CN103214561B - 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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- CN103214561B CN103214561B CN201310078878.2A CN201310078878A CN103214561B CN 103214561 B CN103214561 B CN 103214561B CN 201310078878 A CN201310078878 A CN 201310078878A CN 103214561 B CN103214561 B CN 103214561B
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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 preparation method and application.
Background technology
Hepatitis C infects by viruses of human hepatitis C (HCV, Hepatitis C Virus) transmissible disease caused, and be widely current in the whole world, especially China is one of popular more serious country of HCV, and crowd infection rate is about 3.2%, has the infected of more than 40,000,000.It is a kind of very important route of transmission that haematogenous is propagated, and owing to not having effective preventative vaccine and effective treatment means, therefore early diagnosis and the propagation of blood screening to control HCV have a very important role.
The diagnosis of current HCV clinically and examination are to detect HCV antibody, because HCV antibody test generally exists the window phase in 7-10 week, set up early stage, easy, responsive, special HCV-cAg diagnostic reagent, shortening the window phase detected is the effective way reducing HCV infection.Although RT-PCR can quantitative and qualitative analysis detect HCV RNA effectively shorten window phase, because of RT-PCR complicated operation, easily to pollute and expensive, making it can not widespread use.Research and develop the monoclonal antibody based on hepatitis c core antigen, for early detection infection with hepatitis C virus, by double-antibody sandwich elisa screening energy high-sensitivity detection hepatitis c core antigen, set up hepatitis c core antigen detection system and combine and detect serum antibody to hepatitis C, for antigen-antibody joint inspection application is layed foundation.
Although hepatitis c core antigen well-conserved also has very strong immunogenicity concurrently, but hepatitis c core antigen protein requirement host signal peptidase shear its C hold in signal peptide sequence thus be processed to form ripe core antigen protein (p21), thus cause to go out the natural hepatitis c core antigen albumen of conformation by genetically engineered especially prokaryotic expression and become abnormal difficult.Core antigen protein is very easily combined with nucleic acid and fat egg in addition, and its C end has very strong hydrophobicity, is also all that vivoexpression and the purifying of core antigen protein adds difficulty.In recent years some researchists utilize the white (191aa of the cAg of yeast and mammalian cell successful expression total length, p23), and find that p23 can be sheared by the signal peptidase of cell the p21 albumen forming maturation, and self-assembly can form the nucleocapsid sample particle (nucleocapside1ike particles, NLPs) that diameter is about 35nm in vitro.Utilizing E.coli to express hepatitis c core antigen N holds front 120 amino acid, also can form virus-like particle (VLPs, Virus LikeParticles) under certain condition to have investigator to find.And with producing strong and lasting humoral and cellular immune response response after this VLPs immune mouse, rabbit and goat.Due to immature in method by eucaryon approach great expression purifying HCV core recombinant antigen, therefore Many researchers selection E. coli system expresses core antigen protein, and avoids the amino acid of core antigen 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, cDNA clones HCV-cAg gene, express and the gst fusion protein antigen of separation and purification HCV-cAg, this antigen is for developing HCV diagnostic kit, especially in the immunity stage, the anti-cAg antibody with better avidity and evident characteristics is produced for immunity, for the detection of the HCV-cAg of early stage HCV infection.Therefore, the DNA molecular of genetic engineering fusion protein antigen that the object of the present invention is to provide a kind of core antigen of C type hepatitis virus and its preparation method and application, this fusion rotein antigen of encoding, comprise the expression vector of this DNA molecular, and by prokaryotic host cell that this expression vector transforms.
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, described polypeptide or the aminoacid sequence of protein molecular are as 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 through the conservative mutation of one or more aminoacid addition, deletion, replacement, modification and the conservative variant obtained.
A kind of DNA molecular, it is encoded above-mentioned polypeptide or protein molecular.
In a preferred embodiment of the present invention, the nucleotide sequence of polypeptide or protein molecular described in above-mentioned DNA molecule encode is as 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 including above-mentioned DNA molecular and be connected 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 e. coli bl21.
Test proves, above-mentioned viruses of human hepatitis C's cAg may be used for reagent or the medicine of preparing diagnosis hepatitis C virus.
Prepare a method for above-mentioned viruses of human hepatitis C's cAg, comprise the steps:
(1) expression vector is provided, the expression regulation sequence that this expression vector contains DNA molecular according to claim 2 and is connected with this DNA molecular operability;
(2) with the expression vector transform both prokaryotic host cell described in step (1);
(3) prokaryotic host cell of culturing step (2) gained under the condition being applicable to described polypeptide or protein molecular antigen presentation;
(4) polypeptide described in separation and purification acquisition or protein molecular antigen.
Above-mentioned preparation method, wherein said expression vector is fusion expression vector, the fusion rotein antigen of expressing has correct folding structure, is present in 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 adopt and obtain in intestinal bacteria express with GST protein fusion, and this expression is soluble proteins, its protein structure with close to natural folding mode, thus lays the foundation for the fundamental research of HCV and diagnostic reagent development and application.
Compared with prior art, the viruses of human hepatitis C's cAg that the present invention relates to and preparation method thereof tool has the following advantages and progress significantly:
(1) segmentation and the full-length molecule cloning process of core antigen gene is adopted, 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 closer to the feature of native protein.
Accompanying drawing explanation
Fig. 1 is core antigen of C type hepatitis virus gene coded sequence and primer combinations of pairs schematic diagram.
Fig. 2 is PCR primer clone schematic diagram.
Fig. 3 is the structure schematic diagram of fusion expression vector pET41-HCVCore191.
Embodiment
The preparation method of the genetic engineering fusion protein antigen that the present invention relates to comprises the steps:
1, with the plasmid pM-HCV_Genomeo containing HCV full-length gene group DNA for template, and design primer with for each fragment of core antigen gene, carry out pcr amplification, amplified production is through electrophoretic separation and glue reclaims corresponding specific amplified gene product, and be subcloned into pMD-18 (TaKRa, Inc), in carrier, each gene recombination plasmid is obtained respectively.
2, the primer of design is as shown in table 1
Table 1. hepatitis c core antigen gene each section antigen primer sequence design table
A) adopt primer 1,3 to increase and obtain PCR primer 1, clone's carrier T obtains pMD-H1;
B) adopt primer 2,4 amplification acquisition PCR PCR primer 2, clone obtains pMD-H2;
C) " huge primer two takes turns PCR Chinese patent (ZL95113311.x) " technology is adopted, with the plasmid pM-HCV_Genomeo containing HCV full-length gene group DNA for template, with primer 1+ primer 6, the cAg encoding gene that primer 5+ primer 2 increases respectively, gained two fragment PCR products is after running gel reclaims, take turns the template of PCR reaction as second than mixing by equimolecular, adopt the combination of primer 1+ primer 2 to proceed second and take turns PCR, with the total length core antigen gene encoding sequence that increases, obtain the total length encoding gene in inner Xho I site that suddenlyd change.This PCR primer 3, for building pMD-H3;
D) primer 1,3 increases and obtains PCR primer 4, for building pMD-H4, see Fig. 1.
3, pMD-H1-4 verifies through DNA sequencing.
4, by the restriction endonuclease sites of design, from above-mentioned pMD-H1, pMD-H2, pMD-H3, pMD-H4, double digestion obtains corresponding gene fragment, gene Subcloned technology is adopted to be connected by each gene pET41a obtained, build corresponding HCV-cAg expression vector, obtain the expression vector with aforementioned Seq.6-10 encoding sequence, after Screening and Identification, recombinant plasmid transformed escherichia coli expression host BL21 bacterium, and carry out abduction delivering analysis, set up the fusion rotein antigen engineering bacteria of expression of HCV cAg, for the preparation of recombination fusion protein antigen.
5, express engineering bacteria by fermentation, collect fermentation thalli, 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 is only limitted to following embodiment, all technology realized based on foregoing of the present invention all belong to scope of the present invention.
The acquisition of each segment encodes gene of embodiment 1 core antigen of C type hepatitis virus and qualification
1, the fragment amplification of antigen target gene:
Adopting containing people HCV1b type full-length genome plasmid is template, adopts different pairing combination of primers, and uses the PrimStar of high-fidelity (purchased from TAKARA Inc.) gene to carry out pcr amplification to obtain each genes encoding fragment.Condition is as follows:
50 μ lPCR amplification reaction systems:
Pcr amplification reaction condition:
Wherein combination of primers:
(1) primer 1+ primer 3: amplified production is the sequence of front 120 amino-acid residues of coding core albumen containing inner Xho I point of contact, product name Seq1, as shown in SEQ ID NO:5;
(2) primer 4+ primer 2: amplified production is the encoding sequence that core protein C holds 70 amino-acid residues, product name Seq2, as shown in SEQ ID NO:6;
(3) primer 1+ primer 6, two sections of sequences of the cAg encoding gene that primer 5+ primer 2 increases respectively, product through running gel reclaim after as template, proceed second and take turns PCR, adopt the combination of primer 1+ primer 2 with the total length core antigen gene encoding sequence that increases, obtain the total length encoding gene in inner Xho I site that suddenlyd change.Method continues to use the technology provided in Chinese patent (ZL95113311.x).Product called after Seq3, as shown in SEQ ID NO:7;
(4) primer 1+ primer 3: using product S eq3 as the template of gene amplification, amplification obtains product name Seq4, as shown in SEQ ID NO:8.
2, the electrophoretic separation of PCR primer and purifying:
By 4 kinds of pcr amplification products, 1.2% agarose gel electrophoresis (10V/Cm glue) is separated goal gene band 20 points, and running gel cuts containing goal gene fragments gel bar under gel imaging system; Adopt DNA gel to reclaim test kit (Solarbio Inc) by specification and carry out purifying:
(1) add sol solutions by 1:3 weight/long-pending body ratio, be placed in 55 degree of water-baths 10 minutes, period puts upside down mixing for several times to ensure abundant colloidal sol;
(2) add 7:10 therefrom volume ratio Virahol mixing after be added to adsorption column, 8000rpm, 4 degree centrifugal 1 point, liquid in collection post is added in post again and adsorbs again once, 8000rpm, 4 degree centrifugal 1 point again, abandon or adopt waste liquid;
(3) in adsorption column, add 0.5ml rinsing liquid, 12000rpm, 4 degree are centrifugal 30 seconds, come again, and discard and collect waste liquid in post, adsorption column with 12000rpm, 4 degree centrifugal 3 points;
(4) adsorption column is transferred to a new collection tube, 30 ul sterile water (or 10mMTris-EDTA solution is added to adsorption column central authorities, pH>8.0 is good), to be positioned in 60-70 degree baking box 5 points, 12000rpm after taking out, 4 degree centrifugal 2 points, collect the DNA fragmentation of i.e. purifying in post;
(5), after measuring DNA concentration with Nano-1000 micro-spectrophotometer, be placed in-20 degree refrigerators for subsequent use.
3, PCR primer is cloned into pMD-18T carrier
The PCR primer of purifying illustrates quantitatively by TaKaRa company's T carrier and is connected in pMD-18T 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
After mixing, put 16 degree of water-baths 3 hours, carry out ligation;
(2) colibacillary conversion
Adopt chemoreception bacterium (DH5a, purchased from Solarbio Inc) transform: get 3 microlitre ligation liquid and be added in 50 jjL competent bacterium, be placed in 30 points on ice, transfer to after 42 degree of water-baths carry out thermal shocking in 90 seconds, transfer to and put 5 points on ice, add the LB substratum 1ml without antiauxin, transfer to 37 degree of shaking tables and shake 45 points slowly, 5000rpm, 4 degree centrifugal 10 points, discard unnecessary substratum, LB agar plate containing penbritin (100mg/ml) will be added to after the bacterium leaving about 200 microlitres gently pressure-vaccum number, even spread is to dry, be placed in 37 degree of incubator overnight incubation.
(3) Screening and Identification of recombinant clone
A) the bacterium colony PCR of recombinant clone screens: from transformation plate, picking 10 is separated good single bacterium colony, be seeded in 10 to be added with in the PCR pipe of sterilized water and fully to mix, drawing 5 microlitres respectively adds in the 1ml LB had containing penbritin, makes corresponding numbering centrifuge tube and is used for 37 degree of shaking tables cultivations; 5 microlitres in other 10 PCR pipe, using by the template as bacterium colony PCR, carry out PCR reaction;
Cumulative volume 10 microlitre.
PCR response procedures:
When screening, the extension time can adjust according to expanded target gene fragment length, and cycle index also temporally can arrange adjustment; Carry out 1.2% agarose gel electrophoresis (10V/Cm glue) 20 points after PCR terminates, result gel imaging analysis, amplify gene product and size is be positive colony compared with the bigger 100bp person of target gene fragment; According to the numbering of positive colony bacterium liquid in corresponding centrifuge tube is transferred to and is added with 5ml and spends the night containing shaking in the test tube of penbritin (100mg/ml).
B) littlely put forward plasmid DNA: by plasmid DNA in a small amount extraction agent box illustrate that (Solarbio Inc) carries out: collect and spend the night bacterium 1.5ml in 1.5 Eppendorf tubes, the centrifugal 1 fractional precipitation bacterium of 10000rpm, abandon supernatant, add 0.25ml solution I fully to suspend bacterial sediment, adding solution II 0.25ml topples over for several times with mixing gently, room temperature is put 2 points (solution should be completely limpid), put upside down rapidly after adding 0.35ml solution III and mixedly make protein and genome Precipitation for 5-10 time, 12000rpm, 4 degree centrifugal 10 points, by supernatant gently to in adsorption column, 8000rpm, 4 degree centrifugal 30 seconds, being added in adsorption column by liquid in collection tube adsorbs once again, after centrifugal segregation carves liquid, 0.5ml rinsing liquid is added in adsorption column, 12000rpm, 4 degree centrifugal 1 point, repeat once, discard waste liquid in collection tube, by adsorption column 12000rpm again, 4 degree centrifugal 3 points, 50 ul sterile water (or 10mM Tris-EDTA solution is added to adsorption column central authorities, pH>8.0 is good), to be positioned in 60-70 degree baking box 5 points, 12000rpm after taking out, 4 degree centrifugal 2 points, collect the DNA plasmid of i.e. purifying in post, after measuring DNA concentration with Nano-1000 micro-spectrophotometer, next step endonuclease reaction can be carried out, redundance is placed in-20 degree refrigerators for subsequent use.
C) double digestion qualification recombinant plasmid: the double digestion reaction of recombinant plasmid (with pMD-H1 citing) is composed as follows;
Reaction solution to be placed in 37 degree of water-baths 3 hours through mixing, carries out endonuclease reaction.After reaction terminates, carry out 1.2% agarose gel electrophoresis (10V/Cm glue) 20 points, result gel imaging analysis, can gene product be cut out and size is person consistent with target gene fragment is be positive colony, preserve plasmid; The bacterium that spends the night before this is glycerol stock: after 1.5 bacterium liquid collected by centrifugation, abandon supernatant add containing antibiotic containing 15-25% glycerine, preferentially the LB substratum of 20%, as frozen glycerine pipe (freezing 2 pipes) for subsequent use after suspension.
D) sequencing of recombinant plasmid: sent by frozen glycerine pipe order-checking company (as Shanghai Mei Ji biotechnology company limited) to carry out the order-checking of M13 forward, the gene order and the Genebank sequence that obtain sequencing result and design are compared.
The each segment encodes expression vector of embodiment 2 core antigen of C type hepatitis virus builds and qualification
1, the preparation of gene fragment:
NcoI/XhoI double digestion is adopted respectively through recombinant clone pMD-H1, the pMD-H2 of sequence verification, pMD-H3, pMD-H4 and pET41a, and the Xho I single endonuclease digestion of pET41a, enzyme tangent condition:
Reaction mixing is placed in 37 degree of water-baths, incubation 3 hours.
1.2% agarose gel electrophoresis is carried out after enzyme cuts into, and reclaiming each fragment (method is with embodiment 1-2.) respectively: pMD-H1 enzyme is cut and is obtained Xho I-Xho I (182bp) (being named as M) that coding N holds 60 amino acid Disabled bases in the middle part of Nco I-Xho I fragment (178bp) (being named as N) of 60 amino acid Disabled bases and coding molecule, pMD-H2 enzyme is cut acquisition coding C and is held 70 amino acid Disabled bases Nco I-Xho I fragment (213bp), pMD-H3 enzyme is cut and is obtained encoding full leng 191 amino acid Disabled base Nco I-Xho I fragment (573bp) (being named as 191), pMD-H4 enzyme is cut acquisition coding N and is held 120 amino acid Disabled bases Nco I-Xho I fragment (360bp) and pET41a vector arms (comprising Xho I single endonuclease digestion and NcoI-Xho I double digestion).
2, ligation (method is with embodiment 1)
Ligation forms:
After mixing, put 16 degree of water-baths 3 hours, carry out ligation;
3, colibacillary conversion (method is with embodiment 1)
Adopt chemoreception bacterium (DH5a, purchased from Solarbio Inc) transform: get 3 microlitre ligation liquid and be added in 50 jjL competent bacterium, be placed in 30 points on ice, transfer to after 42 degree of water-baths carry out thermal shocking in 90 seconds, transfer to and put 5 points on ice, add the LB substratum 1ml without antiauxin, transfer to 37 degree of shaking tables and shake 45 points slowly, 5000rpm, 4 degree centrifugal 10 points, discard unnecessary substratum, LB agar plate containing kantlex (50mg/ml) will be added to after the bacterium leaving about 200 microlitres gently pressure-vaccum number, even spread is to dry, be placed in 37 degree of incubator overnight incubation.
4, the Screening and Identification of recombinant clone
A) the bacterium colony PCR of recombinant clone screens: from transformation plate, picking 10 is separated good single bacterium colony, be seeded in 10 to be added with in the PCR pipe of sterilized water and fully to mix, drawing 5 microlitres respectively adds in the 1ml LB had containing kantlex (50mg/ml), makes corresponding numbering centrifuge tube and is used for 37 degree of shaking tables cultivations; 5 microlitres in other 10 PCR pipe, using by the template as bacterium colony PCR, carry out PCR reaction;
Cumulative volume 10 microlitre.
PCR response procedures:
When screening, the extension time can adjust according to expanded target gene fragment length, and cycle index also temporally can arrange adjustment; Carry out 1.2% agarose gel electrophoresis (10V/Cm glue) 20 points after PCR terminates, result gel imaging analysis, amplify gene product and size is person consistent with target gene fragment is be positive colony; According to the numbering of positive colony bacterium liquid in corresponding centrifuge tube is transferred to and is added with 5ml and spends the night containing shaking in the test tube of kantlex (50mg/ml) LB.
B) littlely put forward plasmid DNA: by plasmid DNA in a small amount extraction agent box illustrate that (Solarbio Inc) carries out: collect and spend the night bacterium 1.5ml in 1.5 Eppendorf tubes, the centrifugal 1 fractional precipitation bacterium of 10000rpm, abandon supernatant, add 0.25ml solution I fully to suspend bacterial sediment, adding solution II 0.25ml topples over for several times with mixing gently, room temperature is put 2 points (solution should be completely limpid), put upside down rapidly after adding 0.35ml solution III and mixedly make protein and genome Precipitation for 5-10 time, 12000rpm, 4 degree centrifugal 10 points, by supernatant gently to in adsorption column, 8000rpm, 4 degree centrifugal 30 seconds, being added in adsorption column by liquid in collection tube adsorbs once again, after centrifugal segregation carves liquid, 0.5ml rinsing liquid is added in adsorption column, 12000rpm, 4 degree centrifugal 1 point, repeat once, discard waste liquid in collection tube, by adsorption column 12000rpm again, 4 degree centrifugal 3 points, 50 ul sterile water (or 10mM Tris-EDTA solution is added to adsorption column central authorities, pH>8.0 is good), to be positioned in 60-70 degree baking box 5 points, 12000rpm after taking out, 4 degree centrifugal 2 points, collect the DNA plasmid of i.e. purifying in post, after measuring DNA concentration with Nano-1000 micro-spectrophotometer, next step endonuclease reaction can be carried out, redundance is placed in-20 degree refrigerators for subsequent use.
C) double digestion qualification recombinant plasmid: the double digestion reaction of recombinant plasmid (with pET41-HCVCore_191 citing) is composed as follows;
Reaction solution to be placed in 37 degree of water-baths 3 hours through mixing, carries out endonuclease reaction.After reaction terminates, carry out 1.2% agarose gel electrophoresis (10V/Cm glue) 20 points, result gel imaging analysis, can gene product be cut out and size is person consistent with target gene fragment is be positive colony, preserve plasmid; The bacterium that spends the night before this is glycerol stock: after 1.5 bacterium liquid collected by centrifugation, abandon supernatant add containing antibiotic containing 15-25% glycerine, preferentially the LB substratum of 20%, for subsequent use as frozen glycerine pipe after suspension.
The genetic expression of each segment encodes of embodiment 3 core antigen of C type hepatitis virus
1, the foundation of core antigen of C type hepatitis virus each segment encodes genetic expression bacterial strain
(1) conversion of BL21 bacterium competence bacterium
By through subclone and PCR and enzyme cut each recombinant expression vector pET41-HCVCore_N of qualification, pET41-HCVCore_M, pET41-HCVCore_C, pET41-HCVCore_120, pET41-HCVCore_191 and pET41a empty carrier transformed bacteria BL21: get aforementioned identification each recombinant plasmid 0.5 microlitre accurately, add 50 microlitre BL21 competence bacterias (Solarbio Inc), put 30 points on ice, be transferred to 42 degree of water-bath heat-shockeds after 90 seconds, move to and put 5 points on ice, the LB substratum 1ml without antiauxin is added in conversion tube, transfer to 37 degree of shaking tables and shake 45 points slowly, 5000rpm, 4 degree centrifugal 10 points, discard unnecessary substratum, LB agar plate containing kantlex (50mg/ml) will be added to after the bacterium leaving about 200 microlitres gently pressure-vaccum number, even spread is to dry, be placed in 37 degree of incubator overnight incubation.
(2) cultivation of transformed clone, self-induction are expressed
From growing and being separated good transformation plate, any picking 6 clone, be inoculated into and express substratum (0.02% glucose containing 3ml self-induction, the lactose of 0.2%, 50mM Na2HPO4,50mM NaH2PO4,10mM Mg2SO4,10mMMgCl2,2% peptone, 1.5% yeast extract) in, choose a transformed clone be inoculated in containing in kantlex (50mg/ml) and 2% glucose 3ml LB substratum as negative control, from empty carrier reformer plate choose a clone be inoculated in 3ml self-induction express substratum do positive vector contrast, after spending the night collect express 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 fully suspending, 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, after mixing, more than 85 degree are boiled 5 minutes, and sample is used for acryl amide electrophoresis loading (maybe can freeze in-20 degree for subsequent use).Acrylamide gel electrophoresis: normal compound 12% acrylamide gel, electrophoresis presses albumen Marker, negative control, vehicle Control, clone 1-6, albumen Marker loading, 60 volts of voltage stabilizing electrophoresis 30 points, adjust to 90 volts of voltage stabilizings 90 points end, after carrying out the dyeing of Coomassie light blue and decoloring process, take pictures under gel imaging system and the relative concentration of each albumen of scanning analysis, conclusive each relative expression levels, selects to express recombinant protein the higher person to preserve.
(4) high expression level seed bank is set up
Select the cultivation of the clone that expression of recombinant proteins is relative, switching 50ml substratum (LB of kantlex (50mg/ml) and 2% glucose) is cultivated at 37 degree of shaking tables, when thalline OD600 reaches about 1.5, sterile centrifugation collects bacterium, suspend fully with the LB containing 15% glycerine of 1/4 volume, by often pipe 1ml packing, frozen in-80 degree refrigerator, i.e. seed banks.
2, restructuring core antigen fragment expressing fusion protein
(1) self-induction expresses fermention medium preparation: express by aforementioned self-induction and cultivate preparation containing 0.02% glucose, the lactose of 0.2%, 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 packing.
(2) cultivation of seed liquor: get seed one pipe, access is added with the flask (500ml bottle) of the 100ml LB of the kantlex of 50mg/ml, and 37 degree, 250rpm shaking culture is spent the night.
(3) connect seed liquor by 5% inoculum size, add seed liquor to substratum, 26-32 degree, preferably with 28 degree, 300rpm shaken overnight carries out self-induction expression, expresses and terminates collected by centrifugation thalline, carries out next step purifying (or freeze save backup 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 expresses the chemical cracking of bacterium:
Thalline through abduction delivering adopts chemical cleavage method, adds 5-10ml bacterial lysate (2%Triton-X100,10-50ug/ml N,O-Diacetylmuramidase by every gram of wet bacterium; 1mM DTT; 0.2M PBS, preferably adopts the BugBuster solution of Merck & Co., Inc.) fully suspend thalline, and 37 degree are shaken incubation 1 hour slowly; In 4 degree of 12000 revs/min of high speed centrifugations 20 points, collect supernatant namely for next step affinitive layer purification.
2, adopt gravity method two step affinity chromatography with purification of target recombinant protein, purity is greater than 95%:
A) by above-mentioned after fully centrifugal supernatant, Ni2+-NTA affinity column is through sample-loading buffer (100mMNaH2PO4,10mMTris, 10mM2-ME, pH8.0) fully after balance, drip the supernatant of cracking, collect effluent liquid, get 10ul sample and analyze for SDS-PAGE.With elutriant I(100mM NaH2PO4,10mM Tris, pH6.3) fully wash the OD280=0.01. Fraction collection elutriant of post to effluent liquid, the sample got when 10ul wash-out starts is analyzed for SDS-PAGE.With elutriant II(100mM NaH2PO4,10mMTris, 500mM Imidazole, pH8.0) wash-out target protein: collect every 1mL fraction, get 10ul sample respectively and analyze for SDS-PAGE.Merge each pure component, to carry out next step purifying of GST affinity chromatography gel post.
B) with phosphate buffered saline buffer (the 4.5mM Na2HPO4.12H2O of 10 times of column volumes, 1.5mM KH2PO4,0.15MNaCl(pH7.3)) abundant balance columns, adds the sample after affinity chromatography, collects stream and wears component and be placed on ice.With 10x volume 1xGST Bind/Wash Buffer (4.5mM Na2HPO4.12H2O, 1.5mM KH2PO4,0.15M NaCl, 2.7mM KCl(pH7.3)) wash post, collect stream and wear component and be placed on ice.With 3x volume 1xGSTWash Buffer(4.5mM Na2HPO4.12H2O, 1.5mM KH2PO4,0.15M NaCl, 2.7mM KCl10mM GST (reduced form) (pH7.3)) wash-out target protein.Collection elution fraction is placed in treats following protein electrophoretic analysis on ice, and merges identical protein ingredient, frozen for subsequent use in-20 degree.
Sequence table
SEQUENCE LISTING
<110> Zhejiang Jiahe Pharmaceutical Co., Ltd.
<120> viruses of human hepatitis C cAg and its preparation method 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
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Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu
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Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu
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Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys
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Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn
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Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu
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Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
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Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu
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Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
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Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp
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Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu
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Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr
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Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala
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Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Gly Ser Thr Ser
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Gly Ser Gly His His His His His His Ser Ala Gly Leu Val Pro Arg
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Gly Ser Thr Ala Ile Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu
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Arg Gln His Met Asp Ser Pro Asp Leu Gly Thr Gly Gly Gly Ser Gly
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Asp Asp Asp Asp Lys Ser Pro Met Ala Ser Thr Asn Pro Lys Pro Gln
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Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe
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Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg
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Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser Glu Arg Ser
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Gln Leu Glu His His His His His His His His
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Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu
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Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu
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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
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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
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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 (2)
1. viruses of human hepatitis C's cAg, is characterized in that: its aminoacid sequence is as shown in SEQ ID NO:3, and its protein structure is soluble proteins, and its protein structure is close to natural folding mode.
2. prepare a method for viruses of human hepatitis C's cAg described in claim 1, it is characterized in that comprising the steps:
(1) expression vector is provided, this expression vector contains coding polypeptide according to claim 1 or protein molecular and the expression regulation sequence that is connected with this DNA molecular operability, with the plasmid pM-HCV_Genomeo containing HCV full-length gene group DNA for template, and design primer with for each fragment of core antigen gene, carry out pcr amplification, amplified production is through electrophoretic separation and glue reclaims corresponding specific amplified gene product, and be subcloned in pMD-18 carrier, obtain each gene recombination plasmid respectively, design primer and the cAg encoding gene increased respectively with different primers, gained two fragment PCR products is after running gel reclaims, take turns the template of PCR reaction as second than mixing by equimolecular, adopt combination of primers to proceed second and take turns PCR, with the total length core antigen gene encoding sequence that increases, obtain the total length encoding gene in inner Xho I site that suddenlyd change, wherein said expression vector is fusion expression vector, the fusion rotein antigen of expressing has correct folding structure, be present in kytoplasm with the target protein form of solubility, and target protein can obtain purifying protein by two step affinity chromatographies,
(2) with the expression vector transform both prokaryotic host cell described in step (1);
(3) prokaryotic host cell of culturing step (2) gained under the condition being applicable to described polypeptide or protein molecular antigen presentation, by the restriction endonuclease sites of design, from described fusion expression vector, double digestion obtains corresponding gene fragment, gene Subcloned technology is adopted to be connected by each gene pET41a obtained, build corresponding HCV-cAg expression vector, there is different pairing combination of primers carry out pcr amplification and obtain the expression vector of each genes encoding fragment, after Screening and Identification, recombinant plasmid transformed escherichia coli expression host BL21 bacterium, and carry out abduction delivering analysis, set up the fusion rotein antigen engineering bacteria of expression of HCV cAg, for the preparation of recombination fusion protein antigen,
(4) polypeptide described in separation and purification acquisition or protein molecular antigen.
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| Title |
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| HCV 核心区基因的截短及其表达;彭祥兵;《中华医药杂志》;20061231;第6卷(第11期);摘要，讨论，材料与方法 * |
| Proapoptotic and pronecrosis effect of different truncated hepatitis C virus core proteins;YAN Xue-bing;《Journal of Zhejiang University SCIENCE》;20051231;第6b卷(第4期);第1页右栏第1段 * |
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