CN102260344A - Deletion type human beta interferon and recombination preparation thereof - Google Patents
Deletion type human beta interferon and recombination preparation thereof Download PDFInfo
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- CN102260344A CN102260344A CN2010101826595A CN201010182659A CN102260344A CN 102260344 A CN102260344 A CN 102260344A CN 2010101826595 A CN2010101826595 A CN 2010101826595A CN 201010182659 A CN201010182659 A CN 201010182659A CN 102260344 A CN102260344 A CN 102260344A
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Abstract
The invention discloses a mutant (delta 11-IFN-beta) that 11 amino acids are deleted at an amino terminal of a human beta interferon, and a preparation method thereof. Through recombination preparation, the delta 11-IFN-beta has higher in-vitro anti-virus specific activity and stability than a non-deletion type human beta interferon, and is more suitable for treating diseases such as multiple sclerosis and viral infection.
Description
Technical field the present invention relates to DNA recombinant technology and pharmaceutical field, relate to recombination deficient type human beta interferon, this lacks proteic dna sequence dna to contain coding, the carrier that contains this dna sequence dna, the host cell that contains this carrier, be used for this proteic method of genetically engineered preparation, and this albumen is in the application in treatment disease field.
The background technology human beta interferon is that the class that inoblast and epithelial cell produce has extensive biologic activity, important cytokine (the KozovsKa ME that comprises antiviral, antitumor and immunoregulation effect, et al.Neurology (J), 1999,53:1692-1697), molecular weight with 5 α spirals and 22KDa, interferon--1b sugar basedization wherein, its molecular weight is 18KDa (Arduini, et al.Protein Science (J), 1999,8:1867-1877).Human beta interferon is divided into interferon--1a and interferon--1b at present, wherein interferon--1a is a natural structure, include the glycosylation structure, and interferon--1b is mutated into Ser with 17 Cys to form, and the N end does not contain methionine(Met), forms (Goodkin DE, et al.Lancet (J) by 165 amino acid, 1994,344:1057-1060).Studies show that in a large number, human beta interferon can in intestinal bacteria, yeast, mammalian cell, realize stability and high efficiency expression (Jacobs L, et al.Curr Opin Neuro (J), 1994,7:250-254).Since 1993 the multiple recombinant beta Interferon, rabbit listing of external approved be used for the treatment of multiple sclerosis (multiplesclerosis, MS).
Being used for clinical IFN-β abroad has two types of IFN-β-1a and IFN-β-1b, all is the gene recombination product.The Betaferon that Schering Corp is arranged, Extavia and the Rebif of Xue Lannuo company and the Aronex of Biogen company of europeanized company of Novartis that interferon-has been gone on the market abroad as the medicine for the treatment of multiple sclerosis.Wherein the Extavia of the europeanized company of the Betaferon of Schering Corp and Novartis belongs to IFN-β-1b, by in E.coli with the recombinant human interferon--1b of inclusion body formal representation, need repeatability after the SDS sex change in the technology.And the Aronex of the Rebif of Xue Lannuo company and Biogen company is the recombinant human IFN-β-1a that expresses by CHO (Chinese hamster ovary).There are evident difference in reorganization IFN-β-1b and IFN-β-1a aspect structure and the biological activity, and the extracorporeal antivirus effect specific activity that mainly shows as the IFN-β-1a of CHO expression is higher than IFN-β-1b that inclusion bodies of colibacillus is expressed preparation.
The invention provides a kind of deletion human interferon-mutant Δ 11-IFN-β and express the method for preparing, not only solvability is better for the albumen of preparation, stability is higher, has higher antiviral activity simultaneously.
Present human beta interferon-the 1b of goal of the invention (IFN-β-1b) with the formal representation of inclusion body, needs in the technology with repeatability after the sex change of SDS elder generation in intestinal bacteria, preparation technology's relative complex, and contain the residual of SDS, its antiviral specific activity only is 2 * 10
7IU/mg; Human beta interferon-1a (IFN-β-1a) be to be the glycosylated glycoprotein of having of host cell expression with the Chinese hamster ovary celI, antiviral specific activity is 2 * 10
8IU/mg (Laura Runkel, et al.Pharmaceutical Research (J), 1998,15:641-649).
The present invention recombinates that deletion human interferon-mutant and the natural structure ratio of preparation has that solvability is higher, the better advantage of stability.The recombination deficient type human beta interferon mutant of preparation comprises: Δ 11-IFN-β-1a and Δ 11-IFN-β-1b.Wherein, Δ 11-IFN-β-1b adopts intestinal bacteria to express preparation in the mode of amalgamation and expression, and it is glycine (Gly) or arginine (Arg) or methionine(Met) (Met) that its N holds the 1st amino acids replaceable, and the 6th amino acids is Serine (Ser); And Δ 11-FN-β-1a can adopt yeast cell or expressing cho cell to prepare, and it is glycine (Gly) or arginine (Arg) or methionine(Met) (Met) that its N holds the 1st amino acids replaceable, and the 6th amino acids is halfcystine (Cys).Secondly, can have glycine (Gly) or arginine (Arg) or methionine(Met) (Met) before the 1st amino acids of this recombination deficient type human beta interferon mutant protein.
The engineering bacteria of the expression Δ 11-IFN-β-1b that makes up adds IPTG and induces the back to collect thalline after the fermentor tank enlarged culturing.Centrifugal collection supernatant behind the broken bacterium reclaims fusion rotein by the Ni column purification in specific broken bacterium damping fluid, after specific proteolytic enzyme enzyme is cut by ion-exchange and anti-phase C
18Consummateization of post, the antiviral biology specific activity of reorganization Δ 11-IFN-β-1b of preparation reaches 7.3 * 10
7More than the IU/mg.The expression Δ 11-IFN-β-1a yeast cell that makes up or the Δ 11-IFN-β-1a of Chinese hamster ovary celI strain secreting, expressing after enlarged culturing, the antiviral biology specific activity of Δ 11-IFN-β-1a for preparing through further separation and purification reaches 3.1 * 10 respectively
8IU/mg and 4.3 * 10
8More than the IU/mg.
The object of the present invention is to provide a kind of deletion human interferon-mutant (to comprise Δ 11-IFN-β-1b and the Δ 11-IFN-β-preparation of 1a) and effectively recombinating.The dna molecular of described deletion human interferon-mutant protein of encoding, the host cell that contains the carrier of this dna sequence dna and contain this carrier also are provided.
Another object of the present invention provides a kind of preparation technology of deletion human interferon-mutant, obtains the method for the higher deletion human interferon-mutant protein of antiviral specific activity.
Another object of the present invention provides a kind of can be used for treating virus disease such as viral hepatitis C or hepatitis B, perhaps the recombination deficient type human beta interferon mutant protein of immunological disease such as multiple sclerosis.
Summary of the invention is in a first aspect of the present invention, and it is that 11 amino acid whose Δ 11-IFN-β of N end disappearance (comprise Δ 11-IFN-β-1a and Δ 11-IFN-β-1b) that a kind of deletion human interferon-mutant protein is provided.
In a second aspect of the present invention, provide a kind of in intestinal bacteria by with the fusion rotein of TRX amalgamation and expression, more specifically, comprise different Linker in this fusion rotein, the different proteolytic enzyme restriction enzyme site of design comprises EK, TEV, thrombin, factor Xa etc. in Linker.Described Δ 11-IFN-β-1b albumen has following structure: at first, the 1st amino acids is replaceable to be methionine(Met) (Met) or glycine (Gly) or arginine (Arg), and the 6th amino acids is Serine (Ser); Secondly, can have (Met) or glycine (Gly) or arginine (Arg) before the 1st amino acids.
In a third aspect of the present invention, a kind of reorganization Δ 11-IFN-β-1a albumen of expressing in yeast cell or Chinese hamster ovary celI is provided, described reorganization Δ 11-IFN-β-1a albumen has following structure: at first, the 1st amino acids is replaceable to be methionine(Met) (Met) or glycine (Gly) or arginine (Arg), the 6th amino acids is halfcystine (Cys), and the sky of the 69th amino acids (door) winter acid amides is by glycosylation modified; Secondly, can have (Met) or glycine (Gly) or arginine (Arg) before the 1st amino acids.
In a fourth aspect of the present invention, the above-mentioned recombination deficient type human beta interferon-1a of code book invention and the dna molecular of 1b mutant protein are provided.
In a fifth aspect of the present invention, the carrier that contains above-mentioned dna molecular is provided, and the method that makes up this expression vector.
In a sixth aspect of the present invention, provide a kind of host cell of suitable idiosyncratic carrier (to comprise prokaryotic cell prokaryocyte and eukaryotic cell.Prokaryotic host cell commonly used comprises intestinal bacteria, subtilis etc.; Eukaryotic host cell commonly used comprises yeast cell, Chinese hamster ovary celI, COS cell, C-127 cell, bhk cell, rat HepI cell, rat Hep II cell, TCMK cell, human pneumonocyte, HEK293 cell etc.; Insect cell commonly used such as silkworm culturing cell etc.).And can transfer in the host cell by the carrier that known method will contain the dna sequence dna of goal gene.
In a seventh aspect of the present invention, but the host cell that comprises above-mentioned expression vector and the method for transformation of energy or transfection are provided.
In a eighth aspect of the present invention, provide a kind of the reorganization to produce the method for recombination deficient type human beta interferon mutant protein of the present invention, it may further comprise the steps: be fit to express under the condition of described recombination deficient type human beta interferon mutant, cultivate above-mentioned host cell, give expression to described recombination deficient type human beta interferon mutant protein, prepare the method for recombination deficient type human beta interferon mutant protein through separation and purification.
In a ninth aspect of the present invention, provide a kind of the reorganization to produce the fusion rotein of escherichia coli expression among the present invention, the method for cutting by different proteolytic enzyme enzymes, and separation and purification prepares the method for recombination deficient type human beta interferon-1b mutant protein.
In a tenth aspect of the present invention, provide a kind of the reorganization to produce the deletion human interferon--1a mutant protein of yeast expression among the present invention, prepare the method for deletion human interferon--1a mutant protein by separation and purification.
In a eleventh aspect of the present invention, provide a kind of the reorganization to produce the deletion human interferon--1a mutant protein of expressing cho cell among the present invention, prepare the method for deletion human interferon--1a mutant protein by separation and purification.
In a twelveth aspect of the present invention, provide a kind of this recombination deficient type human beta interferon-1a and-pharmaceutical composition of 1b mutant protein, it comprises pharmaceutically acceptable carrier or vehicle or thinner.
So far the present invention is described in detail, can clearer understanding be arranged to it with reference to following example, described example only is intended to limit the invention for the purpose of illustration and not.
Description of drawings:
Fig. 1: pET-32a-EK-Δ 11-IFN-β-Ser
6Through Kpn I and Hind III double digestion checking (1-4: recombinant plasmid pET-32a-EK-Δ 11-IFN-β-Ser
6Through Kpn I and Hind III double digestion).
Fig. 2: A:SDS-PAGE analysis fusioning protein (TRX-EK-Δ 11-IFN-β-Ser
6) expression level (1: after inducing; 2: before inducing).B: fusion rotein TRX-EK-Δ 11-IFN-β-Ser
6EK enzyme enzyme cut (1: before enzyme is cut; 2: enzyme was cut 10 hours; 3: enzyme was cut 12 hours; 4: enzyme was cut 16 hours).
Fig. 3: the Δ 11-IFN-β-Ser of A:SDS-PAGE analyte preparation
6Albumen.The Δ 11-IFN-β-Ser6 albumen of B:RP-HPLC analyte preparation.
Fig. 4: the active comparison curves of extracorporeal antivirus effect (1: the international active standard substance of interferon-; 2:TRX-EK-Δ 11-IFN-β-Ser
63: Δ 11-IFN-β-Ser
64: Δ 11-IFN-β-Cys
6(yeast expression); 5: Δ 11-IFN-β-Cys
6(CHO expression)).
Embodiment
Example 1 fusion rotein TRX-EK-Δ 11-IFN-β-Ser
6Recombination, amalgamation and expression in intestinal bacteria and target protein Δ 11-IFN-β-Ser
6The preparation of (SEQ ID NO:4).
In the present embodiment, related fusion rotein has TRX-EK-Δ 11-IFN-β-Ser
6Structure.According to the aminoacid sequence of IFN-β, and, entrust precious biological (Dalian) company limited to carry out the cDNA sequence (SEQ IDNO:5) of the synthetic Δ 11-IFN-β of full gene according to the codon that intestinal bacteria are had a preference for.
According to design requirements Synthetic 2 bar primer P1, P2:
P1：5′-CGGGTACCGATGACGATGACAAGTCTTCCAATTTTCAGTCT-3′(SEQ?ID?NO：6)
P2：5′-GCAAGCTTTCATTAGTTTCGGAGGTAACCTGT-3′(SEQ?ID?NO：7)
Annotate: CG is a primer protection base among the P1, and GGTACC is a Kpn I restriction enzyme site; GC is a primer protection base among the P2, and AAGCTT is a Hand III restriction enzyme site.
CDNA sequence (SEQ ID NO:5) with synthetic Δ 11-IFN-β is a template, with primer P1 and P2 amplification EK-Δ 11-IFN-β-Ser
6(SEQ ID NO:8) gene (the P1 head contains the nucleotide sequence of Kpn I restriction enzyme site and coding enteropeptidase restriction enzyme site DDDDK, and the P2 afterbody contains terminator codon and Hand III restriction enzyme site).The EK-Δ 11-IFN-β-Ser of amplification
6(SEQ ID NO:8) gene head and the tail add Kpn I and Hand III restriction enzyme site respectively, insert prokaryotic expression carrier pET-32a, make up recombinant expression vector pET-32a-EK-Δ 11-IFN-β-Ser
6
Extension PCR method amplification EK-Δ 11-IFN-β-Ser
6The reaction system of (SEQ ID NO:8) gene all adopts PCR reaction kit (TAKARA, Dalian), by the specification sheets setting of businessman.The PCR reaction conditions is: 94 ℃ of 1min, and 58 ℃ of 1min, 72 ℃ of 1min, totally 30 circulations, first 94 ℃ of sex change 10min that circulate, last circulation is extended 10min for 72 ℃, and the result shows and has obtained 500bp (EK-Δ 11-IFN-β-Ser
6) target stripe of (SEQ ID NO:8).
The PCR product is behind the low melting-point agarose purifying, behind Kpn I and Hind III double digestion, the purpose fragment that reclaims is connected with the T4DNA ligase enzyme with the plasmid pET-32a (purchasing in Invitrogen) that Hind III double digestion reclaims through Kpn I with same, transformed into escherichia coli cloning host bacterium Top10F ' is with the method screening recombinant plasmid pET-32a-EK-Δ 11-IFN-β-Ser of double digestion checking
6(accompanying drawing 1) shows behind dna sequencing (TAKARA, Dalian), the EK-Δ 11-IFN-β-Ser of amplification
6Gene and design (SEQ ID NO:8) in full accord.The plasmid transformation escherichia coli expressive host bacterium Origami DE3 (Novagen) that above-mentioned order-checking is correct successfully makes up pET-32a-EK-Δ 11-IFN-β-Ser
6/ Origami DE3 recombinant bacterial strain.
To express best engineering strain streak inoculation in the LA agar plate, 37 ℃ of overnight incubation.The picking lawn is inoculated in and contains LB liquid nutrient medium (Tryptones 10g from the LA flat board of incubated overnight, yeast extract 5g, NaCl 10g, add water and be settled to 1000mL, 121 ℃, autoclaving 30min gets final product) in the test tube, cultivated 12 hours for 30 ℃, be transferred in the 1000mL triangular flask that contains 200mL LB nutrient solution in 1% ratio then, 30 ℃ of overnight incubation promptly become and go up a jar seed liquor.Last jar seed liquor is inoculated in the 30L fermentor tank that contains the YT nutrient solution in 5% ratio, 37 ℃ of cultivations, have suffered in the fermenting process, keep dissolved oxygen more than 30% by adjusting rotating speed, blowing air amount, logical pure oxygen amount, the ammoniacal liquor with 28% is regulated pH and is also remained on 7.0.To bacterium liquid OD600=10～14 o'clock, adding final concentration was 0.3mmol/L IPTG, and reducing temperature simultaneously is 32 ℃, continue to cultivate and stop fermentation (accompanying drawing 2:A) after 4 hours, collection bacterium liquid, centrifugal 10 minutes of 8000rpm, abandon supernatant, the collection thalline is put into-20 ℃ of refrigerators and is preserved standby.
From-20 ℃ of refrigerators, take out thalline, put into bacterial lysate (50mmol/LTris-HCl, 1%Zwittergent3-14, pH9.0) dissolving thalline, 37 ℃ are stirred down that to add final concentration after 1 hour be that the DNA enzyme effect 3 hours of 10 μ g/ml is to the broken fully bacterium of microscopy, centrifugal 10 minutes of 12000rpm abandons precipitation.Collect supernatant.
Thick purifying: the supernatant of collecting is reclaimed fusion rotein TRX-EK-Δ 11-IFN-β-Ser by Ni ion chela and purifying
6, dialysed overnight in dialyzate (50mmol/L Tris-HCl, 0.15M Nacl, pH8.0).
Fusion rotein (TRX-EK-Δ 11-IFN-β-Ser
6) EK enzyme enzyme cuts: get the fusion rotein of dialysed overnight, press mass ratio and add EK enzyme at 1: 200, cut in 4 ℃ of following enzymes and got final product in 16 hours, enzyme is cut efficient and is not less than 90% (accompanying drawing 2:B).
Consummateization: the sample after enzyme is cut is through the RP-HPLC (C of Waters company
18Post) purifying (mobile phase A liquid: 0.1% trifluoroacetic acid, 5% acetonitrile, Mobile phase B liquid: 0.1% trifluoroacetic acid, 95% acetonitrile), the 0-100%B linear elution carries out purifying, (buffering is PBS, and pH7.4) gel filtration chromatography obtains reorganization Δ 11-IFN-β-Ser for ion-exchange (SP Sepharose FF) and Superdex-75 (Amersham Biosciences)
6(SEQIDNO:4) albumen, purity is greater than 98.0% (accompanying drawing 3:A, B).
Example 2 absence types reorganization Δ 11-IFN-β-Cys
6Recombinant expressed and the preparation of (SEQ IDNO:1) albumen in yeast.
In the present embodiment, related absence type recombinant protein has Δ 11-IFN-β-Cys
6Structure.CDNA sequence (SEQ ID NO:5) design Synthetic 2 bar primer P3, P4 according to Δ 11-IFN-β:
P3：5′-CGCTCGAGAAAAGATCTTCCAATTTTCAGTGTCAGAAG-3′(SEQ?ID?NO：9)
P4：5′-CGGCGGCCGCTCATTAGTTTCGGAGGTAACC-3′(SEQ?ID?NO：10)
Annotate: CG is a primer protection base among the P3, and CTCGAG is an Xho I restriction enzyme site; CG is a primer protection base among the P2, and GCGGCCGC is a Not I restriction enzyme site.
CDNA sequence (SEQ ID NO:5) with synthetic Δ 11-IFN-β is a template, with primer P3 and P4 amplification Δ 11-IFN-β-Cys
6(SEQ ID NO:11) gene (the P3 head contains Xho I restriction enzyme site, and the P4 afterbody contains terminator codon and Not I restriction enzyme site) is connected construction recombination plasmid pGAPZ α A-Δ 11-IFN-β-Cys with behind Xho I and the Not I double digestion with pGAPZ α A
6, behind dna sequencing (TAKARA, Dalian), show the Δ 11-IFN-β-Cys of amplification
6Gene and design (SEQ ID NO:11) in full accord.
With recombinant plasmid with Avr II linearizing, with electroporation with linearizing recombinant plasmid pGAPZ α A-Δ 11-IFN-β-Cys
6Import in the GS115 pichia spp (Invitrogen),, obtain the height copy clone bacterium of anti-Zeocin1000mg/ml through His+ and Zeocin screening.Picking is expressed bacterium, is inoculated in the 5ml YPD substratum, and 30 ℃ of incubated overnight, switching goes in the 250ml YPD substratum to express 96 hours.Collect supernatant, after ultrafiltration, with SP Sepharose FF, anti-phase C
18, SP Sepharose FF and Superdex-75 purifying prepare recombination fusion protein Δ 11-IFN-β-Cys
6(SEQ ID NO:1), purity is greater than 97.0%.
Example 3 absence types reorganization Δ 11-IFN-β-Cys
6Recombinant expressed and the preparation of (SEQ ID NO:1) albumen in Chinese hamster ovary celI.
In the present embodiment, related absence type recombinant protein has Δ 11-IFN-β-Cys
6Structure.According to design requirements, entrust precious biological (Dalian) company limited to carry out the synthetic Δ 11-IFN-β-Cys of full gene
6CDNA sequence (SEQ IDNO:12).
With the synthetic Δ 11-IFN-β-Cys of total man worker
6Gene order (SEQ ID NO:12), the plasmid pSecTag2A with same double digestion behind Hind III and EcoR I double digestion is connected construction recombination plasmid pSecTag2A-Δ 11-IFN-β-Cys
6Recombinant plasmid transformed intestinal bacteria Top10F ', correct through Hind III and the checking of EcoR I double digestion, dna sequencing (TAKARA, Dalian) result shows, the Δ 11-IFN-β-Cys of amplification
6Gene and design (SEQ ID NO:12) in full accord.The recombinant plasmid transfection CHO-k cell that above-mentioned order-checking is correct, the screening acquisition can be stablized by pressurizeing, efficient secretory expression Δ 11-IFN-β-Cys
6Cell strain.After enlarged culturing, pass through SP Sepharose FF, Blue, anti-phase C
18Purification process such as post, SP Sepharose FF and Superdex-75, the reorganization Δ 11-IFN-β-Cys of preparation
6(SEQ ID NO:1) albumen, purity is greater than 98.0%.
The extracorporeal antivirus effect biological activity test of the absence type recombinant human interferon-of example 4 preparations
Adopt the political reform of VSV (vesicular stomatitis virus) virus attack Wish cytopathy.By 2005 editions the 3rd middle interferon biological activity measuring method of pharmacopeia (appendix VIII), every hole is by 2 * 10
4Individual cell is spread overnight incubation in the flat culture plate in 96 holes, then sample to be checked and different dilution standard substance is added respectively in the culture plate of having inoculated the Wish cell to act on 16-24 hour.All nutrient solutions of sucking-off are cultivated diluted to 100TCID with VSV with attacking
50, add and attacked 24 hours.Violet staining flushing back 70% dissolve with ethanol, OD
570Measure the OD value.By compare the data of the surveying usefulness Softmax pro5.3 of institute software analysis with the international active standard substance of interferon-.By the A value that software Softmaxpro53 calculates respectively, B value, C value, D value and coefficient R ^2 (table 1).Use the antiviral specific activity of absence type recombinant human interferon-of Softmax pro5.3 computed in software simultaneously, the results are shown in Table 2.Wherein the extension rate (Er) of partly imitating of the international active standard substance of beta-interferon (NIBSC) is 90.9, Es be working sample partly imitate extension rate.
Table 1
The antiviral biology specific activity of table 2 result (three mean value)
Sequence table
＜110〉Chongqing Fujin Biological Medicine Co. Ltd
＜120〉deletion human interferon-and reorganization preparation thereof
<130>2010
<160>12
<170>PatentIn?version?3.3
<210>1
<211>155
<212>PRT
＜213〉native sequences
<400>1
Ser?Ser?Asn?Phe?Gln?Cys?Gln?Lys?Leu?Leu?Trp?Gln?Leu?Asn?Gly?Arg
1 5 10 15
Leu?Glu?Tyr?Cys?Leu?Lys?Asp?Arg?Met?Asn?Phe?Asp?Ile?Pro?Glu?Glu
20 25 30
Ile?Lys?Gln?Leu?Gln?Gln?Phe?Gln?Lys?Glu?Asp?Ala?Ala?Leu?Thr?Ile
35 40 45
Tyr?Glu?Met?Leu?Gln?Asn?Ile?Phe?Ala?Ile?Phe?Arg?Gln?Asp?Ser?Ser
50 55 60
Ser?Thr?Gly?Trp?Asn?Glu?Thr?Ile?Val?Glu?Asn?Leu?Leu?Ala?Asn?Val
65 70 75 80
Tyr?His?Gln?Ile?Asn?His?Leu?Lys?Thr?Val?Leu?Glu?Glu?Lys?Leu?Glu
85 90 95
Lys?Glu?Asp?Phe?Thr?Arg?Gly?Lys?Leu?Met?Ser?Ser?Leu?His?Leu?Lys
100 105 110
Arg?Tyr?Tyr?Gly?Arg?Ile?Leu?His?Tyr?Leu?Lys?Ala?Lys?Glu?Tyr?Ser
115 120 125
His?Cys?Ala?Trp?Thr?Ile?Val?Arg?Val?Glu?Ile?Leu?Arg?Asn?Phe?Tyr
130 135 140
Phe?Ile?Asn?Arg?Leu?Thr?Gly?Tyr?Leu?Arg?Asn
145 150 155
<210>2
<211>20
<212>PRT
＜213〉artificial sequence
<400>2
Met?Glu?Thr?Asp?Thr?Leu?Leu?Leu?Trp?Val?Leu?Leu?Leu?Trp?Val?Pro
1 5 10 15
Gly?Ser?Arg?Gly
20
<210>3
<211>60
<212>DNA
＜213〉artificial sequence
<400>3
atggagactga?caccctcct?tctctgggtc?ctccttcttt?gggtcccagg?ttctcgagga 60
<210>4
<211>155
<212>PRT
＜213〉artificial sequence
<400>4
Ser?Ser?Asn?Phe?Gln?Ser?Gln?Lys?Leu?Leu?Trp?Gln?Leu?Asn?Gly?Arg
1 5 10 15
Leu?Glu?Tyr?Cys?Leu?Lys?Asp?Arg?Met?Asn?Phe?Asp?Ile?Pro?Glu?Glu
20 25 30
Ile?Lys?Gln?Leu?Gln?Gln?Phe?Gln?Lys?Glu?Asp?Ala?Ala?Leu?Thr?Ile
35 40 45
Tyr?Glu?Met?Leu?Gln?Asn?Ile?Phe?Ala?Ile?Phe?Arg?Gln?Asp?Ser?Ser
50 55 60
Ser?Thr?Gly?Trp?Asn?Glu?Thr?Ile?Val?Glu?Asn?Leu?Leu?Ala?Asn?Val
65 70 75 80
Tyr?His?Gln?Ile?Asn?His?Leu?Lys?Thr?Val?Leu?Glu?Glu?Lys?Leu?Glu
85 90 95
Lys?Glu?Asp?Phe?Thr?Arg?Gly?Lys?Leu?Met?Ser?Ser?Leu?His?Leu?Lys
100 105 110
Arg?Tyr?Tyr?Gly?Arg?Ile?Leu?His?Tyr?Leu?Lys?Ala?Lys?Glu?Tyr?Ser
115 120 125
His?Cys?Ala?Trp?Thr?Ile?Val?Arg?Val?Glu?Ile?Leu?Arg?Asn?Phe?Tyr
130 135 140
Phe?Ile?Asn?Arg?Leu?Thr?Gly?Tyr?Leu?Arg?Asn
145 150 155
<210>5
<211>465
<212>DNA
＜213〉artificial sequence
<400>5
tcttccaatt?ttcagtctca?gaagctcctg?tggcaattga?atgggaggct?tgaatattgc 60
ctcaaggaca?ggatgaactt?tgacatccct?gaggagatta?agcagctgca?gcagttccag 120
aaggaggacg?ccgcattgac?catctatgag?atgctccaga?acatctttgc?tattttcaga 180
caagattcat?ctagcactgg?ctggaatgag?actattgttg?agaacctcct?ggctaatgtc 240
tatcatcaga?taaaccatct?gaagacagtc?ctggaagaaa?aactggagaa?agaagatttc 300
accaggggaa?aactcatgag?cagtctgcac?ctgaaaagat?attatgggag?gattctgcat 360
tacctgaagg?ccaaggagta?cagtcactgt?gcctggacca?tagtcagagt?ggaaatccta 420
aggaactttt?acttcattaa?cagacttaca?ggttacctcc?gaaac 465
<210>6
<211>41
<212>DNA
＜213〉artificial sequence
<400>6
cgggtaccga?tgacgatgac?aagtcttcca?attttcagtc?t 41
<210>7
<211>32
<212>DNA
＜213〉artificial sequence
<400>7
gcaagctttc?attagtttcg?gaggtaacct?gt 32
<210>8
<211>480
<212>DNA
＜213〉artificial sequence
<400>8
gatgacgatg?acaagtcttc?caattttcag?tctcagaagc?tcctgtggca?attgaatggg 60
aggcttgaat?attgcctcaa?ggacaggatg?aactttgaca?tccctgagga?gattaagcag 120
ctgcagcagt?tccagaagga?ggacgccgca?ttgaccatct?atgagatgct?ccagaacatc 180
tttgctattt?tcagacaaga?ttcatctagc?actggctgga?atgagactat?tgttgagaac 240
ctcctggcta?atgtctatca?tcagataaac?catctgaaga?cagtcctgga?agaaaaactg 300
gagaaagaag?atttcaccag?gggaaaactc?atgagcagtc?tgcacctgaa?aagatattat 360
gggaggattc?tgcattacct?gaaggccaag?gagtacagtc?actgtgcctg?gaccatagtc 420
agagtggaaa?tcctaaggaa?cttttacttc?attaacagac?ttacaggtta?cctccgaaac 480
<210>9
<211>38
<212>DNA
＜213〉artificial sequence
<400>9
cgctcgagaa?aagatcttcc?aattttcagt?gtcagaag 38
<210>10
<211>31
<212>DNA
＜213〉artificial sequence
<400>10
cggcggccgc?tcattagttt?cggaggtaac?c 31
<210>11
<211>491
<212>DNA
＜213〉artificial sequence
<400>11
ctcgagaaaa?gatcttccaa?ttttcagtgt?cagaagctcc?tgtggcaatt?gaatgggagg 60
cttgaatatt?gcctcaagga?caggatgaac?tttgacatcc?ctgaggagat?taagcagctg 120
cagcagttcc?agaaggagga?cgccgcattg?accatctatg?agatgctcca?gaacatcttt 180
gctattttca?gacaagattc?atctagcact?ggctggaatg?agactattgt?tgagaacctc 240
ctggctaatg?tctatcatca?gataaaccat?ctgaagacag?tcctggaaga?aaaactggag 300
aaagaagatt?tcaccagggg?aaaactcatg?agcagtctgc?acctgaaaag?atattatggg 360
aggattctgc?attacctgaa?ggccaaggag?tacagtcact?gtgcctggac?catagtcaga 420
gtggaaatcc?taaggaactt?ttacttcatt?aacagactta?caggttacct?ccgaaactaa 480
tgagcggccg?c 491
<210>12
<211>543
<212>DNA
＜213〉artificial sequence
<400>12
aagcttatgg?agactgacac?cctccttctc?tgggtcctcc?ttctttgggt?cccaggttct 60
cgaggatctt?ccaattttca?gtgtcagaag?ctcctgtggc?aattgaatgg?gaggcttgaa 120
tattgcctca?aggacaggat?gaactttgac?atccctgagg?agattaagca?gctgcagcag 180
ttccagaagg?aggacgccgc?attgaccatc?tatgagatgc?tccagaacat?ctttgctatt 240
ttcagacaag?attcatctag?cactggctgg?aatgagacta?ttgttgagaa?cctcctggct 300
aatgtctatc?atcagataaa?ccatctgaag?acagtcctgg?aagaaaaact?ggagaaagaa 360
gatttcacca?ggggaaaact?catgagcagt?ctgcacctga?aaagatatta?tgggaggatt 420
ctgcattacc?tgaaggccaa?ggagtacagt?cactgtgcct?ggaccatagt?cagagtggaa 480
atcctaagga?acttttactt?cattaacaga?cttacaggtt?acctccgaaa?ctaatgagaa 540
ttc 543
Claims (8)
1. human beta interferon mutant protein (SEQ ID NO:1) that lacks 11 amino acids at N-terminal.
2. the mutant of deletion human interferon-described in the right 1, the halfcystine (Cys) that its aminoacid sequence is the 6th is replaceable to be Serine (Ser).
3. the 1st amino acids of the deletion human interferon-mutant protein described in the claim 1,2 is replaceable is methionine(Met) (Met) or glycine (Gly) or arginine (Arg).
4. can have methionine(Met) (Met) or glycine (Gly) or arginine (Arg) before the 1st amino acids of the deletion human interferon-mutant protein described in the claim 1,2.
5. the mutant of deletion human interferon-described in the claim 1,2,3 is applicable to the recombinant expressed and preparation of genetically engineered, and its recombinant expressed host comprises any of intestinal bacteria, mammalian cell, yeast cell, insect cell or plant, animal.
6. the interferon-of deletion human described in the claim 5 mutant recombinant expressed and preparation refers to that the expression vector of the corresponding cDNA sequence by containing its coding transforms corresponding host cell, through the process of expression screening, cultivation, extraction and purifying target protein.
7. the preferred CHO of the described mammalian cell of claim 5, the recombinant eukaryon expression vector that contains deletion human interferon-mutant gene sequence of structure contains the aminoacid sequence (SEQ ID NO:2) and the dna sequence dna (SEQ IDNO:3) thereof of signal peptide.
8. claim 1,2,3 described deletion human interferon-mutant can be used as a kind of medicine that is used for the treatment of multiple sclerosis or viral infection disease.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112980883A (en) * | 2021-03-03 | 2021-06-18 | 广州达博生物制品有限公司 | Human gamma-interferon virus vector and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101111519A (en) * | 2004-11-02 | 2008-01-23 | 慎英基 | Human interferon-beta mutein |
| CN101633698A (en) * | 2009-08-26 | 2010-01-27 | 北京精益泰翔技术发展有限公司 | Immune fusion protein and gene encoding same and application thereof |
-
2010
- 2010-05-26 CN CN2010101826595A patent/CN102260344A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101111519A (en) * | 2004-11-02 | 2008-01-23 | 慎英基 | Human interferon-beta mutein |
| CN101633698A (en) * | 2009-08-26 | 2010-01-27 | 北京精益泰翔技术发展有限公司 | Immune fusion protein and gene encoding same and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| 《国外医学免疫学分册》 20031230 郭琳 干扰素-beta治疗多发性硬化机制的研究进展 329-332 1-8 第26卷, 第6期 * |
| 《重庆理工大学学报（自然科学）》 20100430 刘日勇 等 重组人beta干扰素-1b的融合表达及制备 第41页右栏第4-5行、第1.3节 2、5-6、8 第24卷, 第4期 * |
| 刘日勇 等: "重组人β干扰素-1b的融合表达及制备", 《重庆理工大学学报（自然科学）》, vol. 24, no. 4, 30 April 2010 (2010-04-30) * |
| 郭琳: "干扰素-β治疗多发性硬化机制的研究进展", 《国外医学免疫学分册》, vol. 26, no. 6, 30 December 2003 (2003-12-30), pages 329 - 332 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112980883A (en) * | 2021-03-03 | 2021-06-18 | 广州达博生物制品有限公司 | Human gamma-interferon virus vector and application thereof |
| CN112980883B (en) * | 2021-03-03 | 2022-04-12 | 广州达博生物制品有限公司 | Human gamma-interferon virus vector and application thereof |
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