CN1208769A - Synthesis and clone of composite polyvalent vaccine gene for type-C hepatitis virus - Google Patents

Abstract

The present invention optimizes five highly preservative T/B cell epi-position from HCV expression products and combines with a tenanus kind toxin (TT) external source exciting T cell epi-position and a proto nucleous translation enhancer into a section 270 bp complex polyvalence antigen gene, so that it establishes a foundation for expressing said vaccine gene effectively and inducing effective immune response.

Description

Synthetic and the clone of composite polyvalent vaccine gene for type-C hepatitis virus

This research belongs to biology field.

Hepatitis C is one of focus of studying in the world at present.Be named as hepatitis C virus from non A non B hepatitis virus in 1988, the research of the molecular biology aspect of pathology, diagnosis, treatment, prevention and the hepatitis C virus of relevant third liver is carried out rapidly, and has obtained a series of progress of advancing by leaps and bounds.In some countries, at present the infection rate of hepatitis C is higher, but does not have truly feasible treatment means, so the research of hepatitis C vaccine is that the control of hepatitis C has brought hope.

About 80% non-A non-B hepatitis is due to the hepatitis C virus (HCV), wherein has 60% hepatitis C to be chronicity again, and easily liver cirrhosis takes place, and in close relations with hepatocellular carcinoma, visible HCV harm is very big.Hepatitis C virus is the sub-thread positive chain RNA virus, is made up of about 9400 Nucleotide, drapes over one's shoulders coating outward, the lipid content height.HCV belongs to the Flavivirus in the Togaviridae on the taxonomy, and gene structure and flavivirus, plague virus are similar.From the analysis of evolving, HCV may be a class recombinant virus, and its C ' terminal amino acid comes from togavirus first section, and N ' brings in from picornavirus section.The HCV whole genome has only an ORF, the amyloid protein precursor of 3010,3011 or 3033 amino-acid residues of coding, and viral polypeptide just originates in its first methionine(Met).In the ORF from 5 ' end be followed successively by C district (core), E1 district (capsid), non-structural protein white area (NS) press 5 '--3 ' five genes of person NS1-NS5 arranged side by side.NS1 claims E2 district or E2/NS1 district again, and there is the hypervariable region of being made up of 25 or 30 amino acid (HVR) in N end in this district.For a long time, the treatment of HCV is never had the ideal way, so the development of hcv vaccine just seems very important, but because the restriction of multiple factor, the development of HCV vaccine is the progress of prominent property also now.

After Houghton in 1988 etc. separated first and cloned the partial sequence of HCV, people had just had more deep understanding to HCV.1991, hope such as Farci obtained the specificity protection by complete HCV virus immunity, but this method of results suggest can not provide the effective protection of IICV (Farci P et al.Hepatitis，1991；14：90A)。Spaete etc. have expressed E2/NS1 albumen in Chinese hamster ovary celI; and with 7 gorillas of a kind of adjuvant immunity; attack with the HCV strain again; wherein there are 2 to obtain protection; control group has then all infected HCV; pointing out this albumen may be a kind of target site of immunne response, can be used as candidate antigens (the Richard RS et al of HCV vaccine.Virology，1992；188：819)。Subsequently; Choo etc. have expressed E1 and the E2 albumen of IICV with vaccinia virus recombinant; and respectively immune 7 chimpanzees of these two kinds of albumen; the result wherein 5 titres that produced anti-HCV-E antibody above 1: 15000; and the attack to homophyletic HCV has excellent protection, and two titres are lower in addition, the of short duration HCV that infects; the anti-HCV-E antibody that prompting is produced may be neutralizing antibody, also need continue to observe (Choo QL et al but whether have intersecting protective or permanent immunity power etc.Proc?Natl?AcadSci?USA，1994；91：1294)。

In China, round pcr is then mainly adopted in the research of IICV vaccine, obtains to express and Studies on Immunogenicity behind the goal gene.China successively has the people to carry out the preliminary trial of HCV recombinant protein.The common people that open of Beijing Nat'l Pharmaceutical ﹠ Biological Products Control Institute wait and use fusion protein expression plasmid pGex, at expression in escherichia coli HCV core area different genes fragment, print and dye and ELISA has proved the antigenicity of HCV core protein with immune.With Qi Zibai etc. also at expression in escherichia coli the non-structure NS4 gene of part of HCV, and expressed IICV part NS4-NS5 gene with the DNA recombination method first at home, through the molecular hybridization analysis, expression product can be specifically and the non-structural area antibody response of anti-HCV.Third Military Medical University's attached southwestern hospital infectious disease training center Shen Ding rosy clouds etc. with HCV adventitia E2/NS1 gene clone to the pattern of fusion expression vector, in intestinal bacteria, express, the recombinant protein of expressing identifies to have the antigenicity of HCV outer membrane protein through Western Blot and ELISA, can with hepatitis c virus infection serum generation specificity association reaction, for the meaning of research outer membrane protein antibody has been created condition.Because the envelope protein of the gene structure of HCV envelope glycoprotein and hydrophobic collection of illustrative plates and flavivirus, pestivirus is closely similar; and flavivirus NS 1 albumen and pestivirus gp53/gp55 have confirmed to stimulate body to produce neutralizing antibody, and the protection body is not subjected to the effect of viral lethal hit.

But adopt round pcr to obtain the means of purpose expression product, can't provide effective protection to the HCV of rapid variation is then possibly, its subject matter is:

(1) HCV highly makes a variation: different with HBV is that the HCV virus strain very easily makes a variation, and has now measured tens of strain HCV complete genome sequences and more HCV RNA cloned sequence all over the world.HCV nucleic acid and amino acid height mutability, the homology of nucleotide sequence is 60%-92% only, so HCV has many different types and hypotype.Homology that it is generally acknowledged nucleotide sequence between a kind of viral different virus body is as adhering to different genotype separately less than 80%.Principle according to this, at present all over the world clone's HCV strain roughly be divided into 4 oligogene types (HCV I, II, III, IV), various amino acid and Nucleotide are formed similarity less than 80%, and almost are difficult to see two identical clones.

Different shaped HCV has certain regional distribution characteristics, the popular HCVI of American-European countries, and Japanese HCVI-IV type all has but based on the HCVII type, the strain in China and South Korea also belongs to the II type.The mutation rate of HCV RNA is 1000000 times that protokaryon and eukaryotic DNA duplicate mutation rate, make the isolating HCV strain in various places, even in same patient's body between isolating HCV clone nucleic acid homology all have necessarily even very big difference, show as the height heterogeneity of HCV.5 ' non-coding region, C, NS3 and NS5 genovariation are less in its genome, and the E2/NS1 gene especially makes a variation very big in the HV district of E2.This prompting has certain special mechanism to keep the relatively stable of HCV critical function gene, keeps the height mutation rate of some gene regions simultaneously again, with the needs that satisfy existence and evolve.RNA viruses is to conform and escape host's immune clearance and easily undergo mutation, and has formed new HCV mutant strain and independent separately the evolution gradually.The relative mutability of relatively stable and some gene of HCV critical function gene, it is highly variable that HCV is shown as, but can divide into typing and many hypotypes, and this also is the mechanism of HCV somatotype.Nucleic acid and viral protein variability are very big between various HCV strain, and especially the variation of E2/NS1 gene regions and coded product thereof is bigger.Weiner finds that E2HV district has in the linearity that is similar in the HIV-1gp120 albumen and antigenic determinant, and different shaped or hypotype even with the HCV clone in also incomplete same with antigenic determinant.The height volatility of HCV is the major reason that the hepatitis C chronicity and the state of an illness are shown effect repeatedly, and the reaction of the virulence of different shaped HCV and antagonism viral therapy is difference to some extent.Therefore, this hcv vaccine that just requires to make up should comprise the HCV strain of various different shaped and hypotype.In addition, because HCV type and hypotype much also mean do not have a kind of hcv vaccine can be general in the whole world probably, it is perhaps more more feasible to make up the HCV vaccine according to various places popular HCV strain.The high sex change of HCV may be to cause HCV to escape host's immunne response, makes body can not produce effective protection antibody, removes the major cause of HCV then.

(2) nonneutralizing antibody produces: still can detect transcribing of HCV RNA among most of anti-HCV male patients serum, obviously point out the relatively poor or basic unprotect of protectiveness of these antibody, and nonneutralizing antibody.Therefore prepared antiserum(antisera) does not usually have tangible anti-hcv activity after the present HCV recombinant vaccine immunity of developing.

(3) the HCV immunity is mainly cellular immunization, but the effect that cytotoxic T cell acts in the HCV vaccine is not subjected to abundant attention.

The objective of the invention is to design a kind of HCV composite polyvalent vaccine gene for type, efficient for preparing, safe, inexpensive, convenient, polyvalent HCV vaccine lays the first stone.

The present invention is according to HCV vaccine and HCV epi-position; the relevant report of determinant; taken into full account the existing problem of HCV vaccine research; the particularly polytropy of HCV; preferred protective epitope's polypeptide of 5 high conservatives in the HCV genome; respectively from core protein (1-16; 132-141); E1 (126-135); NS3 (139-147) and NS5 (768-775); because these epi-positions are equal high conservative in four type HCV; and be proved to be mostly and have good immunogenicity, so the expressed antigen of this complex gene is expected different shaped; the HCV of homophyletic does not provide provide protection, and has added a Toxoid,tetanus (TT) exogenous stimulation epi-position.We have designed the compound polyvalent antigen gene of a segment length for 270bp, and connect these epi-positions with glycine and proline(Pro), machine analysis as calculated, guaranteed that each epi-position is separate substantially, but and under the situation that does not influence amino acid code and intestinal bacteria recognition code, indivedual bases are changed, to delete some deleterious secondary structures.At 5 of gene ' end, designed the prokaryotic translational enhancer (TTAACTTTA) of a 9bp simultaneously, can efficiently express the purpose antigen gene.

The design of whole gene also fully take into account the T cell particularly the CTL effect to the influence of the immunne response of HCV vaccine.CTL is a kind of main defense mechanism that HCV infects, in the HCV chronic infection process, can discern the HCV antigen of expression of liver cell, help body to remove the liver cell that HCV infects, the HLA-I quasi-molecule of CTL by HCV infected liver cell surface combines with TXi Baoshouti and works.These HLA-I quasi-molecules mainly contain the frequency that A2.1, Aw68, B7 etc., particularly HLA-A2.1 occur in the crowd quite high, and Chinese, Japanese, Caucasian etc. are respectively 55%, 43%, 46%.Therefore the present invention in the HCV genome preferred 4 t cell epitopes (wherein 1 epi-position contains the B cell epitope simultaneously, another t cell epitope contains the partial sequence of a B cell epitope) and (5) Toxoid,tetanus (TT) exogenous stimulation epi-position, can be discerned by the CTL of restrictions such as HLA-A2, lay a good foundation for combination vaccine produces effective immunne response.The source of each epi-position, sequence and character are as follows:

Epi-position source epi-position aminoacid sequence epi-position character

C MTSTNPKPQRKTKRNTN(1-16) B

C DLMGYIPLVG(132-141) T

E1 RMAWDMMMW(317-326) T

NS3 TGDFDSVID(1445-1453) B/T

NS5 ELITSCSS(2781-2788) T(B)

TT IYSYFPSVD T

The present invention is by the compound polyvalent antigen gene of synthetic HCV, and reorganization, expression be the proteic antigenicity of research purpose also.Make up compound multivalence protokaryon of HCV and carrier for expression of eukaryon, transform multiple host (intestinal bacteria, attenuated salmonella typhimurium etc.), make up the strain of HCV vaccine candidate.

Preparation method of the present invention mainly realizes by following steps:

1. the splicing of gene: utilize chemical process to synthesize small segment, these gene fragments are spliced into the compound multivalent genetic of HCV of 270bp through minute step annealing.

2. screen positive recombinant clone by conversion, plasmid extraction, enzyme method such as cut, and gene is carried out sequential analysis identify.

3. the structure of expression vector: this gene is cloned into respectively on prokaryotic system floating preteins expression vector pKK223-3, fusion protein expression vector pWR450-1 and eukaryotic system expression vector pSV2 and the pREP9.

4. expression and the ammonium sulfate precipitation preliminary purification product of foreign gene in intestinal bacteria.

5. the recombinant plasmid electrotransformation transforms attenuated salmonella typhimurium, makes up reorganization oral live vaccine candidate strain.

Aforesaid method is method of the prior art, describes in detail among the embodiment.

Innovation part of the present invention is:

(1). at the height variability of HCV; preferred 5 have good immunogenic high conservative epi-position (seeing Table 1) in the HCV genome; respectively from core protein (1-16; 132-141); E1 (126-135); NS3 (139-147) and NS5 (768-775); because these epi-positions are equal high conservative in four type HCV; and be proved to be mostly and had good immunogenicity; therefore the expressed antigen of this complex gene is expected different shaped; the HCV of homophyletic does not provide provide protection; present acquired HCV candidate vaccine gene is then mostly to be to obtain by the PCR method amplification; because the height variability of HCV; therefore these fragments may be effectively to a certain type HCV; but to then possibility unprotect effect of other type HCV; therefore utilize the chemosynthesis gene engineering,, be better than adopting at present round pcr amplification HCV gene approach in theory the multivalent genetic that the high conservative epi-position of HCV gets up to obtain through strictness design heterozygosis.This method of design is not appeared in the newspapers in the HCV vaccine research as yet.

(2). the design of whole gene fully takes into account the characteristics of HCV immunity itself, the CTL effect of lay special stress on HLA restriction is to the influence of HCV vaccine immune response, select 4 can be by the t cell epitope (see figure 1) of HLA or MHC molecular recognition, in addition, also added one and be the t cell epitope of Toxoid,tetanus (IYSYFPSVD), this epi-position has been proved to be a kind of stronger external source t cell epitope.More the stressed cell immunity is ground at the HCV vaccine and may be brought out good immunne response, particularly cellullar immunologic response in the design of this vaccine, and this also is different from present stage HCV vaccine and relatively pays attention to humoral immunization.

(3). prokaryotic translational enhancer can strengthen expression of exogenous gene (tens of times to hundreds of times) to a great extent, the present invention has designed the prokaryotic translational enhancer of a 9bp at 5 of this complex gene ' end, can efficiently express the purpose antigen gene, the application of this prokaryotic translational enhancer in the HCV vaccine do not appear in the newspapers yet, this design has obtained embodying in the expression of this research, and the present invention has obtained to efficiently express the recombinant bacterial strain of HCV fusion rotein.

The compound polyvalent antigen gene of the artificial synthetic of the present invention is compared with the unit price polypeptide vaccine, may have stronger immunogenicity, the easier immune response that evokes body.The synthetic vaccine is attacked specificity because of the incomparable fabulous target site of traditional vaccine is arranged, and safe, inexpensive, multivalence and be easy to store, and is considered to the fresh combatants on the vaccine market in the future.

Fig. 1 is source and the aminoacid sequence and the character of the compound polyvalent antigen gene of HCV epi-position.

Fig. 2 is that the HCV antigen gene divides the step annealing connectivity scenario.

Fig. 3 is that the enzyme of pUCHC is cut evaluation figure.

Fig. 4 is the The sequencing results of HCV antigen gene.

Fig. 5 is the structure and the aminoacid sequence of the compound polyvalent antigen gene of HCV.

Fig. 6 is the structure of multiple recombinant vectors.

Fig. 7 is the amalgamation and expression figure of the compound polyvalent antigen gene of HCV at SL3261.

Fig. 8 is the amalgamation and expression figure of the compound polyvalent antigen gene of HCV at SL5928.

The more detailed implementation method of the present invention can be referring to embodiment.

Embodiment:

1. the splicing of gene: the every pipe 4OD of synthetic dna fragmentation, be dissolved in respectively among the 100 μ l0.5mol/L NaCl, concentration is 90 μ mol/L, carries out the branch step annealing by Fig. 1 scheme and connects.

Except that fragment F1 and fragment F8, all the other each fragments are respectively got 1 μ l, respectively F2 and F13, F3 and F12, F4 and F11, F5 and F10, F6 and F9 are mixed in the pipe, add 14 μ l ddH in each pipe respectively ₂O, 2 μ l, 10 * PNK buffer, 2 μ l 10mmol/L ATP, 0.5 μ l T4PNKinase; Each manages F7, F14 one, adds 7 μ l ddH respectively ₂O, 1 μ l, 10 * PNKbuffer, 1 μ l 10mmol/L ATP, 0.5 μ l T4 PNKinase, all pipes are 37 ℃ of reactions 30 minutes.In F7 and F14, add 1 μ l F8 and F1 respectively, respectively add 9 μ l ddH again ₂O, all 7 managed all boiling water bath 20 minutes, and then naturally cooling is annealed to room temperature (about 15 hours).Mix F1/F14 and F2/F13, F3/F12 and F4/F11, F5/F10 and F6/F9 and F7/F8 respectively, respectively add 1 μ l 10mmol/L ATP, 1 μ l T4 ligase, 20 ℃ of connections are spent the night; Remix F1/F14 and F2/F13 and F3/F12 and F4/F11, restock 1 μ l 10mmol/L ATP, 0.5 μ l T4ligase, 20 ℃ of connections are spent the night; The whole fragments of remix are replenished 1 μ l 10mmol/L ATP, 0.5 μ l T4 ligase, and 20 ℃ of connections are spent the night.Use T4 PNKinase phosphorylation two ends 5 ' end again after walking the final purpose fragment that glue reclaims 270bp by the following method, be connected with pUC119 through HindIII, BamHI double digestion, Transformed E .coli DH5 α screens recombinant clone.

14 single stranded DNA fragment assemblies are become the compound polyvalent antigen gene of third liver of 270bp by the branch step annealing connectivity scenario in the materials and methods part, after 2% agarose gel electrophoresis separation and purification, be connected with plasmid vector pUC119 through the HindIII/BamHI double digestion, transformed into escherichia coli DH5 α bacterial strain is identified containing on the ammonia benzyl LB flat board of IPTG and X-gal picking white colony extracting plasmid.Walk 1% agarose gel electrophoresis behind the HindIII/BamHI double digestion, positive colony has all cut out the band (Fig. 2) of 270bp, illustrates successfully to be cloned into the compound polyvalent antigen gene of third liver, this recombinant plasmid called after pUCHC.

2. insert embrane method and reclaim dna fragmentation: the sample that needs to reclaim purifying is in 2% sepharose after the electrophoretic separation, under ultraviolet lamp before the purpose fragment about 1mm place, cut one with electrophoretic band with wide breach, the place ahead glue is cut along the electrophoresis direction at the breach two ends, insert that a slice is sizeable boiled 10 minutes dialysis membrane through boiling water in advance, film is a little more than the gel face, continues electrophoresis 5-10 minute to target DNA and all is transferred on the film.The taking-up dialysis membrane is put in one and is equipped with in the Eppendorf pipe of 0.2ml TAE, the Vortex vibration, repeat to wash 1 time, collecting cumulative volume is the TAE leach liquor of 0.4ml, add 45 μ l (about 1/10 volume) 3mol/L NaAc, add the extracting of equal-volume chloroform once, the sucking-off supernatant liquor also adds the cold dehydrated alcohol of 2 times of volumes ,-20 ℃ of precipitations 30 minutes.Centrifugal 5 minutes of 12000rpm, the precipitation oven dry is dissolved in 10 μ l ddH ₂Among the O, be the target DNA fragment of recovery.

3. alkaline denaturation method extracting plasmid is routinely cut, connects and transform all to plasmid extraction, enzyme.

4. the screening of positive recombinant clone: with pUC119 is the first preliminary screening of carrying out on the flat board that contains IPTG and X-gal of transformant of carrier, and the picking white colony carries out the evaluation of extracting plasmid enzyme restriction again.With other plasmids is the direct extracting plasmid enzyme restriction evaluation transformant of carrier.

5. insert the fragments sequence analysis: essence is taken out to have and is inserted segmental positive colony plasmid (with pUC119 is carrier, Fig. 3), carry out the DNA automatic sequencing with the fluorescent mark universal primer on the pUC119, the result shows that the complex gene nucleotide sequence of being cloned into meets original design (Fig. 4,5) fully.

6. the structure of expression vector: for study the compound polyvalent antigen of this third liver in the panimmunity approach as the possibility of vaccine, need make it to obtain expressing at multiple expression system.By the figure scheme this gene is cloned into respectively on prokaryotic system floating preteins expression vector pKK223-3, fusion protein expression vector pWR450-1 and eukaryotic system expression vector pSV2 and the pREP9, enzyme is separately cut each recombinant expression vector of assay certificate and is all successfully constructed.

Go up the third liver complex antigen gene that obtains 246bp with EcoR I and the recovery of Pst I double digestion by pUCHC, be connected with same pKK223-3 or pWR450-1 plasmid through EcoR I and Pst I double digestion, transform DH5 α bacterial strain, picking transformant extracting plasmid is identified.Through EcoR I and Pst I double digestion, positive colony has all cut out the purpose fragment of 246bp, respectively called after pKKHC and pWRHC.

Go up the third liver complex antigen gene that obtains 270bp with Hind III and the recovery of BamH I double digestion by pUCHC, with be connected with the pSV2 plasmid of Bgl II double digestion (BamH I is an isoschizomers with Bgl II) through Hind III, transform DH5 α bacterial strain, picking transformant extracting plasmid is identified.Plasmid pSV2 cuts and will only produce band of 5.7kb through EcoR I enzyme, and positive colony is cut through EcoR I enzyme and then cut out 2.6kb and two bands of 3.1kb, proves that the clone is correct.Because the Hind III-Bgl II fragment of losing on the pSV2 just in time also is about 270bp, therefore on the plasmid size, positive colony is the same with vector plasmid.Naming this plasmid is pSVHC.

Go up with Hind III and BamH I double digestion by pUCHC and to reclaim the third liver complex antigen gene that obtains 270bp, and be connected with the pREP9 plasmid of BamH I double digestion through Hind III equally, conversion DH5 α bacterial strain, picking transformant extracting plasmid is identified.Through Hind III and BamH I double digestion, positive colony has all cut out the purpose fragment of 270bp.Plasmid pREP9 will produce 0.7kb and two bands of 9.8kb through Xba I and BamH I double digestion, and positive colony has cut out 0.97kb and two bands of 9.8kb through Xba I and BamH I double digestion, and further the proof clone is correct.Naming this plasmid is pREPHC.

7. fermentation expression and the product analysis of foreign gene in intestinal bacteria: be inoculated in 37 ℃ of activation of 2mlTB+Amp liquid nutrient medium and spend the night, get 50 μ l switching 5ml TB+Amp liquid nutrient medium next day, 37 ℃ of joltings 2 hours, add 5 μ l 2%IPTG, continue to cultivate 3 hours, packing Eppendorf pipe, the centrifuging and taking thalline adds 75 μ l ddH ₂O shakes out, and adds 100 μ l, 2 * Sample Buffer again, boiling water bath 10min, and centrifugal 20 seconds, supernatant was used for the SDS-PAGE electrophoresis detection, separation gel 12%.

8. the fusion rotein of ammonium sulfate precipitation preliminary purification hepatitis C antigen and LacZ: with the centrifugal receipts of the bacterium liquid bacterium that ferments, thalline ultrasonic wave damping fluid (50mmol/L TrisCl pH8.0,10mmol/L EDTA) washing is three times, finally be suspended in the ultrasonic wave damping fluid of 20 times of volumes, the ice bath state is used peak power ultrasonication 30 times down, each 30 seconds and intermittently 30 seconds.The sample of handling well was got supernatant in centrifugal 10 minutes in 4 ℃ of 6000rpm.In the Eppendorf pipe by calculated in advance good will be respectively by on a saturation ratio reach the required volume of ammonium sulfate of 10%, 20%, 30%, 40%, 50% saturation ratio and take by weighing ammonium sulfate, the ultrasonic supernatant liquor that in first pipe, adds 1.5ml, treat to vibrate again 30 minutes after ammonium sulfate dissolves fully, centrifugal 5 minutes of 12000rpm, supernatant is poured second pipe into, the same processing until the 5th pipe.All five pipe precipitations are all used 20 μ l ddH ₂The O dissolving adds 20 μ l, 2 * Sample Buffer, boils cooling back SDS-PAGE electrophoresis detection 5 minutes.

9. electrotransformation transforms attenuated salmonella typhimurium: the picking Salmonellas spends the night in 37 ℃ of activation of 2ml LB liquid nutrient medium, get 40 μ l switching 50ml LB liquid nutrient medium next day, 37 ℃ of jolting to OD values are 0.6-0.7 (about 2-2.5 hour), received bacterium in centrifugal 5 minutes in 4 ℃ of 4000rpm, under ice bath, wash 3 times with 10% glycerine, last thalline is suspended in 2ml 10% glycerine, is competence.Get 200 μ l competence, add 1 μ l plasmid DNA, adsorbed on ice 5 minutes, move in the electric shock cup 2000V electric shock, electric capacity 25 μ F, resistance 200 Ω, 4 milliseconds of discharge times.The electric shock back adds 400 μ l LB, shaking culture 2 hours, coating LB flat board.

10. the fermentation expression of foreign gene in the attenuation salmonella typhi and product analysis: identical with the fermentation expression in intestinal bacteria, just there is not lacIq in the Salmonellas, needn't add IPTG and induce.

Because attenuated salmonella typhimurium expression strain S.typhimurium SL3261 and S.dublin SL5928 are restricted bacterial strain, therefore need pWRHC and pWR450-I plasmid

Transform earlier non-limiting but the bacterial strain S.typhimurium LB5000 of modification property is arranged, and then therefrom the extracting plasmid transforms SL3261 and SL5928.

May be because foreign protein has toxicity (do not have lacIq in the Salmonellas, foreign protein is a constitutive expression) to the host bacterium, or because the vitality problem of host bacterium itself, with usual method such as CaCl ₂Method is difficult to the exogenous protein expression plasmid is imported Salmonellas.Electrotransformation has just solved this problem well, has all obtained transformant in LB5000, SL3261 and three bacterial strains of SL5928.

SL3261/pWRHC and SL5928/pWRHC reorganization bacterium are carried out fermentation expression and product analysis, respectively in contrast with SL3261/pWR450-I and SL5928/pWR450-I, special expressing fusion protein band all occurred at the 60KD place, the LacZ of contrast is 55KD.Expression amount similar in intestinal bacteria is about 10%-20% (Fig. 7,8).

The pWRHC plasmid changed among intestinal bacteria different strains TG1, JM109, the DE3 etc. express, find only in TG1, to express better, and induce with IPTG after 2 hours at switching kind of the daughter bacteria that spends the night, induce after 3 hours and receive bacterium, expression amount is the highest, and fusion rotein accounts for the 10%-20% of tropina.

The pWR450-1 plasmid is the protein expression vector that has strong Lac promotor, the beta-galactosidase enzymes (LacZ) of expressing on it is 55KD, hepatitis C antigen is located beginning at the 49KD of LacZ (447 amino acid) and is merged with it, produces the LacZ-HC fusion rotein of about 60KD.

Be used for the experiment of immunology in order to obtain this fusion rotein from now on, we have tentatively groped LacZ-HC albumen is carried out the condition of separation and purification.The utilization ammonium sulfate precipitation, we by polyacrylamide gel electrophoresis detected respectively the TG1/pWRHC thalline behind ultrasonic disruption soluble proteins 10%, 20%, 30%, 40%, precipitated product under the 50% ammonium sulfate saturation ratio finds that the tunning LacZ-HC of 60KD mainly appears in the throw out of 50% saturation ratio.Therefore, can be earlier once with the ammonium sulfate precipitation of 40% saturation ratio with tropina, get to precipitate at 50% saturation ratio post precipitation again and be further purified with removing about 70% foreign protein like this.

Claims (2)

1. a hepatitis C virus (HCV) vaccine gene; it is characterized in that preferred protective epitope's polypeptide of 5 high conservatives from the HCV genome; comprise 4 t cell epitopes; one of them epi-position contains the B cell epitope simultaneously; another t cell epitope contains partial sequence and B cell epitope of a B cell epitope; the prokaryotic translational enhancer that has added a Toxoid,tetanus (TT) exogenous stimulation epi-position and a 9bp in addition; these epi-positions have been combined into the compound polyvalent antigen gene of a segment length for 270bp; connect with glycine and proline(Pro) between each epi-position, each epi-position is selected as follows:

Epi-position source epi-position aminoacid sequence epi-position character

C MTSTNPKPQRKTKRNTN(1-16) B

C DLMGYIPLVG(132-141) T

E1 RMAWDMMMW(317-326) T

NS3 TGDFDSVID(1445-1453) B/T

NS5 ELITSCSS(2781-2788) T(B)

TT IYSYFPSVD T

2.HCV the cloning and expression method of vaccine gene comprises the splicing of gene, transforms, and the screening of positive recombinant, fermentations etc. is characterized in that:

(1) by dividing step annealing to connect, institute's synthetic gene small segment is spliced into the purpose fragment of designed 270bp;

(2) screen positive recombinant clone by conversion, plasmid extraction, enzyme method such as cut, and gene is identified;

(3) make up multiple eucaryon and prokaryotic expression carrier;

(4) this gene obtains preliminary expression product and expression product is carried out the preliminary purification analysis intestinal bacteria;

(5) make up recombination attenuated salmonella typhimurium oral live vaccine candidate strain.

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