CN1042564A - The preparation method of Hepatitis B virus vaccine and goods thereof - Google Patents

Abstract

The present invention is that a group engineering is produced the method for hepatitis vaccine and its goods.Can effectively block hepatitis B infected vaccinia virus recombinant by making up one, make this virus have complete HBsAg group, can express hepatitis B surface antigen, product has three components of HBsAg simultaneously, promptly main albumen, middle molecule protein and high molecular weight protein, but and can form the excretory particle.This recombinant virus technology can be used to prepare new hepatitis b vaccine, also can be used for producing secretor type total composition HBsAg particle.

Description

The preparation method of Hepatitis B virus vaccine and goods thereof

Type B viral hepatitis is a kind of widely popular disease.Hepatitis B virus (HBV) carrier reaches more than 200,000,000 in the world, and the death toll that causes by hepatitis B or with the hepatitis B diseases associated is huge, very harmful to the mankind.

The infecting virus particle that has 42nm in the serum of some HBV infected patient, it is made up of the surface antigen that constitutes virus coat (HBsAg), virus core antigen (HBcAg) and viral DNA.Except complete virion, also exist in the hepatitis B patients'blood and only contain HBsAg, less, diameter is 20 to 22nm spherical particle and villous themeda shape particle.The HBsAg particle can be induced and be produced single-minded antibody, makes human body produce the immunizing power that opposing HBV infects.But, from blood, separate HBsAg particulate expense costliness, it is also limited to originate, and can't satisfy the people of HBV hotspot and generally inoculate needs.And, also exist the insecurity that unknown etiology may cause in the blood, therefore, people just pass through other approach production hepatitis B surface antigen in active research.

HBsAg is made up of the virus capsid protein of three kinds of different sizes.They are encoded by mutually identical continuous programming code district, position, but initial by different translation initiation site ATG.These three albumen are HBsAg master's albumen (or S albumen), contain 226 amino acid, by the S district coding of HBsAg gene; A spot of middle molecule protein (MS albumen) is to have increased by 55 amino acid at the proteic N end of S, contains 281 amino acid.These 55 amino acid are by gene preS2 district coding, and contain polyerized human serum albumin (pHSA) acceptor; A spot of high molecular weight protein (LS albumen), by complete surface antigen gene, promptly the common coding in preS1, preS2 and S district is made up of about 400 amino acid.MS albumen and LS albumen mainly are present in the shell of virion and have in patients serum's the surface antigen particle of infection activity.

Because the HBsAg particle can make human body produce narrow spectrum antibody, the infection of virus is played immunization, therefore, its biological characteristics and the application in vaccine development cause that people note widely.Overwhelming majority research concentrates on S albumen, still, in recent years MS and LS albumen is also drawn attention gradually.MS and LS albumen have the unexistent antigenic determinant of S albumen, make the Hepatitis B virus vaccine of chemosynthesis vaccine or reorganization with them, can make tire raising (Milich, the D.R. of vaccine, et al., Science[1985] 228 p1195-1199).

Vaccinia virus has been used to express the antigen of multiple virus, comprises HBV.Utilize recombinant DNA technology, required expressed proteins gene is inserted vaccinia virus,, make this protein gene obtain expressing with the suitable cell of resulting recombinant vaccinia virus infection.Recombinant virus itself can be used as living vaccine.On the other hand, recombinant virus can duplicate in cell, express, and may secrete required antigen and as subunit vaccine.

Cheng etc. (J.Virol, [1986] 60, p337-344) structure of a vaccinia virus recombinant has been described.This recombinant virus contains the DNA sequence of coding macromole hepatitis B surface antigen (LS), and promptly complete HBsAg gene is expressed under the regulation and control of a vaccinia virus promotor.They infect the CV-1 cell with recombinant vaccinia, though in cell, there is LS proteic synthetic,, infected cells is not obviously secreted LS albumen.That clearer and more definite is Moss and Flexner(Annual Review[1987] 5 p305-324) point out, the high molecular weight protein of HBsAg can not form particle and secrete, and, if use two recombinant virus-infected cells simultaneously, LS even can influence the proteic secretion of HBsAg master.Paoletli etc. (Proc.Natl.Acad.Sci., USA.[1984] Vol.81, p193-197) be described for the potential living vaccine that uses vaccinia virus recombinant to make up prevention hepatitis B and bleb.They reported that hepatitis B surface antigen can be synthesized and from the cell that infects secretion come out, still, do not report the three kinds of different componentss and their secretion of hepatitis B surface antigen.In their work, it is very low to have the HBsAg that the vaccinia virus recombinant of full surface antigen gene expresses, and detects through RIA, only for only containing several thousandths of the recombinant virus of main protein gene.

The object of the present invention is to provide a novel recombinant vaccine of inhibition of hepatitis b virus infection effectively.A vaccinia virus recombinant that has complete HBsAg gene is provided.It can express justacrine hepatitis B surface antigen particle, and contains all three kinds of components of hepatitis B surface antigen, S, MS albumen and LS albumen.The antigenicity ratio that contains the HBsAg of S, MS and LS simultaneously only contains the much better than of S.It can produce rapidly in vivo and prevent the needed full spectrum antibody of hepatitis B virus infection, thereby provides the foundation for developing efficient, quick-acting Hepatitis B virus vaccines.Vaccinia virus recombinant provided by the present invention also makes people may obtain a large amount of S albumen, MS albumen and LS albumen, prepares novel subunit vaccine.

The present invention also provides one of preparation can express justacrine contain all three kinds of components of HBsAg in zooblast, the method of S albumen, MS albumen and the proteic hepatitis B surface antigen particulate of LS vaccinia virus recombinant, it comprises the vaccinia virus strain of 1. selected low toxicities.2. make up an expression material that contains HBsAg sequence and promoter sequence.3. recombinate to cause in the body with selected vaccinia virus infection cell, and with the expression plasmid transfectional cell that makes up, thereby preparation has certain exogenous antigen gene, as the vaccinia virus recombinant of HBsAg gene.4. the cell that contains vaccinia virus recombinant by the selection markers screening of introducing by certain expression plasmid.5. from the cell that is screened, reclaim vaccinia virus recombinant.

Schematic of the present invention is simply described as follows:

Fig. 1, universal support plasmid pGJP-5 makes up synoptic diagram.

Fig. 2 prepares the synoptic diagram of expression plasmid pLS-1 of the present invention.

Fig. 3 by the reorganization of vaccinia virus and pLS-1, prepares the synoptic diagram of novel vaccinia virus recombinant of the present invention.

Fig. 4, HBsAg protein electrophoresis collection of illustrative plates (A), and corresponding immunoblotting (Western blot) collection of illustrative plates (B).

Fig. 5, HBsAg particulate electron micrograph.

Content of the present invention specifically describes as follows.

We adopt known low toxicity vaccinia virus strain, the Temple of Heaven strain and wide 9 strains. They can obtain from Ministry of Health of the People's Republic of China's Beijing institute of Biological Products and national drug and biological products assay institute, and its advantage is consequent vaccinia virus recombinant during as live vaccine, and its toxicity is very low.

We have made up a suitable expression plasmid. This plasmid also contains an antibiotic resistance genes, such as ampicillin (Ap except containing HBsAg and promoter sequence^r) resistant gene, in Escherichia coli, can carry out the screening of this plasmid with the culture medium that contains antibiotic. In addition, also contain the selected marker of the rear recombinant celo virus of restructuring in the donor on the plasmid, it is the thymidine nucleoside kinase gene (TK of an inactivation^-), it is very similar to natural TK gene, and difference is to have inserted foreign gene. Recombinate in DNA sequence homologous by the TK gene and the vaccinia virus gene group body, HBsAg and initiator sequence can be inserted in the vaccinia virus TK gene in the expression plasmid, recombinant virus causes the insertion deactivation of TK gene in the recombinant virus, so can screen by the deactivation of TK.

As shown in Figure 1, plasmid pGJ-1 is set up behind Hind III enzymolysis by known plasmid vector pWR13 and the wide 9 strain DNA of vaccinia virus to form.PWR13 contains ammonia benzylpenicillin resistant gene (Ap ^r) and a multipurpose joint that comprises Hind III site.Gained plasmid pGJ-1 contains the TK gene of vaccinia virus and the Ap that comes from pWR13 ^rMark.Cut and be connected by suitable enzyme, remove unnecessary order among the pGJ-1, produce plasmid pGJ-2, it still has TK order and Ap ^rMark.PGJ-2 further transforms, and introduces the SalI joint, forms pGJ-7.

Another part pWR13 cuts by the SalI enzyme and is connected with plasmid pVC-1.Plasmid pVC-1 has a bovine vaccine P7.5 promotor, is used for HBsAg genetic expression.P7.5 just is used for of one group of promotor of this purpose.P7.5 is easy to cut out and insert with SalI the multipurpose joint of pWR13 in proper order from pVC-1, produce plasmid pWR/VC1-22 thus.

Use SalI and the EcoRI site of pGJ-7 and pWR/VC1-22, these two plasmids universal support plasmid pGJP-5 that is formed by connecting.In pGJP-5, the P7.5 that comes from pWR/VC1-22 is inserted into the TK order of pGJ-7 in proper order, causes the insertion deactivation of TK gene.Vector plasmid pGJP-5 still keeps AP ^rMark.

As shown in Figure 2, the present invention uses vector plasmid pGJP-5 construction expression plasmid pLS-1.Another parent material that makes up pLS-1 is known plasmid pADR-1, and it contains complete HBsAg order.Represent with S, pS2, pS1 among Fig. 2.This plasmid also has various restriction enzyme sites and Ap ^rMark.At first, we introduce a BamHI restriction enzyme site in the HBsAg of pADR-1 sequence upstream, it is transformed into pADR-2B.Then, the HBsAg sequence is cut out from pADR-2B and with the pGJP-5 pLS-1 that is formed by connecting.As shown in Figure 2, pLS-1 has the promoter sequence P7.5 that comes from pGJP-5, and they are equidirectional with HBsAg in the upstream of HBsAg sequence, and P7.5 and HBsAg sequence are all inserted in the TK gene.Therefore, plasmid pLS-1 is TK ^-, but can be in cell HBsAg expression.TK ^-Reorganization provides useful selection markers in the body in order to carry out with vaccinia virus.The TK sequence that is blocked in the plasmid has very big homology with the TK gene of not transforming, and can carry out reorganization in the body.

This novel vaccinia virus of the present invention can be passed through to make vaccinia virus and pLS-1 carry out recombinating in the body and preparing with the cell of expression plasmid pLS-1 transfection through normal vaccinia virus infection.As previously mentioned, the present invention uses hypotoxic vaccinia virus strain, wide 9 strains and the Temple of Heaven strain.Special needs to be pointed out is that we have used primary cell, as former generation chick-embryo cell as host cell.Passage cell uses primary cell can produce purer living vaccine and subunit vaccine relatively.Because it has avoided being present in the pollution of the impurity in the passage cell.

Fig. 3 is the diagram of preparation vaccinia virus recombinant.With plasmid pLS-1 and wide 9 strains of vaccinia virus or the Temple of Heaven strain (I) introducing chick-embryo cell (II).The TK that the is blocked order (III) of pLS-1 and the complete TK of virus (I) have shown mutual avidity in proper order and have contacted each other.The TK order of plasmid and virus exchanges by regrouping process under rare occasion, produce a spot of vaccinia virus recombinant (IV) thus, be characterized in having and be blocked idle TK order, simultaneously, it contains complete HBsAg gene and regulation and control HBsAg expression promoter.Recombinant virus (IV) can pass through TK ^-Feature screen, and breed to obtain a large amount of virus and use as living vaccine.Three kinds of compositions of all of HBsAg, S, MS and LS albumen all can obtain expressing and forming particle secreting from cell, and Hepatitis B virus vaccine provides new approach in order to produce efficiently.

Content of the present invention is specifically narrated at following embodiment.

Embodiment 1,

Materials and methods:

A.DNA polymerase I Klenow fragment is the Boehringer product; Restriction endonuclease BstE II is the Biolabs product; Low melting-point agarose and BamHI joint are the B.R.L products; Other restriction enzyme and T4 dna ligase are provided by zymin group of Shanghai Inst. of Biochemistry, Chinese Academy of Sciences.

B. thymidine kinase (TK) gene defection type cell Human TK ^-143 cells are provided by doctor B.Moss of NIH, cultivate in the DMEM nutrient solution that contains 25ug/ml 5-bromouracil deoxyribose (BUdR) and 5% foetal calf serum; Vaccinia virus Tiantan strain is provided by Ministry of Health's Beijing institute of Biological Products, and wide 9 strains of vaccinia virus are provided by People's Republic of China's China's medicine and biological products assay institute, and they are bred in former generation chick-embryo cell (CEC).In former generation,, chick-embryo cell prepared with instar chicken embryo on the 10th, cultivated in the DMEM that contains 10% calf serum.

C. the transfection of cell.The CEC cell infects with vaccinia virus Tiantan strain, and the DNA(with coprecipitation of calcium phosphate after two hours contains 10ug plasmid DNA and 15ug milt DNA) transfection.37 ℃ of insulations added fresh medium after 4-6 hour, continued to cultivate harvested cell after 48 hours.

The separation and purification of d.DNA.Vaccinia virus DNA directly separates from infected cells according to the method (document 1) of Espostto; Plasmid DNA is with the method for alkaline denaturation, through Sepharose 2B column purification (document 5).

E. the structure of plasmid DNA.Universal support plasmid pGJP-5 is set up by wide 9 strains of vaccinia virus and is formed (seeing " biological chemistry and Acta Biophysica Sinica " (1987) 19 p397-405 such as Wu Xue).PADR-1 is for containing the complete genomic clone of hepatitis B virus adr hypotype (seeing that Wu Xiang just waits " Chinese science " (B collects) (1983) 26 p162-167).For making up new plasmid pADR-2B and pLS-1, DNA cuts with suitable restriction enzyme, and connects with the T4 dna ligase, then transformed into escherichia coli.When making up pADR-2B, recipient bacterium is selected E.coli HB101 for use.When making up pLS-1, recipient bacterium is selected E.coli JM83 for use.

F. the screening of recombinant virus.TK ^-Recombinant virus is according to the method (document 3) of Weir etc., the selectivity plaque Analysis and Screening under existing by BUdR.

The mensuration of g.HBsAg.The expression of HBsAg is measured with AUSRIA medicine box (Abbott company product), and the reference standard that uses West Germany Paul-Ehrlich-Institut to provide.

H. polypeptide compositional analysis.SDS-polyacrylamide gel electrophoresis (SDS-PAGE) carries out (document 4) according to the method for Laemmli, uses silver staining color.Immunoblotting (Westernblot) is undertaken by the method that document [5] is described.

I. measure preS1 and preS2 with enzyme immunoassay.Protein sample is adsorbed in uses anti-preS1(MA18/7) or anti-preS2(Q19/10) on the micro-spot plate of monoclonal antibody bag quilt, then with the anti-S antibody treatment of enzyme connection.Method is seen document [6].Monoclonal antibody is provided by West Germany doctor W.H.Gerlich; The mensuration of polyerized human serum albumin (pHSA) acceptor is undertaken by the method for document [7].

We describe the concrete steps of enforcement step by step.

(1) preparation of expression plasmid pLS-1 and structure

The first step is to make up universal support pGJP-5, and Fig. 1 is its building process.Second step was preparation expression plasmid pLS-1, and Fig. 2 is its preparation process.

(a) preparation of universal support plasmid pGJP-5 (Fig. 1).

Wide 9 strains of vaccinia virus of low toxicity are cut with Hind III enzyme.Hind III-J fragment cloning produces pGJ-1 to the pWR13 plasmid.PGJ-1 has two EcoR I point of contacts, and one is positioned at the pWR13 part, and another is in Hind III-J fragment of wide 9.The EcoR I point of contact and the wide 9-DNA that remove on the pWR13 go up TK gene partial sequence in addition, obtain pGJ-2.Then, pGJ-2 is gone up the Cla I point of contact adjacent with EcoR I point of contact change Sal I point of contact into, obtain pGJ-7.The proteic promotor gene P7.5 of 7.5K bovine vaccine is inserted between the EcoR I of TK gene among the pGJ-7 and Sal I cut, just constitute pGJP-5.

(b) preparation of expression plasmid pLS-1 (Fig. 2).

Universal support plasmid pGJP-5 contains bovine vaccine TK gene, the proteic promotor of promotor p7.5(7.5K bovine vaccine) and multipurpose joint.Reorganization provides DNA sequence homologous and selection markers to the TK gene order in the body for plasmid and vaccinia virus DNA carry out.The HBV genome that the HBsAg gene is cloned from pADR-1 (adr hypotype) gets.The BstE II restriction enzyme site in preS district among the pADR-1 is transformed into BamH I point of contact.Consequent pADR-2B has two BamH I restriction enzyme sites, the proteic upstream of the LS at HBsAg, and another is in its downstream.Cut out the complete HBsAg gene fragment of 1.8Kb with BamH I enzyme, this fragment is inserted the BamH I site of multipurpose joint among the vector plasmid pGJP-5, by the in situ hybridization screening, obtain containing the expression plasmid pLS-1 of HBsAg LS protein gene.

(2) analysis of expression plasmid pLS-1.

Universal support plasmid pGJP-5 is 4.5Kb.Insert the HBsAg gene fragment of 1.8Kb among the expression plasmid pLS-1 and become 6.3Kb.Plasmid pGJP-5 has single BamH I, EcoR I and Hind III site, therefore, cuts with any one enzyme in these enzymes and all to produce single 4.5Kb band.In the HBsAg gene fragment of inserting, single BamH I, Hind III and Xho I point of contact are arranged, but do not have EcoR I point of contact.Therefore pLS-1 cuts through EcoR I enzyme and still provides a single band, and its length is 6.3Kb, and carrier part contribution 4.5Kb, HBsAg gene insert fragment contribution 1.8Kb.

In pGJP-5, Hind III point of contact is positioned at the P7.5 upstream, and it and BamH I site are at a distance of 0.9Kb.In the HBsAg gene fragment, Hind III site is in downstream, S district, apart from the BamH I site 1.5Kb of upstream, preS district, apart from the BamH I site 0.3Kb in downstream.The small segment that obtains with Hind III enzymolysis is 2.4Kb, rather than 1.2Kb, illustrates that the HBsAg gene that inserts is consistent with promotor P7.5 direction.

Xho I point of contact is positioned at the preS2 district in pLS-1, and near the S district, and vector plasmid pGJP-5 goes up no Xho I point of contact.PLS-1 disappears with BamH I and Xho I double enzymolysis, the BamH I enzymatic fragment of 1.8Kb, produces 0.5kb and two fragments of 1.3kb.0.5Kb fragment contains the preS district, the 1.3kb fragment contains the S district.

(3) structure of vaccinia virus recombinant vTLS-1 and analysis.

Vaccinia virus recombinant is by recombination to construct in the body.After former generation chick-embryo cell (CEC) infected 2 hours with vaccinia virus Tiantan strain, with the pLS-1 transfection of calcium phosphate precipitation, harvested cell after 48 hours, recombinant celo virus.In pLS-1, P7.5 and HBsAg gene are inserted in the vaccinia virus TK gene, thereby, destroyed the activity of TK gene, so vaccinia virus recombinant is TK ^-End user TK ^-143 cells, the plaque select under can existing by BUdR is with TK ^-Recombinant virus screens.By further purifying and screening, obtain vaccinia virus recombinant pLS-1.

We have also used ³²The P mark, the dna fragmentation that contains S and preS is respectively analyzed recombinant virus vTLS-1 with the method for DNA dot blot as probe.

(4) HBsAg that measures vTLS-1 expresses.

We with measured by radioimmunoassay vTLS-1 at people TK ^-The expression of HBsAg in 143 cells.With each cell 0.1PFU(plaque forming unit) virus infection 3X10 ⁶People TK ^-143 cells 48 hours, the HBsAg total amount of expression is 2.1ug, and wherein 1.8ug is in nutrient solution, and only 0.3ug is in cell.

Make and use the same method that we have measured vTLS-1 at people TK ^-The timing relationship of 143 cell expressings.Use 1X10 ⁵People TK ^-143 cells were removed supernatant liquor after 2 hours, added the fresh DMEM nutrient solution that contains 10% foetal calf serum.Collected nutrient solution and cell every 12 hours, and measure HBsAg, the time curve that obtains is presented at and infects that HBsAg increases gradually in back 48 hours, and is saturated after 48 hours.

(5) character of vTLS-1 expression.

Owing in vTLS-1, in the coding framework of HBsAg gene three different translation initiation sites are arranged, therefore, have more than a kind of component in the HBsAg expression product with RIA mensuration.

In order further to analyze vTLS-1 at people TK ^-The polypeptide of expressed HBsAg is formed in 143 cells, and we have carried out the electrophoretic analysis of protein product.Cell 0.1PFU virus infection was collected supernatant liquor after 48 hours, and 1, the centrifugal removal virion of 600rpm.HBsAg in the supernatant liquor Sepharose 4B affinity chromatography column purification of the monoclonal antibody IgG that is associated with anti-S.The SDS-polyacrylamide gel electrophoresis, argentation dyeing.Protein electrophoresis provides 24K, 27K and three protein bands of 30-42K (Fig. 4 A).24K is a S albumen, and 27K is the proteic glucosides product of S, and 30-42K is bigger albumen.Bigger albumen is dispersivity, and it may be because different saccharification degree, can also can be that the S particle has in various degree degraded in the purge process.

We further use Western blot(immunoblotting) identified these HBsAg components.Behind the SDS-PAGE electrophoresis, protein band is transferred on the nitrocellulose filter, with anti-S antibody response, then with ¹²⁵The protein A(A albumen of I mark) hybridization, radioautograph (Fig. 4 B).The basically identical as a result of the result of Western blot and protein electrophoresis has further proved this three components that protein band is HBsAg.The expression product of vTLS-1 can be well and anti-preS1(MA 18/7) and anti-preS2(Q19/10) monoclonal antibody reactive, also having illustrated has bigger albumen to exist in the vTLS-1 expression product, and they can be from people TK ^-Secretion is come out in 143 cells.Simultaneously, expression product has shown pHSA(poly human serum albumin) receptor active, this activity is that the preS2 district by product albumen provides, these the results are shown in following table 1.

The HBsAg character that table 1.vTLS-1 expresses

PreS1 ^aPreS2 ^bThe pHSA acceptor

vTLS-1 1.96 ^C1.60 ＞2.05

vTH-2 ^d0.30 0.31 0.20

Annotate: the people TK that the vTLS-1 of HBsAg infects ^-Purifying in the nutrient solution of 143 cells is with the research of ELISA method.

A. use the monoclonal antibody MA18/7 single-minded to preS1

B. use the monoclonal antibody Q19/10 single-minded to preS2

C.ELISA measures with measuring O.D490, and O.D.490＜0.4 is a negative value

D.TH-2 is made up by the Temple of Heaven strain, only contains the proteic recombinant vaccinia of HBsAg master

Virus, in the table with comparing.

Experimental result shows that we have successfully made up the vaccinia virus recombinant that contains complete HBsAg gene, with its infected person TK ^-Behind 143 cells, can produce all three kinds of components of justacrine HBsAg, i.e. S, preS2 and preS1 albumen.The immunoassay proof preS1 proteantigen determinant of being made of anti-preS1 monoclonal antibody can be secreted in the nutrient solution, the preS2 component of HBsAg product kept the pHSA receptor active and with the ability of anti-preS2 monoclonal antibody reactive.

In our experiment, vTLS-1 infected person TK ^-The expression product of 143 cells comprises total composition HBsAg, i.e. S, MS and LS albumen, and can form the particle of homogeneous.

People TK from the vTLS-1 infection ^-The secretor type particle of collecting in 143 cell culture fluids can be seen (Fig. 5) under electron microscope, and is confirmed by following experimental result.At first, it is super centrifugal that we have carried out the CsCl density gradient, detects particulate with the method for measuring the pHSA acceptor and distribute, and recording particulate density is 1.26g/ml, more bigger than main S particulate density.Secondly, we detect the effective expression of HBsAg with radioimmunology (RIA), have only granular HBsAg to be detected by the RIA method.Same with ELISA sandwich assay detection preS1 and preS2, also have only granular sample ability measured.

It is crucial that the HBsAg particulate forms the hepatitis B subunit vaccine, because the immunogenicity of granular HBsAg is than strong three orders of magnitude of HBsAg antigen monomer.Therefore, secretor type total composition HBsAg particle provides the approach of producing novel subunit vaccine.Such new generation vaccine will produce the anti-HBsAg antibody of full spectrum, and stronger immunity will be arranged.

The proteic preS of the LS of HBsAg district has important biological characteristics.There is stronger antigenicity in the preS district of HBsAg than the S district, it is the generation of immune stimulatory originality quickly, and, the LS albumen that higher proportion is arranged in some acute hepatitis b patient's serum, the proteic preS of LS district also participates in virus to hepatocellular invasion and attack, and it also plays regulating effect in the assembling process of virion.Therefore, can not only be the service of preparation Hepatitis B virus vaccine, and provide new direction for the diagnosis and the treatment of hepatitis B the proteic research of LS.The successful structure of vaccinia virus recombinant vTLS-1 and expression make us can obtain to contain in a large number the HBsAg LS albumen in complete preS district, provide material for further studying the proteic biological characteristics of LS, the new way of preparation new hepatitis b vaccine and subunit vaccine also is provided simultaneously.

(6) toxicity test of vaccinia virus strain

To various vaccinia viruss in mouse LD50 and the plaque number that reaches LD50 measure with ordinary method.The toxicity of the WR strain that Cheng etc. are used and wide 9 strains of low toxicity is listed in table 2.

The toxicity test of table 2, vaccinia virus strain

Virus strain PFU/ML LD50 reaches LD ₅₀The plaque number

WR 1.4X10 ⁸＞5.40 ＜17

Wide 9 1.5X10 ⁸2.90 5692

Annotate: the LD of the Temple of Heaven strain ₅₀Not replicate(determination), it is similar to wide 9 strains.

Reference

1.Espostto，J.et al.，“The Preparation of orthopoxvirus DNA”，J.Virol.Meth.，（1980），2，175.

2.Birnboim，H.C.and J.Doly，“A Rapid Alkaline Extraction Procedure for Screening.Recombinant Plasmid DNA”，Nucleic Acids Research，（1979），7，1513.

3.Weir，J.P.et al.，“Mapping of the Vaccinia Virus Thymidine Kinase Gene by Marker Rescue and by Cell Free Traslation of Selected mRNA”，Proc.Natl.Acd.Sci.U.S.A.，（1982），79，1210.

4.Laemmli，U.K.et al.，“Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4”，Nature，（1970），227，680.

5. Wang Yuan etc., p.623 " vaccinia virus recombinant that has hepatitis b surface antigen gene " Chinese science B collects (1986) 29

6.Marquardt，O.et al.，“Cell-type Dependent Expression and Secretion of Hepatitis B Virus PreS1 Surface Antigen”，Postgraduate Medical J.（1987）63（supp1.2），41.

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8. Wu Xue etc., " establishment of vaccinia virus universal support pGJP-5 " biological chemistry and Acta Biophysica Sinica (1987) 19, p.397

Claims (8)

1, a kind of vaccine production method of the infection of inhibition of hepatitis b virus effectively and its goods, it is characterized in that it being to finish by vaccinia virus recombinant, recombinant virus has complete HBsAg gene, with particulate formal representation justacrine hepatitis B surface antigen, product contains S, MS and three kinds of protein ingredients of LS simultaneously.Therefore, can develop into the preparation method and the goods of novel living vaccine and novel subunit vaccine.

2, the vaccinia virus recombinant in the claim 1 is characterized in that the hypotoxic vaccinia virus strain of hanging oneself and selecting.

3, make up the low strain of vaccinia virus recombinant in the claim 2, it is characterized in that wide 9 strains and the Temple of Heaven strain.

4, the low strain of the vaccinia virus recombinant in the claim 3, it is characterized in that in viable cell, making up, be vaccinia virus and the reorganization formation takes place the expression plasmid that has complete HBsAg order and suitable promotor, HBsAg order and promotor are inserted in reorganization usefulness in one section donor, in the bovine vaccine DNA sequence that plasmid and virus have, obtain recombinant virus at last by screening.

When 5, the vaccinia virus recombinant in the claim 4 made up, it is characterized in that carrying out the interior cell of recombinating usefulness of body was primary cell.

6, make up the used primary cell of vaccinia virus recombinant in the claim 5, it is characterized in that cell is former generation chick-embryo cell.

7, the vaccinia virus recombinant in the claim 4 is characterized in that used expression plasmid contains complete HBsAg order and its expression promoter of regulation and control, and they are inserted in the TK order of vector plasmid, and therefore, vaccinia virus recombinant has TK ^-Feature, this feature can be used as the selection markers of vaccinia virus recombinant.

8, the vaccinia virus recombinant in the claim 7 is characterized in that used expression plasmid is pLS-1.

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