CN1059927C - Reconstituted hepatitis B vaccine with corboxy end having anterior surface antigen 1 determinant - Google Patents

Abstract

The present invention relates to a new hepatitis B vaccine produced by a gene recombination technology. A gene fragment of an antigen determinant of preS1(21-47), which comprises a liver cell conjunction site, and the terminal 3' of a main protein S gene of a hepatitis B virus surface antigen are fused together; the fusion gene is inserted into an expression carrier of pox vaccine, and a recombined vSA-28 virus of pox vaccine is constructed. The recombined virus is capable of stably expressing fusion granules of a hepatitis B surface antigen and a prsS1 antigen and has high double immunogenicity, secretiveness and stability. Used as a new subunit granular hepatitis B vaccine, the hepatitis B vaccine is used for preventing and curing hepatitis B, and the hepatitis B vaccine can also be used for preparing a new hepatitis B live vaccine.

Description

Hepatitis B surface antigen blending gene and the proteins encoded thereof of corboxy end having anterior surface antigen 1 antigenic determinant

Hepatitis B is a kind of serious harm human health and popular disease widely, particularly in developing country.In China, the hepatitis B virus carriers reaches more than 100,000,000, causes death toll huge by hepatitis B or hepatitis B diseases associated, and is very harmful, and general vaccination is the important measures of control hepatitis B.

Having the virion that 42nm has infection in some patients serum that hepatitis B virus (HBV) infects, is to be surface antigen (HBsAg) by viral capsid proteins, cAg (HBcAg) and viral nucleic acid (DNA) composition.Do not have the spherical and rod-shpaed particle of infective 20-22nm in addition in addition, only contain hepatitis B surface antigen.It can be induced and produce special hepatitis B virus resisting antibody, makes human body produce immunizing power.By separating HBsAg among the patients serum, the vaccine from blood of making, since the source difficulty, the cost costliness, poor stability, not ideal enough.

Utilize genetic engineering technique, in different systems: yeast, mammalian cell, recombinant vaccinias etc. are expressed hepatitis B surface antigen, as recombinant vaccine, than vaccine from blood safer, economical, effectively, but much at one, thereby still have the some shortcomings part: as 1) community immunity hyporeactive or the non-responsiveness of 5-10% with regard to its immunogenic essence and vaccine from blood.2) required antigen amount is big.3) immune persistence 4-5.Therefore the hepatitis vaccine of studying efficient economy is very necessary.

Hepatitis B surface antigen has three kinds of protein ingredients: main albumen (S), middle albumen (M) and large protein (L), these three kinds of albumen are by the dna encoding of same reading frame among the HBV, from the different initial translations of initial code ATG, difference is that M albumen Duoed 55 amino acid than S albumen at the ammonia end, be anterior surface antigen 2 (preS2) district, and L albumen Duo 119 amino acid than M albumen at the ammonia end, promptly anterior surface antigen 1 (preS1) is distinguished.M and L albumen mainly are present in the shell of virion.

PreS1 contains in the district antigenic determinant that is different from S.The preS1 district has the immunogenicity on T cell and the b cell level; the amino acid whose peptide section of ammonia end 21-47 wherein; can combine cell 1986:46.429-436 such as () Neurath A.R. with liver plasma membrane and have the ability that produces neutralizing antibody of inducing; thereby can combine with hepatocellular by inhibition of hepatitis b virus, not infected with the protection body.L albumen can activate the T cell special to preS1 in addition, overcomes S albumen and M proteic not reactive.But L albumen is difficult for secreting from cell, and has suppressed the proteic secretion of S (Persing, P.H. etc., Science, 1986,234,1388).18 amino-acid sequences of the Semen Myristicae acidifying of ammonia end and ammonia end all are to influence the excretory reason, and L albumen easily is degraded simultaneously.Therefore will obtain to contain the proteic vaccine of L has certain difficulty.

Studies confirm that with synthetic preS1 (21-47) polypeptide, this zone is contained HBV and is combined necessary site with liver cell, anti-preS1 (21-47) monoclonal antibody MA18/7 and F35.25 can suppress HBV effectively and combine with hepatocellular, ability (Petit M.A. etc. with neutralization virus, Virology 1991,180.483-491).The immunity orangutan can make its attack of resisting HBV (Neurath A.R. etc., Vaccine, 1989,7,234-236).But as vaccine, immunogenicity is low, apart from practical application very big distance is arranged still with synthetic polypeptide.

Recently, utilize hepatitis B surface antigen to make carrier proteins, successfully with exogenous genetic fragment, as: human herpes simplex vicus I type (HSV-I) (Valenzuela, P. etc., Bio-Technology, 1985,3,323.), human immunodeficiency virus (HIV) (Michel, M-L etc., Proc.Natl.Acad.Sci.USA, 1988,85,7957.), poliovirus (polio) (Delpeyroux, F. etc., Science 1986,233,472) all insert the preS2 district, in yeast or mammalian cell, obtain to express.Therefore, by the way that preS1 immunologic determinants and HBsAg master's albumen are merged, the hepatitis vaccine that the obtains band preS1 new technological line of can yet be regarded as.

People such as R.E.Streeck once reported, with preS1 ammonia terminal amino acid order 1-42, the 223rd of hepatitis B surface antigen, added 5 amino acid whose joints and merged with it, and under the control of SV40 promotor, transformant has obtained temporary transient expression.Though they also find in cell and training liquid in the expression of S and preS1 is all arranged, can form particle, fail to obtain stable expression system and to the immunogenicity of expression product study (Machein L, etc., Arch.Virol, 1992,4,133-136).In addition, 20 amino acid of N end may influence the secretion of expression product in the fragment (1-42) that they use.

The object of the present invention is to provide the novel hepatitis B recombinant antigen of the brand new of an energy inhibition of hepatitis b virus infection, can can stably express the fusion particle of justacrine hepatitis B surface antigen and preS1 by a vaccinia virus recombinant (Vaccinia Virus) that contains the epitopes gene fusion of hepatitis B surface antigen S gene and preS1 liver cell binding site.Particle has the dual antigenicity of hepatitis B HBsAg and preS1 simultaneously, the immunogenicity that tool is good, and immune animal can produce the dual antibody of high titre rapidly, and this has just possessed the possibility that develops into efficient, quick-acting hepatitis vaccines.The present invention can secrete the fusion particle that produces HBsAg and preS1 in a large number by a vaccinia virus recombinant.Through separation and purification, can be made into safer novel efficiently hepatitis B gene engineering particle vaccines.This vaccinia virus recombinant also can be used for being prepared into new hepatitis b vaccine.

We utilize round pcr, synthetic the nucleotide sequence of corresponding amino acid 21-47 in the preS1 gene, having removed to influence 20 amino acid of S antigen excretory ammonia end.At hbsag gene carboxylic end, merge mutually with synthetic preS1 nucleotide fragments in the 223rd in corresponding amino acid.Obtained to contain the fusion gene of hepatitis B surface antigen S gene and preS1 (21-47).And made up the vaccinia virus recombinant that contains this fusion gene, successfully expressed the dual antigenic particle of band HBsAg and preS1.The design that experiment showed, us influences neither that the hepatitis B surface antigen particulate forms and from the secretion of cell, does not also influence the immunogenicity of HBsAg, but has the immunogenicity of preS1 simultaneously.Immune animal can produce the antibody of anti-HBsAg and anti-preS1 high-levelly.

This fusion gene also can method routinely changes in the eukaryotic system such as mammalian cell (as CHO) yeast and obtains to express, and the essential property of its expression product should be identical with the expression product in recombinant vaccinia.

This has the epitopes gene of liver cell binding site and the fusion gene of surface antigen, also can dna form, be used to prepare the vaccine of hepatitis B gene immunity and gene therapy.

The invention provides a hepatitis B antigen with the tool novel texture of HBsAg master's albumen carboxylic end and the fusion of preS1 antigenic determinant, can the stably express secretion obtain this novel hepatitis B antigen, be that HBsAg master's albumen and preS1 albumen merge the particulate method by setting up a recombinant vaccinia, comprising:

1. the synthetic and clone (pWR/PS1) of PCR method who contains the preS1 antigenic determinant (21-47) of liver cell binding site.

2. the clone of hbsag gene (S) carboxylic end and synthetic preS1 (21-47) gene fusion makes up (pUC/PSA-28).

3. the structure (pGJPSA-28) that contains the recombinant vaccinia expression plasmid of fusion gene.

4. contain the structure and the screening (vSA-28) of the vaccinia virus recombinant of hepatitis B S gene and preS1 (21-47) gene.

5. the expression of the hepatitis B surface antigen of the propagation of vaccinia virus recombinant and band preS1.

Schematic view illustrating of the present invention is as follows:

Fig. 1. contain the synthetic and clone's synoptic diagram of PCR of preS1 antigenic determinant (21-47) gene of liver cell binding site.

Nomenclature among the figure: St:Stu I (restriction enzyme); X:Xba I; Pv:Pvu II; E:EcoR I; S:Sac I; Sm:Sma I; B:BamH I; Sa:Sal I; Ps:Pst I; H:HindIII.

The fusion of Fig. 2 A. hepatitis B S gene 3 ' end and preS1 (21-47) and clone's synoptic diagram.

Nomenclature among the figure:

The S gene,

PreS1; Ac:Acc I; The same Fig. 1 of surplus person.

Fig. 2 B. hepatitis B S gene 3 ' end and the clone synoptic diagram of preS1 (21-47) fusion on the bovine vaccine expression vector.

Fig. 3. ECL development photo is identified in the Southern hybridization of vaccinia virus recombinant vSA-28.

1. the Temple of Heaven strain, 2.pGJPSA-28,3.vSA-28.

Fig. 4. vaccinia virus recombinant vSA-28 expression product tool S antigen and the dual antigenicity of preS1.

In Fig. 4 A. nutrient solution and the cell pyrolysis liquid ³⁵The hepatitis B surface antigen of S methionine(Met) mark and anti-HBsAg immunoprecipitation are through the radioactive automatic developing figure of protein electrophoresis.

In Fig. 4 B. nutrient solution and the cell pyrolysis liquid ³⁵The hepatitis B preS1 antigen of S methionine(Met) mark and anti-preS1 monoclonal antibody MA18/7 immunoprecipitation are through the autoradiogram(ARGM) of protein electrophoresis.

Among the figure: the Mock contrast; The recombinant vaccinia vTH-2 of S band HBsAg; SA-28 recombinant vaccinia vSA-28.

M. S and the SA-28 albumen of representing glucosidesization and non-glucosidesization for standard molecular weight m. for nutrient solution C. for the cell pyrolysis liquid arrow

Fig. 5. the secretion mensuration of vaccinia virus recombinant vSA-28 expression product.In different cells (CV-1, Vero, CEC) comparison of middle secretion property level.

Nutrient solution; Cell splits Jie's liquid.

Fig. 6. vaccinia virus recombinant vSA-28 expression product graininess is identified.

Fig. 6 A.vTH-2 (on) and the super centrifugal mensuration of vSA-28 (descending) fusion particulate CsCl density.

Fig. 6 B.vSA-28 merges particulate electron microscope observation photo (* 12000).

Fig. 7. the immunogenicity determining of vaccinia virus recombinant vSA-28 expression product.

Content of the present invention specifically describes as follows:

1.preS1 (21-47) the synthetic and clone of the PCR of gene fragment;

Our selected hepatitis B virus adr hypotype contains the preS1 antigenic determinant of liver cell binding site, and template is made with the clone who contains the preS1 gene in amino acid sequence number 21-47 position, has synthesized the corresponding nucleotide sequence with preS1 (21-47) with the PCR method.For ease of the clone, introduce a StuI and XbaI enzyme cutting point respectively at this segmental 5 ' end and 3 ' end.The PCR product behind above-mentioned enzyme double enzymolysis, be cloned into the pWR13 plasmid (Guo Lihe of Shanghai Inst. of Cytobiology, Chinese Academy of Sciences professor institute gives, see Guo Li and etc., biotechnology journal,, the 1st volume, page 4 in 1985 ") PWR/PS1.Verifying the standby (see figure 1) in back by sequential determination.

2. hbsag gene (adr type) carboxylic end and synthetic preS1 (21-47) gene fusion are cloned

On the clone who contains preS1 (21-47), the XbaI point of contact is opened, mend flat reconnecting again, cause a stop code TAG at preS1 (21-47) fragment 3 ' end.After merging at preS1 (21-47) fragment 5 ' end and hbsag gene, still keep reading frame correct, at preS1 fragment 5 ' end, load onto the long SmaI joint of 10 oligonucleoside, and at S gene 3 ' end, corresponding the 223rd amino acid place, it is AccI enzyme point of contact, cut benefit through same enzyme and put down, cause tack to be connected, finish the gene fusion of hbsag gene and preS1 (21-47) with preS1 (21-47) ammonia end SmaI joint.The recombinant plasmid that contains preS1 and HBsAg223 position fusion gene is PUC/SA-28 (seeing Fig. 2 A).

3. the structure of fusion gene on the recombinant vaccinia expression plasmid

For the fusion gene of realization design and the reorganization of vaccinia virus, at first fusion gene must be inserted the bovine vaccine expression plasmid.We select successfully to be used for the bovine vaccine universal expression plasmid pGJP-5 (Wu Xue etc., Acta Biochimica et Biophysica Sinica 1987,19 (5) 395) that various exogenous genes is expressed.This plasmid has bovine vaccine promotor p7.5 and bovine vaccine TK gene order, has several cloning sites therebetween, if insert foreign gene in the centre, can cause the inactivation of TK gene, and can be used as the selective marker of recombinant virus.Also have penbritin (APr) resistance mark in addition, can be used as the recon selective marker when setting up the clone in the intestinal bacteria.To be inserted in the fusion gene that BamHI downcuts on the BamHI site of pGJP-5 plasmid, get recombinant plasmid pGJPSA-28 (seeing Fig. 2 B).We change recombinant plasmid in the intestinal bacteria over to and preserve, and this bacterial classification is that Escherichia coli TG-1/PGJPSA-28 deposits mark CCTCCNO:M93074.

4. contain the reorganization of S and preS1 (21-47) gene fusion and low toxicity vaccinia virus Tiantan strain and the screening of vaccinia virus.

We select known low toxicity vaccinia virus strain for use, and the Temple of Heaven strain (Vaccinia VirusTian Tan) is obtained by Ministry of Health of the People's Republic of China's Beijing institute of Biological Products.Be characterized in that when consequent vaccinia virus recombinant was used as living vaccine, its toxicity was low, this is proved by secular clinical practice.

Recombinant vaccinia expression plasmid pGJPSA-28 is taken out from bacterial classification CCTCC NO:93074, press the plasmid DNA and the Temple of Heaven vaccinia virus of the concrete narration preparation method acquisition of following embodiment and introduce the CV-1 cell, because TK gene and bovine vaccine TK gene on the expression plasmid have DNA sequence homologous and avidity each other, thereby might homology exchange, and reorganization in the generation body, the fusion gene that also is about to TK gene the bovine vaccine promotor p7.5 that is comprised and the hepatitis B surface antigen S that links to each other of inactivation on the expression plasmid and preS1 (21-47) is recombinated in the vaccinia virus gene group, again by to the repeatedly selection of TK-, get final product the vaccinia virus recombinant vSA-28 (seeing Fig. 2 B) of purifying.

5. the propagation of vaccinia virus recombinant and have the expression of preS1 hepatitis B surface antigen.

Through the vaccinia virus recombinant vSA-28 of screening, and behind the analysis verification by Southern hybridization and product, infect Vero, CV-1 or CEC cell can carry out viral propagation, with a large amount of acquisition vaccinia virus recombinants.Can express justacrine behind the recombinant vaccinia virus infection cell and have the hepatitis B surface antigen particle of the fusion of S and preS1 dual immunogenicity, can be used for producing efficient and novel gene engineering Hepatitis B virus vaccine.VSA-28 also can be used as living vaccine to be used.

Content of the present invention is in the concrete narration of following examples:

Material and method:

A. the used restriction enzyme of gene recombination, ligase enzyme, the Klenow polysaccharase, the SmaI joint is all the Boehringer product;

Cell culture medium, the high glucose training of DMEM/HG liquid, foetal calf serum (FCS), no methionine(Met) training liquid (RPM1-1640, Met-Free), (Met-Free FCS) is all the GIBCO product to no methionine(Met) foetal calf serum;

5-bromouracil deoxyribose (BUdR) is the FLUKA product;

³⁵S-mark methionine(Met) (L- ³⁵S-Met) be the Amersham product;

PCR is the Biolabs product with test kit and Tag archaeal dna polymerase;

B. used thymidine kinase (TK) gene defection type cell Human Tk-143 cell is cultivated in the DMEM substratum that contains 25 μ g/ml BUdR and 5% foetal calf serum.Vaccinia virus Tiantan strain (Vaccinia Virus Tian Tan) is provided by Ministry of Health's Beijing institute of Biological Products.Former generation chick-embryo cell (CEC) made the preparation of chicken embryo cultivate in the DMEM of 10% calf serum with 10 days.The CV-1 cell is provided by the general biochemical institute of German horse, and the Vero cell is provided by professor Wen Yumei of Shanghai Medical Univ.

C. the transfection of cell.The CV-1 cell after vaccinia virus Tiantan strain infects two hours, with DNA (containing 10 μ g plasmids and the 10 μ g milt DNA) transfection of coprecipitation of calcium phosphate, is cultivated after 5 hours for 37 ℃, inhales and removes transfection liquid, adds fresh training liquid, continues to cultivate harvested cell after 48 hours.

D. the preparation of plasmid DNA.Use alkaline denaturation, through Sepharose 2B purifying.

E. the reorganization of plasmid and bacterium transform.With reference to Sambrook, the method on people such as J. " molecular cloning " book is carried out, and used recipient bacterium is TG-1 or JM105.

F. the screening of vaccinia virus recombinant and viral DNA extracting.Vaccinia virus DNA preparation is carried out referring to document (J.Virol.Meth 1980,2 for Espostto, J. etc., 175) method.The TK-recombinant virus in the presence of BUdR, selects plaque also to carry out Analysis and Identification according to the method (Weir, J.P. etc., Proc.Natl.Acd.Sci.USA1982,79,1210) of Weir etc.

The mensuration of g.HBsAg and preS1.The mensuration of HBsAg is put inspection-free survey medicine box with AUSRIA medicine box (Abbott company product) or Beijing institute of Biological Products hepatitis B surface antigen.The antigenic mensuration of preS1 adopts Organon Teknika company to detect medicine box, or is undertaken by the ELISA detection reagent that the Zhang Zuchuan of this institute group provides.Secrete the antigen to the nutrient solution, can directly measure with training liquid or intracellular antigen after PBS suspends, with dry ice freeze thawing four times, the supernatant liquor after centrifugal is a frozen-thawed cell liquid sample, also the lysate sample of available cell is measured (referring to method I item).Used anti-preS1 monoclonal antibody (MA18/7) is given by Germany doctor W.H.Gerlich.

H. the protein electrophoresis analysis of expression product.Adopt the sds polyacrylamide electrophoresis to carry out (Laemmli, Nature such as U.K., 1970,227.680) with reference to the Laemmli method.

I. expression product ³⁵S-methionine(Met) mark and immunoprecipitation.Get after the transfection of calcium phosphate precipitation method 20 hours cell, remove to train liquid, after the washing of no methionine(Met) training liquid, in no methionine(Met) FCS and no methionine(Met) training liquid, cultivated 1 hour, again after no methionine(Met) is trained liquid and is washed, adding 50-80uci ³⁵The methionine(Met) of S-mark was cultivated 3 hours, changed DMEM/HG training liquid then into and spent the night.Collect the cell of mark, in the 0.8mlHypotonic damping fluid, add 0.2ml 5 * lysis damping fluid again through the PBS of precooling washing secondary rear overhang, jolting, the supernatant-20 ℃ preservation of centrifugal back is standby to be cell pyrolysis liquid.

The cell pyrolysis liquid of mark or cell training liquid adds S antibody or anti-preS1 antibody respectively, mixing, 0 ℃ after 30 minutes, the centrifugal supernatant of abandoning.Precipitation is given a baby a bath on the third day after its birth inferior with the RIPA damping fluid, be suspended from sample-loading buffer, 100 ℃ 4 minutes, centrifugal collection supernatant carries out electrophoretic analysis.After running gel is drained, the compressing tablet autography.

J.ECL nonradioactive labeling detection reagent and marking operation method, the operation steps that provides referring to Amersham company in detail.

Following substep is described the concrete steps of implementing:

Embodiment 1, contains the synthetic and clone of preS1 (21-47) epitopes gene of liver cell binding site.

We select the preS1 antigenic determinant that contains the liver cell binding site, amino acid sequence number 21-47 position., adopt the PCR method to increase and contain the dna fragmentation of the preS1 of 21-47 position as template (pUC/LS-1) with the recombinant plasmid dna that contains preS1, used 5 ' end primer is: 5 ' CTTTCTGTTCCCAGGCCTCTGGG 3 '

Long altogether 23 Nucleotide of StuI, the StuI enzyme point of contact order that wherein line part is introduced for design.3 ' end primer is: 5 ' CTGGCCAGTGATCTCTAGAGGGTTGAAG 3 '

Long altogether 29 Nucleotide of XbaI, the XbaI point of contact order that wherein line part is introduced for design.The PCR reaction system is referring to the Biolabs method.The fragment of pcr amplification is inserted on the StuI and XbaI position of plasmid pWR13 after using SmI and XbaI double enzymolysis, must contain the recombinant clone pWR/PS1 of preS1 (21-47), and preS1 (21-47) measures the checking (see figure 1) through DNA sequence.

Embodiment 2, the establishment of preS1 (21-47) and S gene fusion clone pUC/SA-28.

For realizing the fusion of preS1 (21-47) fragment and S antigen carboxylic end, we at first will contain the plasmid (pWR/PS1) of preS1 (21-47) gene fragment, use the XbaI enzymolysis, with big fragment polysaccharase it be mended flat back again and connect, cause a stop code TAG, simultaneously the XbaI point of contact is lost.For keeping correct to preS1 (21-47) fragment reading frame by the S gene, we go up place, StuI enzyme point of contact behind the StuI enzymolysis at preS1 (21-47), add that 10 Nucleotide SmaI joints get pUC/10 preS1 (21-47) TAG in addition.

It is the segmental merging point of preS1 (21-47) that selected S gene pairs is answered AccI point of contact, amino acid sequence number the 223rd place.The recombinant plasmid pWR/HBS-4 △ SalI that will have the S gene cuts the AccI enzyme earlier and opens, again through the klenow fragment polishing.Again 3 ' end being mended flat S gene downcuts with the SacI enzyme, be inserted on the site of the SacI of pUC/10 preS1 (21-47) TAG and SmaI double enzymolysis, produce pUC/SA-28, through sequential determination, checking S gene and preS1 (21-47) fusion place framework are correct, and after preS1 (21-47) carboxylic end contains stop code, make up the recombinant vaccinia expression plasmid (seeing Fig. 2 A) that contains fusion gene again.

Embodiment 3, contain the structure of the recombinant vaccinia expression plasmid pGJPSA-28 of S gene and preS1 (21-47) gene fusion.

Recombinant plasmid pUC/SA-28, behind the BamHI enzymolysis, intactly tell the dna fragmentation that contains S gene and preS1 (21-47) fusion, directly insert the recombinant vaccinia expression plasmid pGJP-5 that cuts through the BamHI enzyme, get recombinant plasmid pGJPSA-28, return through BamHI and to cut, must be about the fusion gene of 0.78Kb.The upstream of this recombinant plasmid is followed successively by the TK gene like this, bovine vaccine promotor p7.5, and ATGS (1-223)+Val, Trp, Gly+preS1 (21-47)+Ser, Ser+TAG, plasmid use the enzyme point of contact more, the TK gene.Recombinant plasmid pGJPSA-28 also has APr resistance mark, can transform in intestinal bacteria (as TG-1), selects and amplification, with preparation recombinant plasmid dna (seeing Fig. 2 B).The intestinal bacteria Escherichia coliTG-1/pGJPSA-28 that contains recombinant plasmid pGJPSA-28 is stored in and is numbered CCTCC NO:93074.

Embodiment 4, contain the screening of vaccinia virus recombinant of S and preS1 (21-47) gene fusion and the evaluation of vSA-28.

The bovine vaccine of reorganization is by recombinant vaccinia expression plasmid pGJPSA-28 and the Temple of Heaven strain virus reorganization to take place in the CV-1 cell paste to produce.By infecting the TK-143 cell, in the presence of BUdR, select the recombinant vaccinia plaque again.Owing to be inserted with p7.5 promotor and fusion gene in the expression plasmid TK gene, TK loses activity, be TK-, after the reorganization, vaccinia virus recombinant also is TK-in body takes place, and also is that BUdR exists down, have only the TK-of reorganization to survive, through twice plaque purifying, and through HBsAg and preS1 antigen presentation be determined as the positive after, select the vaccinia virus vSA-28 obtain recombinating.

Vaccinia virus recombinant vSA-28 has been carried out Southern blot on dna level identify.

Preparation vSA-28 DNA is contrast with the expression plasmid pGJPSA-28 DNA of Tiantan strain vaccinia virus DNA and fusion gene, all behind BamHI enzymolysis electrophoresis, transfers on the nitrocellulose filter, and be that probe is hybridized with the S gene, the result is by shown in Figure 3.Known recombinant expression plasmid pGJPSA-28, the BamHI enzymatic fragment includes complete S and the preS1 fusion gene is about 0.78Kb, and recombinant vaccinia vSA-28DNA, in the fragment of BamHI enzymolysis can with the S gene recombination also be the fragment of 0.78Kb, the Temple of Heaven strain contrast then is negative, this shows that what fusion gene was correct is inserted among the vaccinia virus recombinant DNA.

Embodiment 5, and vaccinia virus recombinant vSA-28 expression product has S and the dual antigenic evaluation of preS1.

We use ³⁵S-methionine(Met) mark vSA-28 infects the CV-1 cell after 20 hours, follow the trail of and collect 1ml cell pyrolysis liquid and 3ml training liquid after 20 hours, get 100 μ l cell pyrolysis liquids and 300 μ l training liquid, with exempting from anti-S serum post precipitation electrophoresis, compare with the CV-1 cell of vTH-2 (only containing the proteic vaccinia virus recombinant of S master) infection with without the CV-1 cell that infects.By Fig. 4 A as seen, the special 24KD precipitation band and the 27KD band of glucosidesization are arranged in vTH-2, and in vSA-28, also have can and the 29KD band of special sedimentary 27KD of S antibody and glucosidesization, also having in training liquid equally can be by the band of S antibodies, but the ratio by glucosidesization is intracellular bigger, this may be in secretion process, the cause that the degree of glucosidesization increases.

In addition, expression product carries out the immunoprecipitation rear electrophoresis with anti-preS1 (29-36) monoclonal antibody MA18/7, the results are shown in Figure 4B, because vTH-2 only expresses S antigen, thereby can not precipitate with monoclonal antibody MA18/7 specific combination.And in the vSA-28 expression product no matter in training liquid and cell, all have special band 24KD and 29KD to occur with preS1 (29-36) monoclonal antibody MA18/7, usefulness S antiserum(antisera) is consistent in conjunction with sedimentary stripe size among result and Fig. 4 A.This expression product that shows vSA-28 can be discerned and can be discerned by anti-preS1 antiserum(antisera) by anti-S antibody, and promptly the expression product SA-28 albumen of vSA-28 has the antigenic dual antigenicity of S antigen and preS1 simultaneously.

Embodiment 6, the proteic secretion of vSA-28 expression product SA-28.

Follow the trail of by pulse labelling--the experimental result of immunoprecipitation is found out, can detect SA-28 albumen and exist in training liquid, and this shows that product has secretion.

We have further measured SA-28 albumen at different cell CV-1, the secretion situation among Vero and the CEC.Sampling in 96 hours after infecting Vero and CEC cell, the S antigen amount of measuring respectively in training liquid and the cell pyrolysis liquid is the SA-28 amount of index, the results are shown in Figure 5.Similar to vTH-2, vSA-28 secreting, expressing in CV-1 is relatively poor, and is higher, best with the Vero cell especially at Vero cell and CEC cell expressing and secretion level, and SA-28 compares with vTH-2 and expresses the antigenic level of S and secretion property is closely similar.Detected antigen amount is near quite in nutrient solution and cell pyrolysis liquid, this show nearly account for altogether S antigenic half be secreted in the training liquid.

The result who detects preS1 with the ELISA method is consistent (data are not listed as) with the antigenic result of S.

Embodiment 7, the analysis of the SA-28 protein grain that vaccinia virus recombinant vSA-28 expresses.

We use vTH-2 and vSA-28 vero cells infection respectively, and the S antigen of the S antigen of expressing in the training liquid after 96 hours and the SA-28 of fusion is measured behind cesium chloride density gradient centrifugation.In vTH-2 and vSA-28 training liquid, can find density to be respectively the positive peak (seeing Fig. 6 B) of S antigen ria-determination of 1.20g/ml and 1.23g/ml, removing cesium chloride, after concentrated and negative staining, visible diameter is about the SA-28 particle (seeing Fig. 6 A) of 25 (μ m) under Electronic Speculum again.The particle of fusion rotein and S antigen particles do not have fairly obvious difference on density and size.

Embodiment 8, the immunogenicity analysis of the SA-28 that vSA-28 expresses

We infect 48 hours training liquid of CV-1 cell with vSA-28, centrifugal go bovine vaccine after, through hydrophobic medium JL-QT6S-C4 (A) column chromatography and Sepharose 4B column purification, collect S antigenic activity peak, obtain partially purified SA-28 albumen.Immunity Balb/C mouse, getting 1.9 μ g SA-28 albumen is adjuvant with aluminium hydroxide, be contrast through HBsAg 3.2 μ g with the quadrat method purifying, every group of mouse 6-7 abdominal injection, same amount is strengthened after 21 days, at the 21st day and the 49th day, and promptly strengthened the 28th day, anti-S antibody and anti-preS1 anti-body contg are measured in blood sampling, the results are shown in Figure 7.SA-28 immunity Balb/C mouse has good immunogenicity, no matter is after once immunity or twice immunity, the SA-28 sample with compare with standard vaccine, produce anti-S antibody and almost be on the same level.

After immunity once, promptly there is the anti-preS1 antibody of higher level to produce, this points out the anti-preS1 antibody may be early than anti-S production of antibodies.And by a larger margin raising is arranged after twice immunity, shown that SA-28 also has good preS1 immunogenicity.

Our result shows, compares with the synthetic polypeptide, and the advantage of our invention is that the proteic graininess of SA-28 that vSA-28 expresses is the good immunogenicity of SA-28, provides the foundation.And the good secretion of SA-28 particle, for the separation and purification for preparing novel hepatitis B gene engineering subunit vaccine provides favourable condition.

Known anti-preS1 (21-47) antibody has the viral ability of neutralization and the tool provide protection.Thereby SA-28 antigen, develop into hepatitis vaccine and might have following superiority:

1. except that producing anti-S antibody, can also produce anti-preS1 antibody.Can combine with liver cell on two links at neutralize virus and blocking virus and work.Therefore can obtain protecting more completely.

2. can provide provide protection more early than the anti-preS1 antibody of high-caliber generation early.

3. have 10% crowd can not produce responsing reaction to S antigen clinically approximately, application SA-28 produces anti-preS1 antibody blocking virus particle and combines with hepatocellular, thereby also might induce the anti-S antibody of generation to reach provide protection.

4. the chronic viral hepatitis B patient is on the T cell levels reactionless to S antigen, but the immune response of preS1 antigen on the T cell levels will be much higher than S antigen in vivo.Thereby might under the SA-28 of high dosage effect, make t cell responses help to produce preS1 antibody and anti-S antibody, with neutralization and removing virion, reach the purpose of treatment.

Claims (5)

1, the fusion gene of the hepatitis B surface antigen of a kind of corboxy end having anterior surface antigen S1 antigenic determinant of encoding is characterized in that it is the gene fragment 3 ' end and anterior surface antigen S1 antigenic determinant fusion corresponding to the gene fragment of 21-47 amino acids of hepatitis B surface antigen S corresponding to the 1-223 amino acids.

2, fusion gene as claimed in claim 1 is characterized in that this fusion gene inserts the bovine vaccine expression vector, expresses in the recombinant vaccinia system, obtains its proteins encoded.

3, fusion gene as claimed in claim 1 is characterized in that this fusion gene expresses in mammalian cell or Yeast system, obtain its proteins encoded.

4, the hepatitis B surface antigen of a kind of corboxy end having anterior surface antigen S1 antigenic determinant, it is characterized in that it being to merge the fusion rotein that anterior surface antigen S1 21-47 amino acids peptide section is arranged, the i.e. proteins encoded of the described fusion gene of claim 1 at hepatitis B surface antigen S carboxylic end the 223rd amino acids place.

5, hepatitis B surface antigen as claimed in claim 4 is characterized in that this hepatitis B surface antigen is to have S antigenicity and the antigenic fusion rotein particle of S1 simultaneously, can produce anti-S antibody and anti-S1 antibody behind the immune mouse.

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