CN103014048A - Preparation method and application of target protein - Google Patents
Preparation method and application of target protein Download PDFInfo
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- CN103014048A CN103014048A CN2012104652787A CN201210465278A CN103014048A CN 103014048 A CN103014048 A CN 103014048A CN 2012104652787 A CN2012104652787 A CN 2012104652787A CN 201210465278 A CN201210465278 A CN 201210465278A CN 103014048 A CN103014048 A CN 103014048A
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Abstract
The invention relates to a preparation and application of target protein. The preparation method comprises the following steps: obtaining a nucleotide sequence fragment of the target protein; constructing a recombinant expression vector of the target protein; inductively expressing and purifying the target protein; cleaving; introducing a single cleavage site to the downstream of the nucleotide sequence fragment of the target protein; synthesizing a coding strand and a template strand; and hybridizing the coding strand and the template strand, thus obtaining the target protein, wherein a recognition site for monomer cleavage is introduced while synthesizing the coding strand and the template strand; and preferably, the target protein can be a human nerve growth factor, thymosin, Huwentoxin-XI and Huwena analgesic peptide. The target protein prepared by the preparation method provided by the invention is high in expression quantity and easy to purify; the gene expression product is the target protein monomer and brings no extra amino acid residue, and thus the high biological activity of the target protein is ensured, and the multi-copy cleavage cost is saved; and the target protein is simple to purify and easy for mass production.
Description
Technical field
The present invention relates to genetic engineering technique, particularly a kind of target protein preparation method and its usage.
Background technology
Along with the development of genetic engineering technique, some have bioactive micromolecule polypeptide such as Zadaxin etc. and are paid much attention to.Zadaxin is a kind of proteins and peptides hormone that is produced by thymus gland.As far back as the sixties in 20th century, the people such as Miller (Lancet, 1961,2:748-749.) just find that Zadaxin plays an important role to immunity system, and the polypeptide mixture that therefrom to be separated to one group of molecular weight be 1000-15000D, be called Zadaxin component 5(thymosin fraction 5, TF5); People (the Proc. Natl. Acad. Sci. USA such as Goldstein subsequently, 1966,56:1010 – 1017) TF5 from ox thymus gland extract isolates a kind of Acid polypeptide α 1 component (thymosin α 1, T α 1), T α 1 is the main active component of TF5, unusually conservative in Mammals, be distributed widely in mammiferous thymus gland, spleen, lung, kidney, brain, blood and its hetero-organization, concentration is the highest in thymus gland.
At present, the methods that prepare most employing chemosynthesis or biological tissue extracted of active polypeptide, no exception as the Zadaxin of active polypeptide.The Thymic preparation of the various modification methods of the usefulness of widespread use preparation is the mixture of the multiple peptide class extracted from animal tissues mostly clinically; the activity of the Thymic preparation that different company produces differs greatly; and have because production technique or quality problems make albumen such as containing ox or pig in the product, can produce anaphylaxis during injection.Therefore, the someone adopts people's embryonic thymus as raw material, prepares human thymosin alfa 1 component (thymosin α 1, T α 1), and curative effect is pretty good.But because people's embryonic origin difficulty is expensive, and T α 1 content seldom.Along with the continuous enforcement of the Human Genome Project, the aminoacid sequence of Zadaxin order can obtain easily, makes the active polypeptide of the similar Zadaxin of chemosynthesis become possibility.Domestic and international a plurality of study group, such as Wang S S etc., Huang attain brightness, glycosides just like etc., (Int J Pept Res, 1992,40 (3-4): 344 such as Cheng Hu; Pharmaceutical Biotechnology, 2001,8 (4): 207; Journal of Chemical Industry and Engineering, 2004,55 (2): 305; Nanjing University of Technology's journal (natural science edition), 2004,26 (2): 78) adopt different solid phase methods and suitable technology to carry out the synthetic and process optimization of T α 1, but the difficult problem that chemical synthesis process can't resolve all the time the cost height, yields poorly, purity difference is low with activity.
In order to obtain quality height, eutherapeutic Thymic preparation, scientist begins to explore the using gene engineering method and prepares Zadaxin, and has caused the broad research for T α 1
[1,2,7]Compare with chemical synthesis process or tissue extraction method, gene engineering method is reducing cost, raising output, minimizing toxic by-products or is recovering to have certain advantage and improvement aspect the biological activity for the production of T α 1 goods, but, because amalgamation and expression makes purpose peptide proportion in fusion rotein little, cause the waste of host expresses potential, reduce fusion rotein stability and reduce label protein length.Therefore, no matter be single expression or form with label protein and to merge the phraseology of improving cytotoxic effect, all can not fundamentally solve production problems, be difficult to further promote as the Technology of scale operation.
Above-mentioned single copy or the existing problem of amalgamation and expression, polypeptide gene can be carried out tandem expression and solved: (1) series connection has increased polypeptide gene quantity, is conducive to improve expression amount; (2) expression of series connection polymer form can effectively shield host toxicity; (3) the polymer expression product is more stable.Therefore, adopt the polymeric method of structure to have outstanding advantage for improving expression amount to T α 1 polypeptide.
So far, the polymer of multiple peptide, the multiple copied form expression that succeeded
[8-11]Mention the directed multiple copied clone preparation scheme of T α 1 in domestic two pieces of reports that minority only arranged, one adopts gene fragment is annealed at random and round pcr combines method to make up the T α 1 series connection construct of 3 copies
[28]Its two employings expression cassette series process has made up the construct that contains 1-8 copy T α 1 expressed intact box
[29]In first method, although traditional PCR method is simple and easy to do, the author reckons without the series connection construct and expresses monomer cutting problem afterwards, has only inquired into merely the feasibility of the method from technological method.Although second method can obtain the series connection construct of higher copy, each expression cassette independence express polypeptide product and evaded follow-up polypeptide cutting step, but T α 1 monomer expression product molecular weight is too little, is degraded easily and is difficult to realize the lifting of expression amount.
Therefore, need at present a kind of structure to efficiently express the gene engineering method of Zadaxin goods.
Summary of the invention
The technical problem to be solved in the present invention is: the gene engineering method that a kind of high efficiency expressing destination protein is provided.
For achieving the above object, the invention provides following technical scheme, may further comprise the steps:
The acquisition of the nucleotide sequence fragment of target protein;
The structure of target protein recombinant expression vector;
The abduction delivering of target protein;
The purifying of target protein;
The cutting of purified product;
Single cleavage site is introduced in the downstream of the nucleotide sequence fragment of described target protein;
Described target protein is hybridized acquisition by composite coding chain and template strand with both, introduces the recognition site of monomer cutting when composite coding chain and template strand;
Preferably, described target protein is growth factor of human nerve, Zadaxin, brave line gram pancreas peptide, Huwenatoxin-I.
Behind cutting step, can also comprise the step that is further purified, be preferred for and take the HPLC purifying.
Can also comprise the target protein determination of activity.
Described target protein gene is one or more copy; Preferably, the multiple copied number of described target protein gene is realized by the target protein gene fragment is carried out external being connected in series.
Described single cleavage site is the aminoacid sequence according to the codon rule encoding; Preferably, described aminoacid sequence is R or W or M or D or E or K.Described English capitalization is amino acid whose code, represents arginine such as R, and W represents tryptophane, and M represents methionine(Met), and D represents aspartic acid, and E represents L-glutamic acid, and K represents Methionin, observes the password sublist of 20 seed amino acids.
The acquisition of the nucleotide sequence fragment of described target protein gene is synthetic target protein gene template chain and coding strand; Through the sex change anneal coding strand and template strand hybridization are obtained heteroduplex, heteroduplex has in reaction system one by one that orientation is connected to form polymeric feature, and described polymer contains 2 and above copy number target protein gene fragment;
Preferably, can also comprise that the polymer that will form carries out the acquisition of more target protein gene copy numbers as the template of polymerase chain reaction.
Described expression vector is prokaryotic expression system, is preferably pET system, pGEX system, pMAL system;
The purifying of described target protein is preferably for adopting affinity column to carry out purifying, and the label of affinity column is His label, GST label, MBP label.
The cutting of described purified product can be chemical reagent or enzyme cutting, preferably BrCN cutting.Concrete steps can be to add the rearmounted 4 ℃ of refrigerators of BrCN solution in the protein behind purifying to process 24 hours, add the two H of steaming of equal-volume
2The O lyophilize.
The present invention also protects the target protein goods by the method gained.
The present invention also protects the purposes that is used for medicine by the target protein goods of the method gained.
The present invention take Zadaxin as example illustrates method of the present invention.
Method provided by the invention has following design:
1, obtain the gene coding region base sequence of coding target protein according to genome database, and the synthetic template strand of commercialization and coding strand oligonucleotide chain;
2, synthetic oligonucleotide chain has following feature: single cleavage sequences is all introduced in the downstream of 3 ' end downstream sequence of coding strand and template strand, and described single cleavage sequences has following feature: the aminoacid sequence according to the codon rule encoding can be by chemical reagent or the single cutting of enzyme.Preferably, described single cleavage sequences coding single amino acids, i.e. R or W or M or D or E or K;
3, described single cleavage sequences by the cutting of chemical reagent and enzyme after, target protein carboxy terminal and downstream all produce a unnecessary amino acid, definitely, this amino acid is R or W or M or D or E or K;
4, described coding strand and template strand obtain heteroduplex through after the sex change anneal, and described heteroduplex has following feature: double-stranded 5 ' end all has the sticky end of 3 bases, and sticky end has base complement, and complementarity has again directional property;
5, described directional property refers to the complementary pairing that the double-stranded complementary sticky end of linear DNA occurs and only 5 ' end sticky end and 3 ' is held sticky end;
6, described heteroduplex has in reaction system one by one that orientation is connected to form polymeric feature, and described polymer contains 2 and above copy number target protein gene fragment.The mixture that described polymer forms can be used as the template of polymerase chain reaction (polymerase chain reaction, PCR);
7, described template can be by the primer amplification designed according to the polypeptide of interest gene fragment, and described amplified production contains the directed connector element of target protein gene fragment of high copy;
8, order-checking was identified and is obtained copy number construct interested after dna gel reclaimed described high copy connector element and carries out the TA clone;
9, described construct subclone forms expression construct to expression vector.Can be prokaryotic expression carrier such as expression vector, described construct can carry out prokaryotic expression in prokaryotic expression system, and described prokaryotic expression system comprises pET system, pGEX system, pMAL system etc., does not get rid of other prokaryotic expression systems;
10, the product behind the prokaryotic expression can be by the affinity tag affinity purification in the prokaryotic expression system in described prokaryotic expression system for described prokaryotic expression construct, and described affinity tag comprises His label, GST label, MBP label etc., does not get rid of other affinity tag;
11, described prokaryotic expression product can by chemical reagent or enzyme cutting, form the mixture that contains the target protein monomer behind affinity purification;
12, described target protein monomer mixture carries out degree of depth purifying through high performance liquid chromatography (HPLC) method;
13, the determination of activity of the target protein monomeric products behind the described purifying.
One embodiment of the present of invention provide the step for preparing the Zadaxin goods with method of the present invention, and compared with prior art, preparation method of the present invention is simple and efficiently express Zadaxin.
One embodiment of the present of invention have proved that the Zadaxin monomeric products that the method obtains has remarkable promotion kunming mice proliferation of splenocytes;
One of purpose of the present invention is the directed multiple copied clone who makes up target protein, and then realizes efficient, the high yield preparation of T α 1.Two of purpose of the present invention is to set up the method that the directed multiple copied clone of a kind of target protein gene makes up.
In the present invention, term " orientation " refers to that the target protein gene DNA fragment interconnects according to coding strand 5 '-3 ' direction, also refers to express the target protein product and interconnects by aminoterminal-carboxyl terminal direction.
In the present invention, term " multiple copied " refers to that the target protein gene DNA fragment is more than or equal to two copy numbers.
Embodiment
Below in conjunction with embodiment embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only is used for explanation the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person among the embodiment carry out according to the described technology of the document in this area or condition or according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
Following target protein be take Zadaxin as example describes method of the present invention, other target protein is similar.
The sequence that relates in following examples is synthetic, order-checking is finished by the Invitrogen bio tech ltd.
The directed multiple copied construct of embodiment 1:T α 1 gene makes up
1, the synthetic T α 1 gene oligonucleotide fragment of commercialization:
Coding strand: T α 1-F:5 '-AGCGATGCCGCCGTGGATACCAGCAGCGAAATTACCACCAAAGATCTGAAAGAAAA AAAAGAAGTGGTGGAAGAAGCCGAAAAC
ATG-3 ' (SEQ ID NO:1)
Template strand: T α 1-R:5 '-GTTTTCGGCTTCTTCCACCACTTCTTTTTTTTCTTTCAGATCTTTGGTGGTAATTT CGCTGCTGGTATCCACGGCGGCATCGCT
CAT-3 ' (SEQ ID NO:2)
The single restriction enzyme site of coding strand 3 ' terminal ATG trinucleotide (see underscore indicate) for introducing wherein, coding methionine(Met) (M) can be by chemical reagent cyanogen bromic acid (BrCN) in the single incision in the carboxyl terminal site of methionine(Met).Template strand CAT trinucleotide (see underscore indicate) is the reverse complementary sequence of described ATG trinucleotide, and described complementary sequence can obtain the two copies concatermer of T α 1 encoding gene through external ligation.
2, the external connection of T α 1 gene fragment
The described coding strand of step 1 and template strand after processing, T4 polynueleotide kinase (TAKARA) are carried out hybridization in hybridization solution (50mM NaCl, 10mM Tris-Cl, 1mM EDTA).Gained hybridization product carries out ligation with T4 ligase enzyme (TAKARA) and obtains connecting product.
3, T α 1 gene fragment multiple copied directed cloning
The resulting connection product of step 2 is through following primer: T-F:5 '-ATGAGCGATGCCGCCGCCGT-3 ' (SEQ ID NO:3)
T-R：5’-CTAGTTTTCGGCTTCTTCCACC-3’ （SEQ ID NO:4）
(TAKARA) increases with the Pyrobest archaeal dna polymerase, the PCR reaction system is: 10 * PCR damping fluid, 5 μ L, dNTP4 μ L, TaqDNA polysaccharase 0.25 μ L, each 5 μ L of T-F and T-R (10 μ mol/L), template (the connection product of step 2) 1 μ L adds distilled water to 50 μ L.The PCR response procedures: 94 ℃ of denaturation 5min of elder generation, 94 ℃ of sex change 30s that then carry out 35 circulations, 60 ℃ of annealing 30s, 72 ℃ are extended 30s; After the loop ends again 72 ℃ keep 5min, cool off 4 ℃.Reclaim amplified production fragment and and the pMD19-T(TAKARA of multiple copied) carry out the TA clone operations.Bacterium colony PCR mode screening positive clone, screening obtains to contain the construct of 1,2,4,5,7 copies.Described construct subclone is to prokaryotic expression carrier pET-28b(+) (Merck), difference called after pET-28b-T1, pET-28b-T2, pET-28b-T4, pET-28b-T5 and pET-28b-T7.The construct order-checking is identified correct.
Embodiment 2:T α 1 gene multiple copied directed cloning construct prokaryotic expression and purifying
1, T α 1 gene multiple copied directed cloning construct prokaryotic expression
Described pET-28b-T2, pET-28b-T4, pET-28b-T5 and pET-28b-T7 construct transform BL21(DE3) competent cell, the single bacterium colony of picking is containing 37 ℃ of overnight incubation on the 5mL LB substratum of 50mg/mL kantlex respectively, culture 1 ︰ 100 enlarged culturing to the OD value of 600nm wavelength is 0.6 ~ 0.8, add respectively 0.5mM IPTG abduction delivering, shaking table was cultivated 4 hours, centrifugal collection thalline, centrifugal acquisition total bacterial protein solution behind the ultrasonic degradation;
2, the preliminary purification of prokaryotic expression product
Described total bacterial protein solution progressively elutes in connection with the target protein on post as elutriant with imidazoles behind the flush away foreign protein after affinity column (Ni2+-charged IDA his-bind column, Merck) combination;
3, the cutting of purified product
The product of described preliminary purification is measured protein content with BCA method (Priece) method, and every gram protein adds 1mL BrCN solution (50mg/mL, the preparation of 70% formic acid) and puts 4 ℃ of refrigerators processing 24 hours, adds the two H of steaming of equal-volume
2The O lyophilize obtains T α 1 polypeptide monomer product mixtures except formic acid removal and BrCN;
4, the degree of depth purifying of cleaved products
Described T α 1 polypeptide monomer cleaved products mixture is further purified through HPLC.
The research of embodiment 3:T α 1 polypeptide monomer its lytic activity
Carry out T α 1 polypeptide monomer product to the increment experiment of mouse boosting cell (being purchased from the true Bioisystech Co., Ltd in Shanghai) with conventional mtt assay.The result: T α 1 polypeptide monomer product has significant promotion kunming mice proliferation of splenocytes.
SEQUENCE LISTING
＜110〉Xiamen Beidazhilu Biological Engineering Co., Ltd
＜120〉a kind of target protein preparation method and its usage
<130> P6976
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 87
<212> DNA
＜213〉synthetic
<400> 1
agcgatgccg ccgtggatac cagcagcgaa attaccacca aagatctgaa agaaaaaaaa 60
gaagtggtgg aagaagccga aaacatg 87
<210> 2
<211> 87
<212> DNA
＜213〉synthetic
<400> 2
gttttcggct tcttccacca cttctttttt ttctttcaga tctttggtgg taatttcgct 60
gctggtatcc acggcggcat cgctcat 87
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<212> DNA
＜213〉synthetic
<400> 3
atgagcgatg ccgccgccgt 20
<210> 4
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<212> DNA
＜213〉synthetic
<400> 4
ctagttttcg gcttcttcca cc 22
Claims (10)
1. the preparation method of a target protein is characterized in that: comprises the steps,
The acquisition of the nucleotide sequence fragment of target protein;
The structure of target protein recombinant expression vector;
The abduction delivering of target protein;
The purifying of target protein;
The cutting of purified product;
Single cleavage site is introduced in the downstream of the nucleotide sequence fragment of described target protein;
Described target protein is hybridized acquisition by composite coding chain and template strand with both, introduces the recognition site of monomer cutting when composite coding chain and template strand;
Preferably, described target protein is growth factor of human nerve, Zadaxin, brave line gram pancreas peptide, Huwenatoxin-I.
2. the construction process of claim 1 is characterized in that, also comprises the step that is further purified behind cutting step;
Randomly, also comprise the target protein determination of activity.
3. claim 1 or 2 construction process is characterized in that, described target protein gene is copy more than 1 or 1; Preferably, 1 of described target protein gene above copy number is realized by the target protein gene fragment is carried out external being connected in series.
4. claim 1 or 2 construction process is characterized in that described single cleavage site is the aminoacid sequence according to the codon rule encoding; Preferably, described aminoacid sequence is R or W or M or D or E or K.
5. claim 1 or 2 construction process is characterized in that, the acquisition of the nucleotide sequence fragment of described target protein gene is synthetic target protein gene template chain and coding strand; Through the sex change anneal coding strand and template strand hybridization are obtained heteroduplex, heteroduplex has in reaction system one by one that orientation is connected to form polymeric feature, and described polymer contains 2 and above copy number target protein gene fragment;
Preferably, can also comprise that the polymer that will form carries out the acquisition of more target protein gene copy numbers as the template of polymerase chain reaction.
6. claim 1 or 2 construction process is characterized in that described expression vector is prokaryotic expression system, are preferably pET system, pGEX system, pMAL system;
Randomly, the purifying of described target protein is preferably for adopting affinity column to carry out purifying, and the label of affinity column is His label, GST label, MBP label.
7. claim 1 or 2 construction process is characterized in that, the cutting of described purified product can be chemical reagent or enzyme cutting, preferably BrCN cutting.
8. the construction process of claim 2 is characterized in that, described being further purified as taking the HPLC purifying.
9. the target protein of claim 1-8 either method gained.
10. the target protein of claim 9 is used for the purposes of medicine.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103614377A (en) * | 2013-10-28 | 2014-03-05 | 华大基因杨凌创新研究院有限公司 | Promoter OsP001, and preparation method and application thereof |
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| CN101041818A (en) * | 2006-03-24 | 2007-09-26 | 中国人民解放军第三军医大学第一附属医院 | Method for recombinant production of pancreatic glucagons sample peptide-2 |
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2012
- 2012-11-19 CN CN2012104652787A patent/CN103014048A/en active Pending
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| CN1273248A (en) * | 1999-11-24 | 2000-11-15 | 刘建宁 | Efficient gene engineering process for preparing polypeptide medicines |
| CN1307900A (en) * | 2000-04-11 | 2001-08-15 | 厦门北大之路生物工程有限公司 | Application of tigroid spider toxic extract in preparing analgesic |
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Non-Patent Citations (1)
| Title |
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| LIANG ZHOU ET AL: "Expression and Hydroxylamine Cleavage of Thymosin Alpha 1 Concatemer", 《JOURNAL OF BIOMEDICINE AND BIOTECHNOLOGY》, 31 December 2008 (2008-12-31), pages 1 - 8 * |
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| CN103614377A (en) * | 2013-10-28 | 2014-03-05 | 华大基因杨凌创新研究院有限公司 | Promoter OsP001, and preparation method and application thereof |
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Application publication date: 20130403 |