl (pH 7,6), 10 mM MgCl2, 10 mM DTT i 1 mM ATP/ i reakcija je izvedena preko noći na 4°C u prisutnosti 180 jedinica T4DNA ligaze. Fragments (a), (b) and (c), each weighing 0.1 µg, were dissolved in 20 µl reaction solution /50 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 10 mM DTT and 1 mM The ATP/i reaction was performed overnight at 4°C in the presence of 180 units of T4DNA ligase.
Reakcijska otopina je zatim tretirana sa rubidij-kloridom postupkom opisanim također u Molecular Cloning, tako da se transformiraju E. coli vrste Dhl. Dobivene Tetr kolonije su testirane na sadržaj PD26SVCE3. The reaction solution was then treated with rubidium chloride by the procedure also described in Molecular Cloning, so that E. coli species Dhl were transformed. The resulting Tetr colonies were tested for PD26SVCE3 content.
Kao što je prikazano na slici 22, plazmid pD26SVCE3α ima CSF gen vezan na rani gen SV40, i dhfr gen vezan ispod principjelno posljednjeg promotora adenoviroze. As shown in Figure 22, plasmid pD26SVCE3α has the CSF gene linked to the SV40 early gene, and the dhfr gene linked below the principally last adenovirus promoter.
Plazmid pAdD26SVpA je tretiran sa EcoRI i BamHi kao u 2) primjera 25, tako da se dobije DNA fragment (oko 2 kb) koji sadrži dhfr gen. Ovaj fragment je vezan na fragment (a) i EcoRI-SaII fragment pHGA410 (H), tako da stvara Ampr vektor ekspresije pDRCE3α (slika 22). Plasmid pAdD26SVpA was treated with EcoRI and BamHi as in 2) of example 25, so that a DNA fragment (about 2 kb) containing the dhfr gene was obtained. This fragment was ligated to fragment (a) and the EcoRI-SaII fragment of pHGA410 (H), thus creating the Ampr expression vector pDRCE3α (Figure 22).
CHO stanice su trasnformirane sa tako dobivenim plazmidima, pD26SVC3α i pDRCE3α, kao u primjeru 25. Ponovljenom selekcijom kros rast u prisutnosti MTX klonova G-CSF dobivena je produkcijska vrsta. CHO cells were transformed with the thus obtained plasmids, pD26SVC3α and pDRCE3α, as in example 25. By repeated cross-growth selection in the presence of MTX clones of G-CSF, a production species was obtained.
Primjer 40: Example 40:
Ispitivanje G-CSF aktivnosti transformanata (koji ekspresira humani kromosomski gen) Examination of G-CSF activity of transformants (expressing a human chromosomal gene)
Supernatanti kultura C127 stanmica i CHO stanica koji su dobiveni u primjerima 38 i 39, obrađeni su kao u primjeru 26 tako da se dobije humani G-CSF i ispitana je njegova aktivnost. Rezultati su prikazani u tablici 11. Culture supernatants of C127 cells and CHO cells obtained in Examples 38 and 39 were processed as in Example 26 to obtain human G-CSF and its activity was tested. The results are shown in table 11.
Tablica 11 Table 11
Ispitivanje humane G-CSF aktivnosti Assay of human G-CSF activity
[image] [image]
Primjer 41: Example 41:
Molekularna masa i izoelektrična točka transformanata Molecular mass and isoelectric point of transformants
Pročišćeni CSF uzorci korišteni u analizama sastava aminokiselina u primjerima 16, 20, 27, 33 i 37 su podvrgnuti mjerenju molekularnih masa i izoelektričnih točaka prateći sljedeće postupke> The purified CSF samples used in the amino acid composition analyzes in Examples 16, 20, 27, 33 and 37 were subjected to molecular mass and isoelectric point measurements following the following procedures>
1) Molekularna masa 1) Molecular mass
Molekulama masa CSF-a je određena natrij dodecilsulfat-poliakrilamid gel elektro-forezom (SDE-PAGE). Oprema za elektroforezu bila je PROTEANTM (16 cm, proizvod BioRad Corporation) koristeći gel načinjen od gela poliakrilamid ploče (T=15%,C=2,6%) dimenzija 140 mmx160 mmx1,5 mm i koncentrata gela (T=3%, C=20). Denaturirani CSF uzorak je dobiven prateći sljedeći postupak: CSF je kuhan 3 minute u otopini koja sadrži 2% natrij dodecilsulfata u 0,46 M 2-merkaptoetanolu. Nakon vršenja elektroforeze sa 4 µg uzorka sa konstantnom strujom od 30 mA tijekom 4 sata, gel je odvojen i obojen sa 0,24% Coomassie Brilliant Blue R 250 (proizvod tvrtke Sigma Chemical Co.) za detekciju vrpce. Sljedeće supstance su upotrijebljene kao markeri molekularne mase poslije sličnih tretmana: fosfoforilaza B (mol. mase 92500), albumin serum goveda (BSA, mol. m. 67000), ovalbumin (OVA, mol.m. 45000), ugljena anhidraza (mol. m. 31000), sojin tripsin inhibitor (mol. m. 21500) i lizozim (mol. m. 14400). The mass of CSF molecules was determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDE-PAGE). The electrophoresis equipment was PROTEANTM (16 cm, a product of BioRad Corporation) using a gel made from a polyacrylamide plate gel (T=15%, C=2.6%) measuring 140 mmx160 mmx1.5 mm and a gel concentrate (T=3%, C=20). A denatured CSF sample was obtained following the following procedure: CSF was boiled for 3 minutes in a solution containing 2% sodium dodecyl sulfate in 0.46 M 2-mercaptoethanol. After electrophoresis with 4 µg of sample at a constant current of 30 mA for 4 hours, the gel was separated and stained with 0.24% Coomassie Brilliant Blue R 250 (product of Sigma Chemical Co.) for band detection. The following substances were used as molecular mass markers after similar treatments: phosphophorylase B (mol. mass 92500), bovine serum albumin (BSA, mol. m. 67000), ovalbumin (OVA, mol. m. 45000), carbonic anhydrase (mol. m. m. 31000), soy trypsin inhibitor (mol. m. 21500) and lysozyme (mol. m. 14400).
Na ovaj način, jednostruka traka koja odgovara molekularnoj masi od 185000±1000 je identificirana za svaki od CSF uzoraka dobivenih u primjeru 18 /E. coli/cDNA (+VSE)/ i primjeru 20 /E. coli/cDNA (-VSE)/, i jednostruka traka koja odgovara molekularaoj masi od 19000±1000 je identificirana za svaki CSF uzorak dobiven u primjeru 27 /C127,CHO/cDNA (+VSE)/, primjeru 33 /C127,CHO/cDNA (-VSE)/ i primjeru 37 /COS/gDNA/. In this way, a single band corresponding to a molecular weight of 185000±1000 was identified for each of the CSF samples obtained in Example 18 /E. coli/cDNA (+VSE)/ and example 20 /E. coli/cDNA (-VSE)/, and a single band corresponding to a molecular weight of 19000±1000 was identified for each CSF sample obtained in Example 27 /C127,CHO/cDNA (+VSE)/, Example 33 /C127,CHO/cDNA (-VSE)/ and example 37 /COS/gDNA/.
2) Izoelektrična točka 2) Isoelectric point
Izoelektrična točka CSF ovog izuma je određena pomoću ravnog postolja, izoelektrične aparature, FBE-3000 (Proizvod Pharmacia Fine Chemicals). Poslije 2 sata elektroforeze sa konstantnom snagom od 30 vati (Vmax=2000 volti) na poliakrilamid gelu (T=5%, C=3%, 115 mmx230 mm) koji sadrži Framelit (pH= 4-6,5, Pharmacia Fine Chemicals) i 4M uree, CSF je fiksiran sa 30% metanol/10% trikloroctena kiselina/35% sulfoalicilna kiselina, i obojen sa Coomassie Brilliant Blue R 250. Nizak pI kit (pH=2,5-6,5, proizvod Pharmacia Fine Chemi-cals) je upotrijebljen kao marker izoelektrične točke. The isoelectric point of the CSF of this invention was determined using a flat stand, isoelectric apparatus, FBE-3000 (Product of Pharmacia Fine Chemicals). After 2 hours of electrophoresis with a constant power of 30 watts (Vmax=2000 volts) on a polyacrylamide gel (T=5%, C=3%, 115 mmx230 mm) containing Framelit (pH= 4-6.5, Pharmacia Fine Chemicals) and 4M urea, CSF was fixed with 30% methanol/10% trichloroacetic acid/35% sulfoalylic acid, and stained with Coomassie Brilliant Blue R 250. Low pI kit (pH=2.5-6.5, product of Pharmacia Fine Chemi- cals) was used as an isoelectric point marker.
Analiza razdvojenja traka na pH od 4-6,5 daje jednostruku vrpcu koja odgovara pI=6,1 za svaki od CSF uzoraka dobivenih u primjerima 16 i 20, i dalje tri razdvojene vrpce, koje odgovaraju za pI=5,5, 5,8 i 6,5 za svaki od uzoraka dobivenih u primjerima 27, 33 i 37. Band separation analysis at pH 4-6.5 gives a single band corresponding to pI=6.1 for each of the CSF samples obtained in Examples 16 and 20, and further three separated bands, corresponding to pI=5.5, 5, 8 and 6.5 for each of the samples obtained in examples 27, 33 and 37.
Primjer 42: Example 42:
Zaštitni efekt humanog G-CSF protiv mikrobioloških infekcija Protective effect of human G-CSF against microbiological infections
Metoda ispitivanja Test method
1. Zaštita protiv infekcije sa Pseudomonas aeruginosa 1. Protection against infection with Pseudomonas aeruginosa
Endoksan (trgovačko ime Shionogi & Co., Ltd.) je unesen interperiotenalno u 8-9 tjedana stare miševe (mužjaci; 35,3±1,38 g tjelesne težine) u dozi od 200 mg/kg. Endoxan (trade name Shionogi & Co., Ltd.) was administered intraperitoneally to 8-9-week-old mice (males; 35.3±1.38 g body weight) at a dose of 200 mg/kg.
Miševi su tada podijeljeni u tri grupe /dvije grupe su dobile potkožno 4 injekcije (deze 0,1 ml) na svaka 24 sata otapala /1% propanol i 0,5% (m/v) albumin seruma miša u fiziološkoj otopini) koji sadrži humani G-CSF (25000 ili 50000 jedinica po mišu), dok je drugoj grupi dano samo otapalo prema istom rasporedu. Tri sata nakon posljednje injekcije, miševi u svakoj grupi su inficirani sa Pseudomonas aeruginosa GNB-139 potkožnom injekcijom (3,9x105 CFU/miš). Dvadeset jedan sat nakon inficiranja, miševima prve grupe je dana druga potkožna injekcija otapala koje sadrži humani G-CSF (25000 ili 50000 jedinica/miš) a drugoj grupi je dano samo otapalo. Mice were then divided into three groups / two groups received subcutaneously 4 injections (deze 0.1 ml) every 24 hours of solvent /1% propanol and 0.5% (m/v) mouse serum albumin in saline) containing human G-CSF (25,000 or 50,000 units per mouse), while the other group was given solvent alone according to the same schedule. Three hours after the last injection, mice in each group were infected with Pseudomonas aeruginosa GNB-139 by subcutaneous injection (3.9x105 CFU/mouse). Twenty-one hours after infection, the mice of the first group were given a second subcutaneous injection of a solvent containing human G-CSF (25,000 or 50,000 units/mouse) and the second group was given only solvent.
Zaštitni efekt humanog G-CSF je ispitan brojanjem miševa koji su ostali živi 10 dana nakon inficiranja. The protective effect of human G-CSF was tested by counting mice that remained alive 10 days after infection.
Pripremanje suspenzije stanica Preparation of cell suspension
Pseudomonas aeruginosa. GNB-139 je kultivirana preko noći uz mućkanje na 37°C u kardijalnom infuzijskom tekućem mediju (trgovačko ime Difco). Kultura je suspendirana u fiziološku slanu otopinu. Pseudomonas aeruginosa. GNB-139 was cultured overnight with shaking at 37°C in cardiac infusion liquid medium (trade name Difco). The culture was suspended in physiological saline solution.
2. Zaštita protiv infekcije sa Candida 2. Protection against Candida infection
Endoksan (trgovačko ime Shinogi & Co., Ltd.) je unesen interperitonealno 8 tjedana starim ICR štakorima (mužjaci; 40,5±1,60 g tjelesne težine) u dozi od 200 mg/kg. Miševi su podijeljeni u dvije grupe; jednoj grupi su dane 4 potkožne injekcije, (doze 0,1 ml) na svaka 24 sata, otapala /1% propanol i 10% m/v) ICR seruma miša u fiziološkoj otopini/ koja sadrži humani G-CSF (50000 jedinica po mišu), dok je drugoj grupi dano samo otapalo prema istom rasporedu. Četiri sata nakon posljednje injekcije, miševi u svakoj grupi su inficirani sa Candida albicans U-50-1 (vrsta izolirana iz urina ili leukemičkih pacijenata; zahvaljujući ljubaznosti Bakteriološkog laboratorija, Tohoku University, School of Medicine) intravenskom injekcijom (5,6x105 CFU/miš). Zaštitni fekt humanog G-CSF je ispitivan brojanjem miševa koji su ostali živi deset dana nakon inficiranja. Endoxan (trade name Shinogi & Co., Ltd.) was administered intraperitoneally to 8-week-old ICR rats (male; 40.5±1.60 g body weight) at a dose of 200 mg/kg. The mice were divided into two groups; one group was given 4 subcutaneous injections, (doses 0.1 ml) every 24 hours, of a solvent (1% propanol and 10% m/v) of ICR mouse serum in saline/ containing human G-CSF (50,000 units per mouse ), while the other group was given only the solvent according to the same schedule. Four hours after the last injection, mice in each group were infected with Candida albicans U-50-1 (species isolated from urine or leukemic patients; courtesy of the Laboratory of Bacteriology, Tohoku University, School of Medicine) by intravenous injection (5.6x105 CFU/mouse ). The protective effect of human G-CSF was examined by counting mice that remained alive ten days after infection.
Pripravljanje suspenzije stanica Preparation of cell suspension
Candida albicans U-50-1 je kultivirana preko noći uz mućkanje na 37°C u ekstraktu kvasca koji sadrži Sabouraud-tekući medij (2% dekstroza iz Junsei Pure Chemicals Co., Ltd.; triptokase peptona, trgovačko ime BBL; 5% esktrakta kvasca od Difco; pH 5,6). Kultura je dva puta isprana sa fiziološkom otopinom i suspendirana u fiziološku otopinu. Candida albicans U-50-1 was cultured overnight with shaking at 37°C in yeast extract containing Sabouraud-liquid medium (2% dextrose from Junsei Pure Chemicals Co., Ltd.; tryptocase peptone, trade name BBL; 5% extract yeast from Difco; pH 5.6). The culture was washed twice with saline and suspended in saline.
3. Zaštita protiv infekcije sa intracelularnom parazitskom Listeria 3. Protection against infection with intracellular parasitic Listeria
Endotoksan (trgovačko ime Shionogi & Co., Ltd.) je unesen intraperitonealno 7 tjedana starim ICR miševima; 34,7±1,24 g tjelesne težine) u dozi od 200 mg/kg. Miševi su podijeljeni u dvije grupe; jednoj grupi su dane 4 potkožne injekcije, (0,1 ml doza) na svaka 24 sata, otapalo /1% propanol i 10% (m/v) ICR seruma miša u fiziološkoj otopini/ koja sadrži humani G-CSF (50000 jedinica/miš) dok je drugoj grupi dano samo otapalo prema istom rasporedu. Četiri sata nakon posljednje injekcije, miševi svake grupe su inficirani sa Listeria monocytogenes 46 (zahvaljujući ljubaznosti Mikrobiološkog laboratorija, Tohoku University, School of Medicine) intravenskim injekcijama od 1,0x107 CFU/miš. Zaštitni efekt humanog G-CSF je ispitivan brojanjem miševa koji su ostali živi 12 dana nakon inficiranja. Endotoxan (trade name Shionogi & Co., Ltd.) was administered intraperitoneally to 7-week-old ICR mice; 34.7±1.24 g of body weight) in a dose of 200 mg/kg. The mice were divided into two groups; one group was given 4 subcutaneous injections, (0.1 ml dose) every 24 hours, solvent /1% propanol and 10% (m/v) ICR mouse serum in saline/ containing human G-CSF (50000 units/ mouse) while the other group was given only solvent according to the same schedule. Four hours after the last injection, mice of each group were infected with Listeria monocytogenes 46 (courtesy of the Microbiology Laboratory, Tohoku University, School of Medicine) by intravenous injections of 1.0x107 CFU/mouse. The protective effect of human G-CSF was examined by counting mice that remained alive 12 days after infection.
Priređivanje suspenzije stanica Preparation of cell suspension
Listeria monocytes 46 je kultivirana preko noći uz mućkanje na 37°C u moždano-kardijalnom infuzijskom tekućem mediju (trgovačko ime Difco). Kultura je suspendirana u fiziološku otopinu. Listeria monocytes 46 were cultured overnight with shaking at 37°C in brain-cardiac infusion liquid medium (trade name Difco). The culture is suspended in physiological solution.
Rezultati: The results:
i) Ispitivanja 1, 2 i 3 su vršena sa E. coli G-CSF (+VSE) polipeptidom dobivenim u primjeru 16. Rezultati su prikazani u tablicama 12, 13 i 14. i) Tests 1, 2 and 3 were performed with the E. coli G-CSF (+VSE) polypeptide obtained in Example 16. The results are shown in Tables 12, 13 and 14.
Tablica 12 Table 12
Efekt protiv Pseudomonas aeruginosa Effect against Pseudomonas aeruginosa
[image] [image]
Tablica 13 Table 13
Efekt protiv Candida albicans Effect against Candida albicans
[image] [image]
Tablica 14 Table 14
Efekt protiv Listeria monocytogenes Effect against Listeria monocytogenes
[image] [image]
ii) Ispitivanje 1 je izvršeno sa E. coli G-CSF (-VSE) polipeptidom dobivenim u primjeru 20. Rezultati su prikazani u tablici 15. ii) Test 1 was performed with the E. coli G-CSF (-VSE) polypeptide obtained in Example 20. The results are shown in Table 15.
Tablica 15 Table 15
Efekt protiv Pseudomonas aeruginosa Effect against Pseudomonas aeruginosa
[image] [image]
iii) Ispitivanje protiv Pseudomonas aeruginosa iii) Testing against Pseudomonas aeruginosa
[image] Suštinski, isti rezultati su dobiveni kada se ispitivanje 1 vrši sa C127 izvedenom stanicom, pročišćenog G-CSF uzorka istog koji je upotrebljen u analizi sastava aminokiseline u primjeru 27. [image] Substantially the same results were obtained when assay 1 was performed with a C127 cell-derived, purified G-CSF sample of the same one used in the amino acid composition analysis in Example 27.
iv) Ispitivanje 1 je izvršeno sa CHO izvedenim stanicama, pročišćenog humanog G-CSF uzorka (+VSE) koji je isto kao onaj upotrebljen u analizi sastava aminokiseline u primjeru 33. iv) Test 1 was performed with CHO derived cells, a purified human G-CSF sample (+VSE) which is the same as that used in the amino acid composition analysis in Example 33.
Rezultati su prikazani u tablici 17. The results are shown in table 17.
Tablica 17. Table 17.
Efekt protiv Pseudomonas aeruginosa Effect against Pseudomonas aeruginosa
[image] [image]
Suštinski, isti rezultati su dobiveni kada je ispitivanje 1 izvršeno sa C127 izvedenim stanicama, pročišćenog humanog G-CSF uzorka koji je bio isti kao onaj koji je upotrebljen za analizu sastava aminokiseline u primjeru 33. Essentially the same results were obtained when assay 1 was performed with C127 cell-derived, purified human G-CSF sample that was the same as that used for the amino acid composition analysis in Example 33.
Claims (10)
Applications Claiming Priority (10)
Application Number | Priority Date | Filing Date | Title |
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JP20606685 | 1985-09-17 | ||
JP20963885 | 1985-09-20 | ||
JP21715085 | 1985-09-30 | ||
JP26945685 | 1985-12-02 | ||
JP60269455A JPS62129298A (en) | 1985-12-02 | 1985-12-02 | Novel polypeptide |
JP27083985 | 1985-12-03 | ||
JP60270838A JPH06102021B2 (en) | 1985-12-03 | 1985-12-03 | Novel polypeptide |
JP61166709A JPH0657152B2 (en) | 1985-09-17 | 1986-07-17 | CSF genes |
JP61166710A JPS62236497A (en) | 1985-09-17 | 1986-07-17 | Novel glycoprotein and production thereof |
YU161086A YU48546B (en) | 1985-10-25 | 1986-09-16 | Human granulocyte colony stimulating factor |
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HRP920628A2 true HRP920628A2 (en) | 1996-06-30 |
HRP920628B1 HRP920628B1 (en) | 2001-04-30 |
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