CN103214561A - Human hepatitis c virus core antigen and preparation method and application thereof - Google Patents
Human hepatitis c virus core antigen and preparation method and application thereof Download PDFInfo
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- CN103214561A CN103214561A CN2013100788782A CN201310078878A CN103214561A CN 103214561 A CN103214561 A CN 103214561A CN 2013100788782 A CN2013100788782 A CN 2013100788782A CN 201310078878 A CN201310078878 A CN 201310078878A CN 103214561 A CN103214561 A CN 103214561A
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Abstract
The invention discloses a human hepatitis c virus core antigen, and the antigen protein can be applied to generation of a monoclonal antibody by immunization aiming at the human hepatitis c virus core antigen. The invention employs a gene engineering method for developing a recombinant fusion protein soluble antigen, which is characterized in that the antigen has a relatively complete natural protein space structure for generating the anti-core antigen antibody by immunization. The invention has the characteristics of linear synthetic peptide antigen, high antibody specificity, better specificity and endophilicity of the core antigen in a natural sample.
Description
Technical field
The invention belongs to medicine and gene engineering technology field, be specifically related to viruses of human hepatitis C's cAg and its production and application.
Background technology
Hepatitis C is to infect the transmissible disease that causes by viruses of human hepatitis C (HCV, Hepatitis C Virus), is widely current in the whole world, and especially China is one of popular more serious country of HCV, and the crowd infection rate is about 3.2%, and the infected more than 4,000 ten thousand is arranged.It is a kind of very important route of transmission that haematogenous is propagated, owing to do not have effective preventative vaccine and effective treatment means, so early diagnosis and blood screening have a very important role to the propagation of control HCV.
The diagnosis of present HCV clinically and examination are to detect HCV antibody, because generally there is the window phase in 7-10 week in the HCV antibody test, set up early stage, easy, responsive, special HCV cAg diagnostic reagent, shortening the window phase that detects is to reduce the effective way that HCV infects.Though RT-PCR can qualitatively and quantitative detect HCV RNA and effectively shorten window phase, because of the RT-PCR complicated operation, pollute easily and cost an arm and a leg, making it can not widespread use.Research and development are based on the monoclonal antibody of hepatitis C cAg, for the early detection infection with hepatitis C virus, by double-antibody sandwich elisa screening energy high-sensitivity detection hepatitis C cAg, set up hepatitis C cAg detection system and combination detection serum antibody to hepatitis C, use for the antigen-antibody joint inspection and lay foundation.
Though the high conservative of hepatitis C cAg also has very strong immunogenicity concurrently, but hepatitis C cAg protein requirement host signal peptase shears that thereby signal peptide sequence is processed to form sophisticated cAg albumen (p21) in its C end, thereby causes to go out structure by genetically engineered especially prokaryotic expression and become difficult unusually as natural hepatitis C cAg albumen.CAg albumen very easily combines with nucleic acid and fat egg in addition, and its C end has very strong hydrophobicity, also all is that proteic vivoexpression of cAg and purifying have increased difficulty.In recent years some researchists white (191aa of cAg of total length that utilized yeast and mammalian cell successful expression, p23), and find that p23 can be sheared the sophisticated p21 albumen of formation by the signal peptidase of cell, and can external self-assembly form the about 35nm of diameter nucleocapsid sample particle (nucleocapside1ike particles, NLPs).There is the investigator to find to utilize E.coli to express hepatitis C cAg N and holds preceding 120 amino acid, also can form virus-like particle (VLPs, Virus Like Particles) under certain condition.And with producing strong and persistent body fluid and cellullar immunologic response behind this VLPs immune mouse, rabbit and the goat.Because on method and immature, so Many researchers selects with intestinal bacteria system expression cAg albumen by eucaryon approach great expression and purifying HCV core recombinant antigen, and avoids the amino acid of cAg PROTEIN C end signal peptide moiety.
Summary of the invention
In view of the deficiencies in the prior art, the present invention passes through gene recombination technology, segmentation clone HCV cAg gene, express the also gst fusion protein antigen of separation and purification HCV cAg, this antigen is used to develop the HCV diagnostic kit, especially in the immunity stage, be used for immunity and produce anti-cAg antibody, be used for the detection of the HCV cAg of early stage HCV infection with better avidity and evident characteristics.Therefore, the object of the present invention is to provide genetic engineering fusion protein antigen of a kind of core antigen of C type hepatitis virus and its production and application, the antigenic dna molecular of this fusion rotein of encoding, comprise the expression vector of this dna molecular, and the prokaryotic host cell that is transformed by this expression vector.
The object of the present invention is achieved like this:
A kind of viruses of human hepatitis C's cAg, it is polypeptide or protein molecular, the aminoacid sequence of described polypeptide or protein molecular is shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, or this sequence is suddenlyd change and the conservative property varient of acquisition through the conservative property of one or more aminoacid addition, deletion, replacement, modification.
A kind of dna molecular, above-mentioned polypeptide or the protein molecular of its coding.
In a preferred embodiment of the present invention, the nucleotide sequence of described polypeptide of above-mentioned dna molecule encode or protein molecular is shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10.
A kind of expression vector, the expression regulation sequence that includes above-mentioned dna molecular and link to each other with this dna molecular operability.
A kind of prokaryotic host cell, it is transformed by above-mentioned expression vector.Preferably, described prokaryotic host cell is intestinal bacteria.Further preferably, described prokaryotic host cell is an e. coli bl21.
Evidence, above-mentioned viruses of human hepatitis C's cAg can be used to prepare reagent or the medicine of diagnosing hepatitis C virus.
A kind of method for preparing above-mentioned viruses of human hepatitis C's cAg comprises the steps:
(1) provides an expression vector, the expression regulation sequence that this expression vector contains the described dna molecular of claim 2 and links to each other with this dna molecular operability;
(2) transform prokaryotic host cell with the described expression vector of step (1);
(3) prokaryotic host cell of culturing step (2) gained under the condition that is fit to described polypeptide or protein molecular antigen presentation;
(4) separation and purification obtains described polypeptide or protein molecular antigen.
Above-mentioned preparation method, wherein said expression vector is a fusion expression vector, the fusion rotein antigen of expressing has correct folding structure, be present in the kytoplasm with the target protein form of solubility, and target protein can obtain purifying protein by two step affinity chromatographies.
The present invention adopts a kind of brand-new partitioned representation mode, and employing and GST albumen fusion acquisition expression in intestinal bacteria, and this expression is a soluble proteins, and its protein structure is with near the natural folding mode, thereby for the fundamental research of HCV and diagnostic reagent development and use and lay the foundation.
Compared with prior art, viruses of human hepatitis C's cAg that the present invention relates to and preparation method thereof has following advantage and obvious improvement:
(1) segmentation and the full-length molecule cloning process of employing cAg gene, one group of genetic engineering fusion protein antigen of acquisition;
(2) adopt GST amalgamation and expression technology, the fusion rotein antigen of acquisition has solubility expression, and its constitutional features is more near the characteristics of native protein.
Description of drawings
Fig. 1 is core antigen of C type hepatitis virus gene coded sequence and primer combinations of pairs synoptic diagram.
Fig. 2 is a PCR product cloning synoptic diagram.
Fig. 3 is the structure synoptic diagram of fusion expression vector pET41-HCVCore191.
Embodiment
The genetic engineering fusion protein that the present invention relates to is antigenic preparation method comprise the steps:
1, be template with the plasmid pM-HCV_Genomeo that contains HCV full-length gene group DNA, and use at each fragment design primer of cAg gene, carry out pcr amplification, amplified production reclaims corresponding specific amplified gene product through electrophoretic separation and glue, and subclone is to pMD-18 (TaKRa, Inc) in the carrier, obtain each gene recombination plasmid respectively.
2, She Ji primer is as shown in table 1
Each section antigen primer sequence design table of table 1. hepatitis C cAg gene
A) adopt primer 1,3 amplifications to obtain PCR product 1, clone T carrier gets pMD-H1;
B) adopt primer 2,4 amplifications to obtain PCR PCR product 2, the clone obtains pMD-H2;
C) adopt " huge primer two is taken turns PCR Chinese patent (ZL95113311.x) " technology, with the plasmid pM-HCV_Genomeo that contains HCV full-length gene group DNA is template, with primer 1+ primer 6, the cAg encoding gene that primer 5+ primer 2 increases respectively, gained two fragment PCR products are after running gel reclaims, take turns the template that PCR reacts by equimolecular than mixing as second, adopt the combination of primer 1+ primer 2 to proceed second and take turns PCR, with amplification total length cAg gene coded sequence, the total length encoding gene in the inner Xho I site that obtains to have suddenlyd change.This PCR product 3 is used to make up pMD-H3;
D) primer 1,3 amplifications obtain PCR product 4, are used to make up pMD-H4, referring to Fig. 1.
3, pMD-H1-4 is through the dna sequencing checking.
4, by the restriction endonuclease sites that designs, double digestion obtains corresponding gene fragment from above-mentioned pMD-H1, pMD-H2, pMD-H3, pMD-H4, each gene pET41a that adopts gene subclone technology to obtain connects, make up corresponding HCV cAg expression vector, acquisition has the expression vector of aforementioned Seq.6-10 encoding sequence, through after the Screening and Identification, recombinant plasmid transformed escherichia coli expression host BL21 bacterium, and carry out the abduction delivering analysis, set up the fusion rotein antigen engineering bacteria of expression of HCV cAg, be used to prepare recombination fusion protein antigen.
5, express engineering bacteria by fermentation, collect fermentation thalline, broken bacterium, separation, two step affinity chromatographys obtain purified fusion protein antigen.
Form is described in further detail foregoing of the present invention again by the following examples, but this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following embodiment, all technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
The acquisition and the evaluation of each section encoding gene of embodiment 1 core antigen of C type hepatitis virus
1, the fragment amplification of antigen target gene:
It is template that employing contains the full geneome plasmid of people HCV1b type, adopts different pairing combination of primers, and uses PrimStar (available from the TAKARA Inc.) gene of high-fidelity to carry out pcr amplification to obtain each genes encoding fragment.Condition is as follows:
50 μ lPCR amplification reaction systems:
The pcr amplification reaction condition:
Combination of primers wherein:
(1) primer 1+ primer 3: amplified production is the sequence that contains preceding 120 amino-acid residues of coding core protein at inner Xho I point of contact, and product name Seq1 is shown in SEQ ID NO:5;
(2) primer 4+ primer 2: amplified production is the encoding sequence of 70 amino-acid residues of core protein C end, and product name Seq2 is shown in SEQ ID NO:6;
(3) primer 1+ primer 6, two sections sequences of the cAg encoding gene that primer 5+ primer 2 increases respectively, product after running gel reclaims as template, proceed second and take turns PCR, adopt the combination of primer 1+ primer 2 with amplification total length cAg gene coded sequence, the total length encoding gene in the inner Xho I site that obtains to have suddenlyd change.Method is provided by the technology that is provided in the Chinese patent (ZL95113311.x).Product called after Seq3 is shown in SEQ ID NO:7;
(4) primer 1+ primer 3: with the template of product S eq3 as gene amplification, amplification obtains product name Seq4, shown in SEQ ID NO:8.
2, the electrophoretic separation of PCR product and purifying:
With 4 kinds of pcr amplification products, 1.2% agarose gel electrophoresis (10V/Cm glue) separated the goal gene band 20 minutes, and running gel cuts under gel imaging system and contains the target gene fragment gel strips; Adopt dna gel to reclaim test kit (Solarbio Inc) by specification and carry out purifying:
(1) press 1:3 weight/long-pending body than adding sol solutions, place 55 degree water-baths 10 minutes, during put upside down and mix for several times to guarantee abundant colloidal sol;
(2) add 7:10 and therefrom be added to adsorption column behind the Virahol mixing of volume ratio, 8000rpm, centrifugal 1 minute of 4 degree will be collected in the post liquid and be added in the post absorption more once, 8000rpm, 4 spend centrifugal again 1 minute, abandon or adopt waste liquid;
(3) in adsorption column, add the 0.5ml rinsing liquid, 12000rpm, centrifugal 30 seconds of 4 degree come again, and discard to collect waste liquid in the post, and adsorption column is with 12000rpm, and 4 spend centrifugal 3 minutes;
(4) adsorption column is transferred to a new collection tube, added 30 microlitre sterilized waters (or 10mMTris-EDTA solution, pH〉8.0 be good) to adsorption column central authorities, be positioned over 60-70 degree baking box interior 5 minutes, take out back 12000rpm, centrifugal 2 minutes of 4 degree, collecting in the post is the dna fragmentation of purifying;
(5) with after the Nano-1000 micro-spectrophotometer mensuration DNA concentration, place in-20 degree refrigerators standby.
3, the PCR product cloning is to the pMD-18T carrier
The PCR product of purifying illustrates quantitatively and is connected in the pMD-18T carrier by TaKaRa company's T carrier, obtains corresponding 4 kinds of recombinant plasmids, names pMD-H1, pMD-H2, pMD-H3, pMD-H4 respectively.
(1) ligation
The composition of ligation is:
pMD-18T 0.5ul
PCR(>100ng) 4.5ul
Sol I(T4Ligase,Buffer) 5.0ul
Behind the mixing, put 16 degree water-baths 3 hours, carry out ligation;
(2) colibacillary conversion
Adopt chemoreception bacterium (DH5a, available from Solarbio Inc) transform: get 3 microlitre ligation liquid and be added in the 50 microlitre competence bacterias, placed 30 minutes on ice, after transferring to 42 degree water-baths carrying out thermal shocking in 90 seconds, transfer to and put 5 fens on ice, the LB substratum 1ml that adds no antiauxin, transferring to 37 degree shaking tables shook 45 fens slowly, 5000rpm, centrifugal 10 minutes of 4 degree discard unnecessary substratum, and the bacterium that stays about 200 microlitres is added to the LB agar plate that contains penbritin (100mg/ml) behind the pressure-vaccum number gently, evenly be applied to driedly, place 37 degree incubator overnight incubation.
(3) Screening and Identification of recombinant clone
A) the bacterium colony PCR of recombinant clone screening: separate good single bacterium colony from transforming dull and stereotyped 10 of the pickings of going up, be seeded in 10 PCR pipes that are added with sterilized water and carry out abundant mixing, drawing the adding of 5 microlitres respectively has among the 1ml LB that contains penbritin, makes the corresponding numbers centrifuge tube and is used for 37 degree shaking tables cultivations; 5 microlitres in other 10 PCR pipes will be used as the template of bacterium colony PCR, carry out the PCR reaction;
Cumulative volume 10 microlitres.
The PCR response procedures:
When screening, the extension time can be adjusted according to expansion target gene fragment length, and cycle index also can arrange to adjust by the time; After finishing, PCR carried out 1.2% agarose gel electrophoresis (10V/Cm glue) 20 minutes, gel imaging analysis as a result, and amplifying gene product and size and be than the bigger 100bp person of target gene fragment is positive clone; According to the numbering of positive colony bacterium liquid in the corresponding centrifuge tube is transferred to and is added with 5ml and contains to shake in the test tube of penbritin (100mg/ml) and spend the night.
B) put forward plasmid DNA for a short time: undertaken: collect and spend the night bacterium 1.5ml in 1.5 Eppendorf tubes by plasmid DNA a small amount of extraction agent box explanation (Solarbio Inc), the centrifugal 1 fractional precipitation bacterium of 10000rpm, abandon supernatant, add the 0.25ml solution I bacterial sediment that fully suspends, adding solution II 0.25ml topples over for several times gently with mixing, room temperature is put 2 minutes (solution should be limpid fully), add to put upside down rapidly after the 0.35ml solution III to mix and protein and genome are precipitated separate out, 12000rpm, centrifugal 10 minutes of 4 degree, with supernatant gently to adsorption column, 8000rpm, centrifugal 30 seconds of 4 degree, liquid in the collection tube is added in the adsorption column absorption once again, after liquid is carved in centrifugal removal, in adsorption column, add the 0.5ml rinsing liquid, 12000rpm, centrifugal 1 minute of 4 degree, repeat once, discard waste liquid in the collection tube, with adsorption column 12000rpm again, centrifugal 3 minutes of 4 degree, central authorities add 50 microlitre sterilized waters (or 10mM Tris-EDTA solution to adsorption column, pH〉8.0 be good), be positioned over 60-70 degree baking box interior 5 minutes, and took out back 12000rpm, centrifugal 2 minutes of 4 degree, collecting in the post is the DNA plasmid of purifying, after Nano-1000 micro-spectrophotometer mensuration DNA concentration, can carry out next step endonuclease reaction, redundance places in-20 degree refrigerators standby.
C) double digestion is identified recombinant plasmid: the double digestion reaction of recombinant plasmid (giving an example with pMD-H1) is composed as follows;
Reaction solution through mixing be placed on 37 the degree water-baths in 3 hours, carry out endonuclease reaction.After reaction finishes, carried out 1.2% agarose gel electrophoresis (10V/Cm glue) 20 minutes, gel imaging analysis as a result, can cut out gene product and size and be with the consistent person of target gene fragment is positive clone, the preservation plasmid; The bacterium that spends the night before this is a glycerol stock: after the centrifugal collection of 1.5 bacterium liquid, abandon the supernatant adding and do not contain the antibiotic 15-25% of containing glycerine, preferentially 20% LB substratum suspends afterwards as frozen glycerine pipe standby (freezing 2 pipes).
D) sequencing of recombinant plasmid: send order-checking company (as biotechnology company limited of U.S. season of Shanghai) to carry out the order-checking of M13 forward frozen glycerine pipe, the gene order and the Genebank sequence that obtain sequencing result and design are compared.
Embodiment 2 core antigen of C type hepatitis virus each section encoding gene expression vector establishment and evaluation
1, the preparation of gene fragment:
Recombinant clone pMD-H1, pMD-H2, pMD-H3, pMD-H4 and the pET41a of process sequence verification adopts the Xho I single endonuclease digestion of NcoI/XhoI double digestion and pET41a respectively, the enzyme tangent condition:
The reaction mixing is placed in the 37 degree water-baths, incubation 3 hours.
After cutting, enzyme carries out 1.2% agarose gel electrophoresis, and reclaim each fragment (method is with embodiment 1-2.) respectively: the pMD-H1 enzyme is cut the Xho I-Xho I (182bp) (being named as M) of 60 the amino acid Disabled bases of Nco I-Xho I fragment (178bp) (being named as N) and coding molecule middle part that obtain 60 amino acid Disabled bases of coding N end, the pMD-H2 enzyme is cut and is obtained 70 amino acid Disabled bases of coding C end Nco I-Xho I fragment (213bp), the pMD-H3 enzyme is cut and is obtained coding total length 191 amino acid Disabled base Nco I-Xho I fragment (573bp) (being named as 191), and the pMD-H4 enzyme is cut and obtained 120 amino acid Disabled base Nco I-Xho I fragments (360bp) of coding N end and pET41a vector arms (comprising Xho I single endonuclease digestion and NcoI-Xho I double digestion).
2, ligation (method is with embodiment 1)
Ligation is formed:
Behind the mixing, put 16 degree water-baths 3 hours, carry out ligation;
3, colibacillary conversion (method is with embodiment 1)
Adopt chemoreception bacterium (DH5a, available from Solarbio Inc) transform: get 3 microlitre ligation liquid and be added in the 50 microlitre competence bacterias, placed 30 minutes on ice, after transferring to 42 degree water-baths carrying out thermal shocking in 90 seconds, transfer to and put 5 fens on ice, the LB substratum 1ml that adds no antiauxin, transferring to 37 degree shaking tables shook 45 fens slowly, 5000rpm, centrifugal 10 minutes of 4 degree discard unnecessary substratum, and the bacterium that stays about 200 microlitres is added to the LB agar plate that contains kantlex (50mg/ml) behind the pressure-vaccum number gently, evenly be applied to driedly, place 37 degree incubator overnight incubation.
4, the Screening and Identification of recombinant clone
A) the bacterium colony PCR of recombinant clone screening: separate good single bacterium colony from transforming dull and stereotyped 10 of the pickings of going up, be seeded in 10 PCR pipes that are added with sterilized water and carry out abundant mixing, drawing the adding of 5 microlitres respectively has among the 1ml LB that contains kantlex (50mg/ml), makes the corresponding numbers centrifuge tube and is used for 37 degree shaking tables cultivations; 5 microlitres in other 10 PCR pipes will be used as the template of bacterium colony PCR, carry out the PCR reaction;
Cumulative volume 10 microlitres.
The PCR response procedures:
When screening, the extension time can be adjusted according to expansion target gene fragment length, and cycle index also can arrange to adjust by the time; After finishing, PCR carried out 1.2% agarose gel electrophoresis (10V/Cm glue) 20 minutes, gel imaging analysis as a result, and amplifying gene product and size and be with the consistent person of target gene fragment is positive clone; According to the numbering of positive colony bacterium liquid in the corresponding centrifuge tube is transferred to and is added with 5ml and contains to shake in the test tube of kantlex (50mg/ml) LB and spend the night.
B) put forward plasmid DNA for a short time: undertaken: collect and spend the night bacterium 1.5ml in 1.5 Eppendorf tubes by plasmid DNA a small amount of extraction agent box explanation (Solarbio Inc), the centrifugal 1 fractional precipitation bacterium of 10000rpm, abandon supernatant, add the 0.25ml solution I bacterial sediment that fully suspends, adding solution II 0.25ml topples over for several times gently with mixing, room temperature is put 2 minutes (solution should be limpid fully), add to put upside down rapidly after the 0.35ml solution III to mix and protein and genome are precipitated separate out, 12000rpm, centrifugal 10 minutes of 4 degree, with supernatant gently to adsorption column, 8000rpm, centrifugal 30 seconds of 4 degree, liquid in the collection tube is added in the adsorption column absorption once again, after liquid is carved in centrifugal removal, in adsorption column, add the 0.5ml rinsing liquid, 12000rpm, centrifugal 1 minute of 4 degree, repeat once, discard waste liquid in the collection tube, with adsorption column 12000rpm again, centrifugal 3 minutes of 4 degree, central authorities add 50 microlitre sterilized waters (or 10mM Tris-EDTA solution to adsorption column, pH〉8.0 be good), be positioned over 60-70 degree baking box interior 5 minutes, and took out back 12000rpm, centrifugal 2 minutes of 4 degree, collecting in the post is the DNA plasmid of purifying, after Nano-1000 micro-spectrophotometer mensuration DNA concentration, can carry out next step endonuclease reaction, redundance places in-20 degree refrigerators standby.
C) double digestion is identified recombinant plasmid: the double digestion reaction of recombinant plasmid (giving an example with pET41-HCVCore_191) is composed as follows;
Reaction solution through mixing be placed on 37 the degree water-baths in 3 hours, carry out endonuclease reaction.After reaction finishes, carried out 1.2% agarose gel electrophoresis (10V/Cm glue) 20 minutes, gel imaging analysis as a result, can cut out gene product and size and be with the consistent person of target gene fragment is positive clone, the preservation plasmid; The bacterium that spends the night before this is a glycerol stock: after the centrifugal collection of 1.5 bacterium liquid, abandon the supernatant adding and do not contain the antibiotic 15-25% of containing glycerine, preferentially 20% LB substratum is standby as frozen glycerine pipe after suspending.
The genetic expression of each section coding of embodiment 3 core antigen of C type hepatitis virus
1, the foundation of each section encoding gene expression strain of core antigen of C type hepatitis virus
(1) conversion of BL21 bacterium competence bacterium
To cut each recombinant expression vector pET41-HCVCore_N of evaluation through subclone and PCR and enzyme, pET41-HCVCore_M, pET41-HCVCore_C, pET41-HCVCore_120, pET41-HCVCore_191 and pET41a empty carrier transformed bacteria BL21: get aforementioned evaluation each recombinant plasmid 0.5 microlitre accurately, add 50 microlitre BL21 competence bacterias (Solarbio Inc), put 30 minutes on ice, be transferred to 42 degree water-bath heat-shockeds after 90 seconds, move to and put 5 fens on ice, in conversion tube, add the LB substratum 1ml of no antiauxin, transfer to 37 degree shaking tables and shook slowly 45 fens, 5000rpm, centrifugal 10 minutes of 4 degree discard unnecessary substratum, and the bacterium that stays about 200 microlitres is added to the LB agar plate that contains kantlex (50mg/ml) behind the pressure-vaccum number gently, evenly be applied to driedly, place 37 degree incubator overnight incubation.
(2) cultivation of transformed clone, self-induction are expressed
From growing and separating on the good conversion flat board, any 6 clones of picking, be inoculated into and contain 3ml self-induction expression substratum (0.02% glucose, 0.2% lactose, 50mM Na2HPO4,50mM NaH2PO4,10mM Mg2SO4,10mMMgCl2,2% peptone, 1.5% yeast extract) in, chooses a transformed clone and be inoculated in and contain in kantlex (50mg/ml) and the 2% glucose 3ml LB substratum as negative control, choose a clone from the empty carrier reformer plate and be inoculated in the 3ml self-induction and express substratum and make positive vehicle Control, the back of spending the night is collected and is expressed thalline.
(3) the acrylamide gel electrophoresis analysis of expression of recombinant proteins level
Collect sample preparation: 100ul PB solution (50mM Na2HPO4 in bacterial sediment, 50mM NaH2PO4, pH7.2) after abundant the suspension, add 2* sample-loading buffer (2xSDS gel loading buffer: 100mmol/LTris-HCl (pH6.8), 200mmol/L dithiothreitol (DTT) (DTT), 4%SDS (electrophoresis level), 0.2% tetrabromophenol sulfonphthalein, 20% glycerine) 100 microlitres boiled 5 minutes more than 85 degree behind the mixing, and sample is used for sample on the acrylamide electrophoresis (maybe can freeze in-20 degree standby).Acrylamide gel electrophoresis: conventional preparation 12% acrylamide gel, electrophoresis is pressed upward sample of albumen Marker, negative control, vehicle Control, clone 1-6, albumen Marker, 60 volts of voltage stabilizing electrophoresis 30 minutes, adjust to 90 volts of voltage stabilizings finished in 90 minutes, after examining dyeing of Ma Shi light blue and decolouring program, take pictures under gel imaging system and each proteic relative concentration of scanning analysis, conclusive each relative expression's level selects express recombinant protein the higher person to preserve.
(4) set up the high expression level seed bank
Select the relative clone's of expression of recombinant proteins cultivation, switching 50ml substratum (LB of kantlex (50mg/ml) and 2% glucose) is cultivated at 37 degree shaking tables, aseptic centrifugal collection bacterium when treating that thalline OD600 reaches 1.5 left and right sides, after the LB suspension fully that contains 15% glycerine with 1/4 volume, by every pipe 1ml packing, frozen in-80 degree refrigerator, i.e. seed banks.
2, reorganization cAg fragment expressing fusion protein
(1) self-induction is expressed the fermention medium preparation: express to cultivate to prepare by aforementioned self-induction and contain 0.02% glucose, 0.2% lactose, 50mM Na2HPO4,50mM NaH2PO4,10mM Mg2SO4,10mM MgCl2,2% peptone, the fermention medium of 1.5% yeast extract carries out conventional high pressure bacterium after the packing.
(2) culture of seed liquid: get seed one pipe, insert the flask (500ml bottle) of the 100ml LB of the kantlex that is added with 50mg/ml, 37 degree, the 250rpm shaking culture is spent the night.
(3) connect seed liquor by 5% inoculum size, add seed liquor, 26-32 degree to substratum, preferably with 28 degree, the 300rpm shaken overnight is carried out self-induction and is expressed, and expresses and finishes centrifugal collection thalline, carries out next step purifying (perhaps freeze and preserve standby in-20 degree) after claiming Sheng heavy.
The isolation and purification of each section fusion rotein of embodiment 4 core antigen of C type hepatitis virus
1, e. coli bl21 is expressed the chemical cracking of bacterium:
Thalline through abduction delivering adopts the chemical cracking method, adds 5-10ml bacterial lysate (2%Triton-X100,10-50ug/ml N,O-Diacetylmuramidase by the wet bacterium of every gram, 1mM DTT, 0.2M PBS preferably adopts the BugBuster solution of Merck ﹠ Co., Inc.) thalline that fully suspends, 37 degree shook incubation 1 hour slowly; In 12000 rev/mins of high speed centrifugations of 4 degree 20 minutes, collect supernatant and promptly be used for next step affinitive layer purification.
2, adopt two step of gravity method affinity chromatography with the purification of target recombinant protein, purity is greater than 95%:
A) with the abundant centrifugal back of above-mentioned process supernatant, the Ni2+-NTA affinity column is through sample-loading buffer (100mMNaH2PO4,10mMTris, 10mM2-ME pH8.0) after the abundant balance, drips the cracked supernatant, collect effluent liquid, get the 10ul sample and be used for the SDS-PAGE analysis.With elutriant I(100mM NaH2PO4,10mM Tris, pH6.3) fully to wash post and collect elutriant step by step to the OD280=0.01. of effluent liquid, the sample when getting the 10ul wash-out and beginning is used for SDS-PAGE and analyzes.With elutriant II(100mM NaH2PO4,10mMTris, 500mM Imidazole, pH8.0) wash-out target protein: collect every 1mL fraction, get the 10ul sample respectively and be used for the SDS-PAGE analysis.Merge each pure component, to carry out next step purifying of GST affinity chromatography gel post.
B) with the abundant balance columns of the phosphate buffered saline buffer of 10 times of column volumes (4.5mM Na2HPO4.12H2O, 1.5mM KH2PO4,0.15MNaCl(pH7.3)), add, collect stream and wear component and place on ice through the sample after the nickel post affinity chromatography.With 10x volume 1xGST Bind/Wash Buffer (4.5mM Na2HPO4.12H2O, 1.5mM KH2PO4,0.15M NaCl, 2.7mM KCl(pH7.3)) wash post, collection stream is worn component and is placed on ice.With 3x volume 1xGSTWash Buffer(4.5mM Na2HPO4.12H2O, 1.5mM KH2PO4,0.15M NaCl, 2.7mM KCl10mM GST (reduced form) are (pH7.3)) the wash-out target protein.The collection elution fraction places treats follow-up protein electrophoresis analysis on ice, and merges identical protein ingredient, frozen standby in-20 degree.
Sequence table
SEQUENCE LISTING
＜110〉Zhejiang man and pharmaceutical Co. Ltd
＜120〉viruses of human hepatitis C's cAg and its production and application
<160> 10
<170> Patent Version3.5
<210> 1
<211> 347
<212> Prt
<213> GST-HCVCore_N
<400> 1
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro
1 5 10 15
Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu
20 25 30
Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu
35 40 45
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys
50 55 60
Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn
65 70 75 80
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu
85 90 95
Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
100 105 110
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu
115 120 125
Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
130 135 140
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp
145 150 155 160
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu
165 170 175
Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr
180 185 190
Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala
195 200 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Gly Ser Thr Ser
210 215 220
Gly Ser Gly His His His His His His Ser Ala Gly Leu Val Pro Arg
225 230 235 240
Gly Ser Thr Ala Ile Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu
245 250 255
Arg Gln His Met Asp Ser Pro Asp Leu Gly Thr Gly Gly Gly Ser Gly
260 265 270
Asp Asp Asp Asp Lys Ser Pro Met Ala Ser Thr Asn Pro Lys Pro Gln
275 280 285
Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe
290 295 300
Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg
305 310 315 320
Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser Glu Arg Ser
325 330 335
Gln Leu Glu His His His His His His His His
340 345
<210> 2
<211> 378
<212> Prt
<213> GST-HCVCore_ M
<400> 2
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro
1 5 10 15
Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu
20 25 30
Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu
35 40 45
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys
50 55 60
Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn
65 70 75 80
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu
85 90 95
Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
100 105 110
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu
115 120 125
Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
130 135 140
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp
145 150 155 160
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu
165 170 175
Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr
180 185 190
Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala
195 200 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Gly Ser Thr Ser
210 215 220
Gly Ser Gly His His His His His His Ser Ala Gly Leu Val Pro Arg
225 230 235 240
Gly Ser Thr Ala Ile Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu
245 250 255
Arg Gln His Met Asp Ser Pro Asp Leu Gly Thr Gly Gly Gly Ser Gly
260 265 270
Asp Asp Asp Asp Lys Ser Pro Met Gly Tyr Arg Gly Ser Glu Phe Cys
275 280 285
Thr Gly Leu Gly Ala Pro Ala Gly Glu Leu Arg Arg Gln Ala Cys Gly
290 295 300
Arg Thr Arg Gly Arg Arg Gln Pro Ile Pro Lys Ala Arg Arg Pro Glu
305 310 315 320
Gly Arg Thr Trp Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn
325 330 335
Glu Gly Cys Gly Trp Ala Gly Trp Leu Leu Ser Pro Arg Gly Ser Arg
340 345 350
Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly
355 360 365
Leu Glu His His His His His His His His
370 375
<210> 3
<211> 362
<212> Prt
<213> GST-HCVCore_C
<400> 3
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro
1 5 10 15
Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu
20 25 30
Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu
35 40 45
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys
50 55 60
Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn
65 70 75 80
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu
85 90 95
Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
100 105 110
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu
115 120 125
Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
130 135 140
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp
145 150 155 160
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu
165 170 175
Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr
180 185 190
Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala
195 200 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Gly Ser Thr Ser
210 215 220
Gly Ser Gly His His His His His His Ser Ala Gly Leu Val Pro Arg
225 230 235 240
Gly Ser Thr Ala Ile Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu
245 250 255
Arg Gln His Met Asp Ser Pro Asp Leu Gly Thr Gly Gly Gly Ser Gly
260 265 270
Asp Asp Asp Asp Lys Ser Pro Met Ala Lys Val Ile Asp Thr Leu Thr
275 280 285
Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro
290 295 300
Leu Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu
305 310 315 320
Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser
325 330 335
Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Val Pro Ala Ser Ala
340 345 350
Leu Glu His His His His His His His His
355 360
<210> 4
<211> 410
<212> Prt
<213> GST-HCVCore120
<400> 4
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro
1 5 10 15
Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu
20 25 30
Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu
35 40 45
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys
50 55 60
Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn
65 70 75 80
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu
85 90 95
Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
100 105 110
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu
115 120 125
Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
130 135 140
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp
145 150 155 160
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu
165 170 175
Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr
180 185 190
Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala
195 200 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Gly Ser Thr Ser
210 215 220
Gly Ser Gly His His His His His His Ser Ala Gly Leu Val Pro Arg
225 230 235 240
Gly Ser Thr Ala Ile Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu
245 250 255
Arg Gln His Met Asp Ser Pro Asp Leu Gly Thr Gly Gly Gly Ser Gly
260 265 270
Asp Asp Asp Asp Lys Ser Pro Met Ala Ser Thr Asn Pro Lys Pro Gln
275 280 285
Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe
290 295 300
Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg
305 310 315 320
Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser Glu Arg Ser
325 330 335
Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro Lys Ala Arg Arg Pro Glu
340 345 350
Gly Arg Thr Trp Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn
355 360 365
Glu Gly Cys Gly Trp Ala Gly Trp Leu Leu Ser Pro Arg Gly Ser Arg
370 375 380
Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly
385 390 395 400
Leu Glu His His His His His His His His
405 410
<210> 5
<211> 481
<212> Prt
<213> GST-HCVCore191
<400> 5
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro
1 5 10 15
Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu
20 25 30
Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu
35 40 45
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys
50 55 60
Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn
65 70 75 80
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu
85 90 95
Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
100 105 110
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu
115 120 125
Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
130 135 140
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp
145 150 155 160
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu
165 170 175
Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr
180 185 190
Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala
195 200 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Gly Ser Thr Ser
210 215 220
Gly Ser Gly His His His His His His Ser Ala Gly Leu Val Pro Arg
225 230 235 240
Gly Ser Thr Ala Ile Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu
245 250 255
Arg Gln His Met Asp Ser Pro Asp Leu Gly Thr Gly Gly Gly Ser Gly
260 265 270
Asp Asp Asp Asp Lys Ser Pro Met Ala Ser Thr Asn Pro Lys Pro Gln
275 280 285
Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe
290 295 300
Pro Gly Gly Val Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg
305 310 315 320
Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser Glu Arg Ser
325 330 335
Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro Lys Ala Arg Arg Pro Glu
340 345 350
Gly Arg Thr Trp Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn
355 360 365
Glu Gly Cys Gly Trp Ala Gly Trp Leu Leu Ser Pro Arg Gly Ser Arg
370 375 380
Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly
385 390 395 400
Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu Met Gly Tyr
405 410 415
Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg Ala Leu Ala
420 425 430
His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn
435 440 445
Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys
450 455 460
Leu Thr Val Pro Ala Ser Ala Leu Glu His His His His His His His
465 470 475 480
His
<210> 6
<211> 1052 bp
<212> DNA
<213> GST-HCVCore_N DNA
<400> 6
atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60
ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120
tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180
ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240
atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300
gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360
gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420
acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480
gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540
aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600
tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660
ggttcaacta gtggttctgg tcatcaccat caccatcact ccgcgggtct ggtgccacgc 720
ggtagtactg caattggtat gaaagaaacc gctgctgcta aattcgaacg ccagcacatg 780
gacagcccag atctgggtac cggtggtggc tccggtgatg acgacgacaa gagtcccatg 840
gccagcacga atcctaaacc tcaaagaaaa accaaacgta acaccaaccg tcgcccacag 900
gacgtcaagt tcccgggtgg cggtcagatc gttggtggag tttacttgtt gccgcgcagg 960
ggccctagat tgggtgtgcg cgcgacgagg aagacttccg agcggtcgca actcgagcac 1020
caccaccacc accaccacca ctaattgatt aa 1052
<210>7
<211>1137bp
<212>DNA
<213>GST-HCVCore_M DNA
<400>7
atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60
ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120
tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180
ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240
atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300
gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360
gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420
acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480
gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540
aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600
tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660
ggttcaacta gtggttctgg tcatcaccat caccatcact ccgcgggtct ggtgccacgc 720
ggtagtactg caattggtat gaaagaaacc gctgctgcta aattcgaacg ccagcacatg 780
gacagcccag atctgggtac cggtggtggc tccggtgatg acgacgacaa gagtcccatg 840
ggatatcggg gatccgaatt ctgtacaggc cttggcgcgc ctgcaggcga gctccgtcga 900
caagcttgcg gccgcactcg aggtagacgt cagcctatcc ccaaggcacg tcggcccgag 960
ggcaggacct gggctcagcc cgggtaccct tggcccctct atggcaatga gggttgcggg 1020
tgggcgggat ggctcctgtc tccccgtggc tctcggccta gctggggccc cacagacccc 1080
cggcgtaggt cgcgcaattt gggtctcgag caccaccacc accaccacca ccactaa 1137
<210>8
<211>1097bp
<212>DNA
<213>GST-HCVCore_C DNA
<400>8
atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60
ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120
tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180
ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240
atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300
gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360
gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420
acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480
gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540
aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600
tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660
ggttcaacta gtggttctgg tcatcaccat caccatcact ccgcgggtct ggtgccacgc 720
ggtagtactg caattggtat gaaagaaacc gctgctgcta aattcgaacg ccagcacatg 780
gacagcccag atctgggtac cggtggtggc tccggtgatg acgacgacaa gagtcccatg 840
gccaaggtca tcgataccct tacgtgcggc ttcgccgacc tcatggggta cataccgctc 900
gtcggcgccc ctcttggagg cgctgccagg gccctggcgc atggcgtccg ggttctggaa 960
gacggcgtga actatgcaac agggaacctt cctggttgct ctttctctat cttccttctg 1020
gccctgctct cttgcctgac tgtgcccgct tcagccctcg agcaccacca ccaccaccac 1080
caccactaat tgattaa 1097
<210>9
<211>1301bp
<212>DNA
<213>GST-HCVCore120DNA
<400>9
atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60
ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120
tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180
ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240
atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300
gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360
gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420
acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480
gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540
aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600
tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660
ggttcaacta gtggttctgg tcatcaccat caccatcact ccgcgggtct ggtgccacgc 720
ggtagtactg caattggtat gaaagaaacc gctgctgcta aattcgaacg ccagcacatg 780
gacagcccag atctgggtac cggtggtggc tccggtgatg acgacgacaa gagtcccatg 840
gccagcacga atcctaaacc tcaaagaaaa accaaacgta acaccaaccg tcgcccacag 900
gacgtcaagt tcccgggtgg cggtcagatc gttggtggag tttacttgtt gccgcgcagg 960
ggccctagat tgggtgtgcg cgcgacgagg aagacttccg agcggtcgca acctcgaggt 1020
agacgtcagc ctatccccaa ggcacgtcgg cccgagggca ggacctgggc tcagcccggg 1080
tacccttggc ccctctatgg caatgagggt tgcgggtggg cgggatggct cctgtctccc 1140
cgtggctctc ggcctagctg gggccccaca gacccccggc gtaggtcgcg caatttgggt 1200
ctcgagcacc accaccacca ccaccaccac taattgatta atacctaggc tgctaaacaa 1260
agcccgaaag gaagctgagt tggctgctgc caccgctgag c 1301
<210>10
<211>1514bp
<212>DNA
<213>GST-HCVCore191DNA
<400>10
atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60
ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120
tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180
ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240
atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300
gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360
gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420
acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480
gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540
aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600
tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660
ggttcaacta gtggttctgg tcatcaccat caccatcact ccgcgggtct ggtgccacgc 720
ggtagtactg caattggtat gaaagaaacc gctgctgcta aattcgaacg ccagcacatg 780
gacagcccag atctgggtac cggtggtggc tccggtgatg acgacgacaa gagtcccatg 840
gccagcacga atcctaaacc tcaaagaaaa accaaacgta acaccaaccg tcgcccacag 900
gacgtcaagt tcccgggtgg cgttcagatc gttggtggag tttacttgtt gccgcgcagg 960
ggccctagat tgggtgtgcg cgcgacgagg aagacttccg agcggtcgca accacgaggt 1020
agacgtcagc ctatccccaa ggcacgtcgg cccgagggca ggacctgggc tcagcccggg 1080
tacccttggc ccctctatgg caatgagggt tgcgggtggg cgggatggct cctgtctccc 1140
cgtggctctc ggcctagctg gggccccaca gacccccggc gtaggtcgcg caatttgggt 1200
aaggtcatcg atacccttac gtgcggcttc gccgacctca tggggtacat accgctcgtc 1260
ggcgcccctc ttggaggcgc tgccagggcc ctggcgcatg gcgtccgggt tctggaagac 1320
ggcgtgaact atgcaacagg gaaccttcct ggttgctctt tctctatctt ccttctggcc 1380
ctgctctctt gcctgactgt gcccgcttca gccctcgagc accaccacca ccaccaccac 1440
cactaattga ttaataccta ggctgctaaa caaagcccga aaggaagctg agttggctgc 1500
tgccaccgct gagc 1514
Claims (10)
1. viruses of human hepatitis C's cAg, be polypeptide or protein molecular, it is characterized in that: the aminoacid sequence of described polypeptide or protein molecular is shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, or this sequence is suddenlyd change and the conservative property varient of acquisition through the conservative property of one or more aminoacid addition, deletion, replacement, modification.
2. dna molecular, coding described polypeptide of claim 1 or protein molecular.
3. dna molecular according to claim 2 is characterized in that: the nucleotide sequence of its coding said polypeptide or protein molecular is shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ IDNO:10.
4. expression vector, the expression regulation sequence that includes the described dna molecular of claim 2 and link to each other with this dna molecular operability.
5. prokaryotic host cell, it is transformed by the described expression vector of claim 4.
6. prokaryotic host cell according to claim 5 is characterized in that: it is intestinal bacteria.
7. prokaryotic host cell according to claim 5 is characterized in that: it is an e. coli bl21.
8. the described viruses of human hepatitis C's cAg of claim 1 is in the reagent of preparation diagnosis hepatitis C virus or the purposes in the medicine.
9. a method for preparing the described viruses of human hepatitis C's cAg of claim 1 is characterized in that comprising the steps:
(1) provides an expression vector, the expression regulation sequence that this expression vector contains the described dna molecular of claim 2 and links to each other with this dna molecular operability;
(2) transform prokaryotic host cell with the described expression vector of step (1);
(3) prokaryotic host cell of culturing step (2) gained under the condition that is fit to described polypeptide or protein molecular antigen presentation;
(4) separation and purification obtains described polypeptide or protein molecular antigen.
10. method according to claim 9, it is characterized in that: described expression vector is a fusion expression vector, this expression vector can express have correct folding, the target protein of solubility, and target protein can obtain effective purifying by two step affinity chromatographys.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108530521A (en) * | 2018-04-18 | 2018-09-14 | 南京京达生物技术有限公司 | Recombinate the preparation and application of hepatitis C antigen |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102072957A (en) * | 2011-01-18 | 2011-05-25 | 威海威高生物科技有限公司 | Hepatitis C virus antibody diagnostic kit and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102072957A (en) * | 2011-01-18 | 2011-05-25 | 威海威高生物科技有限公司 | Hepatitis C virus antibody diagnostic kit and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| YAN XUE-BING: "Proapoptotic and pronecrosis effect of different truncated hepatitis C virus core proteins", 《JOURNAL OF ZHEJIANG UNIVERSITY SCIENCE》 * |
| 彭祥兵: "HCV 核心区基因的截短及其表达", 《中华医药杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108530521A (en) * | 2018-04-18 | 2018-09-14 | 南京京达生物技术有限公司 | Recombinate the preparation and application of hepatitis C antigen |
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