CN1109784A - Hepatitis B surface antigen protein having ammonia end with pre-surface antigen determinant - Google Patents

Abstract

A new hepatitis B vaccine, HBsAg protide having ammonia end with preS antigenic determinant, produced by means of gene engineering technique for preventing and curing hepatitis B features that its ammona end is partially deficient preS region and its carboxy end is complete S region. The HBsAg protide has the advantage that it is expressed in the form of particles able to secrete in cells of mammal, and becomes particle in yeast cell. After the cell is broken, it may be purified conventionally.

Description

Hepatitis B surface antigen protein having ammonia end with pre-surface antigen determinant

The present invention is the hepatitis B vaccine that a class is produced with technique for gene engineering, and International Classification of Patents is C12N15/09.

Hepatitis B is a kind of infectious disease of serious harm human health.The pathogenic former of it is hepatitis B virus (HBV).China's hepatitis b virus carrier reaches more than 100,000,000 people according to investigations.Hepatitis B, particularly chronic persistent hepatitis B can cause liver cirrhosis, and directly relevant with bringing out of primary hepatocarcinoma.The effective Hepatitis B virus vaccine of development and design comes extensive immune Susceptible population and treatment HBV carrier, to ensureing that people's health has great strategic importance.

It is the HBV virion of 42nm that diameter is not only arranged in the hepatitis B patient blood, and the much more diameter of quantity is spherical or fibrous surface antigen (HBsAg) granule of 22nm in addition, the latter two do not contain HBV DNA, thereby do not have infectivity, but can bring out the antibody that human body produces the HBV of neutralization invasion.From the surface antigen of hepatitis B virus carriers blood separation preparation is first generation Hepatitis B virus vaccine.But the blood source is limited, and it also has other viruses as HIV, pollutions such as HCV.And the genetic engineering Hepatitis B virus vaccine is expressed in zooblast or yeast, the albumen of granulopotent HBsAg.The production of this class vaccine is not limited by material source, and is with low cost when preparing in a large number, and got rid of the pathogenic former contamination of heavy of other haematogenous.Clinic trial shows that recombinant hepatitis b vaccine is identical to the protective effect of Susceptible population with blood source Hepatitis B virus vaccine.

The long reading frame of the HBsAg gene of HBV has a plurality of translation initiation codons, can form three kinds of HBsAg molecules that vary in size from the polypeptide of the initial translation of different AUG.Their carboxylic end has common order, but in ammonia end difference.Long 226 aminoacid of surface antigen master albumen (S albumen); Surface antigen middle protein (MS albumen) ammonia end contains the preS2 district, long 281 aminoacid; Surface antigen large protein (LS albumen) ammonia end contains preS1 and preS2 district, long 389 or 400 aminoacid.The latter two mainly are present in the HBV virion.These two albumen, particularly LS albumen play an important role in HBV granule assembling with to hepatocellular identification.PreS1 and preS2 district also have a plurality of antigenic determinants, and be wherein particularly important with the antigen decision family in preS1 district.Because the preS district is exposed to the surface of HBsAg protein molecular, is the target area of body immune system, thereby good immunogenicity is arranged.People such as Neurath find can both block HBV and hepatocellular combination (Neurath corresponding to the section of synthesized peptide and the antibody thereof of hepatocyte receptor binding domain (preS aa21-47), A.R.etal., " Identification and chemical synthesis of a host cell receptor binding site on hepatitis Bvirus ", Cell, (1986), 46,429).Many HBV researcheres propose as vaccine following advantage is arranged with the HBsAg albumen that contains the preS district according to animal model and human clinical trial's result.1, because the preS district can transfer the immunoreation of body to HBV with different mechanism with the S district, use the hepatitis vaccine of being with the preS district can improve the immunogenicity of vaccine greatly, still can reach same or better immune effect after reducing vaccine consumption and immune time, recombinant vaccine is more widely used.2, existing commercial blood source and recombinant vaccine only contain S albumen.Hepatitis vaccine helps to overcome hypoergia or the anergy to existing Hepatitis B virus vaccine of some people among the crowd.3, can develop into a class therapeutic hepatitis B vaccine, with helping to overcome body to the chronically infected immunologic tolerance of HBV, thus the new way of opening up the treatment chronic hepatitis B.

But at zooblast, that carries out in the transgenic mice studies show that, because the particularly existence in preS1 district of preS district, LS albumen can not be secreted from zooblast, and it also suppresses the proteic secretion of MS and S consumingly.LS albumen exists with the embrane-associated protein form that is insoluble to endochylema in zooblast, can not form the HBsAg granule separately.PreS district, particularly preS2 district also have the site of protein degradation enzyme identification.This also is that extraction and purification LS albumen have brought difficulty (Persing, P.H., et al., " Inhibition of secretion of hepatitis B surface antigen by a related presurface polypeptide ", Science, (1986), 234,1388; Molnar-kimber, K.L., et al. " Distinctive properties of the hepatitis B virus envelope proteins ", J.Virol., (1988), 62,407).Though S albumen and a spot of LS albumen can form blended HBsAg granule when expressing simultaneously.But LS only accounts in this granule about 5%, can not give full play to the immunogenicity in preS district.Report from external research, utilize recombinant DNA technology that the preS district is transformed, particularly optionally behind the proteic preS of the excalation LS district, the important antigenic determinant that can keep the preS district, improve LS albumen again and formed granule and excretory performance and stability, improving the proteic expression of HBsAg, is the feasible method that obtains Hepatitis B virus vaccine of new generation.The Hexal biotech company of Germany has reported with behind the vaccine immunity crowd (comprising that some contain the proteic vaccine nonresponder of S to existing) who is with the preS district, absolutely produce the antibody (Thoma of anti-S and anti-preS, H.A., et al., " Does preS2 have the same effect in improving the HBV immune response as preS1 ", in Viral Hepatitis and Liver Disease, F.B.Hollinger, S, M, Lemon and H.S.Margolis(ed.) The Williamsand Wilkins Co., Baltmore).But their vaccine is the mixture of three kinds of albumen (be S albumen, there is the MS and the LS albumen of disappearance in the S district), thereby preparation process is complicated.People such as Prange find that then preS1 district or wherein a part of fusion rotein that is connected S district carboxylic end gained can form the granule justacrine and go out mammalian cell (Streeck, R.E., et al., " Analysis of HBsAg assembly and secretion using site-directed mutagenesis ", in " Viral Hepatitis and Liver Disease ", F.B.Hollinger, S.M.Lemon and H.S.Margolis(ed.), The Williams and Wilkins Co., Baltimore).The HBsAg gene in band preS district has been expressed by the Merck company of the U.S. in yeast, and has applied for patent (Chinese patent CN 87100465 A) in American-European and China.Their designed HBsAg albumen comprises a part of preS1 district and preS2 district and part or all of S district at least.Belgian Smith Kline company has also reported the HBsAg albumen of expressing band preS1 and preS2 antigenic determinant in yeast, L albumen (Cabezon, T., et al., " A new hepatitis Bvaccine containing preS1 and preS2 epitopes from saccharomyces cerevisiae ", Vaccine, (1990), 199).But such albumen is difficult to form separately granule in yeast.And can seriously influence output and the secretion of HBsAg albumen in zooblast according to our the long ammonia end preS district that studies show that.Therefore unlikelyly reach and produce these albumen at zooblast system invading the exterior.

The objective of the invention is has the HBsAg gene of disappearance by making up the preS coding region, these genes is expressed the HBsAg albumen that produces band preS district in mammalian cell or yeast cells.Their characteristics are: 1, only contain the preS1 district or the preS2 district of excalation, thereby the LS albumen of molecular weight ratio wild type is much smaller.Make by single protein molecular and present the preS of exposure simultaneously and the antigenic determinant in S district becomes possibility.2, kept in the producing of preS1 district or preS2 district and the antigenic determinant of the antibody of HBV at protein molecular ammonia end, helped giving full play to their immunogenicity.3, can form granule separately, also can form the HBsAg granule with S albumen or other HBsAg albumen.4, most of granule that forms can secrete mammalian cell.5, in cell and culture fluid, good stable is arranged.6, expression is close with S albumen in mammalian cell.Though can not secrete in yeast cells, albumen exists so that soluble protein is particle stabilized in endochylema mostly, is easy to conventional yeast protein extracting method purification.

Schematic view illustrating of the present invention is as follows:

The structural representation of the section H BsAg gene that Fig. 1, usefulness PCR and vitro recombination method make up.

All plasmids all comprise the coding region of preS1 except pS100 and pS200.

(S300 is a LS albumen, and S200 is a MS albumen, and S100 is a S albumen).

Fig. 2, SV40 expression plasmid structural representation.

Fig. 3, ³⁵There is the HBsAg albumen of excalation in the preS district of S labelling by the autoradiograph of anti-HBsAg or anti-preS1 antibody mediated immunity post precipitation.

In Fig. 3 a, the Cytoplasm extract ³⁵The autoradiograph of the HBV surface antigen of S labelling behind anti-HBsAg antibody mediated immunity precipitation and protein electrophoresis.

In Fig. 3 b, the Cytoplasm extract ³⁵The autoradiograph of the HBV surface antigen of S labelling behind anti-preS1 antibody mediated immunity precipitation and protein electrophoresis.

(S100-S albumen, the arrow indication is respectively the surface antigen protein of saccharifying type and non-saccharifying type in each row, the left side is a standard molecular weight).

The proteic secreted of Fig. 4, S311, S311 albumen and S(S100 in different cell strains) comparison of proteic expression and secretion level.

There is the protein electrophoresis figure of HBsAg albumen behind immunoprecipitation of deletion mutation in Fig. 5, the preS district of expressing in yeast.

As shown in Figure 1, we adopt polymerase chain reaction (PCR) and other extracorporeal recombination to lack the code area of part preS from the wild-type HBsAg gene, have made up a series of HBsAg genes. These recombinant HBsAg genes can be cloned into various eucaryon bio-carriers and express. Our choice for use SV40 expression plasmid-mammalian cell and ADH1 expression plasmid-yeast cells system screen and can express the recombinant HBsAg gene with above-mentioned a plurality of advantages.

We have made up a SV40 expression plasmid.As shown in Figure 2, this plasmid is the derivant of plasmid pSP65.It has ampicillin resistance gene and dna replication dna starting point, thereby can screening and cloning and amplification in escherichia coli.The HBsAg gene is inserted between SV40 early promoter and the HBV poly-adenosine signal.The SV40 early promoter can drive the HBsAg expression of gene in mammalian cell.We use monkey-kidney cells COSM6, CV-1, Vero and the human hepatoma cell strain HepG2 and the Huh7 of the mammalian cell of SV40 expression plasmid transient transfection.Since the COSM6 cell can constitutive expression the T antigen of SV40 virus, thereby the SV40 expression plasmid that changes over to can increase, thereby increased the expression of HBsAg.The cell that the SV40 expression plasmid changes over to is used ³⁵S methionine pulse labeling is also followed the trail of back results culture fluid and cell, the cytoplasm protein that extracts after the cracking or be directly used in radioimmunoassay or separate with the SDS-protein electrophoresis through the antibody mediated immunity post precipitation of anti-HBsAg or anti-preS1 respectively, and autoradiography, study the stability of expression product with this, form particulate performance, secreted and the expression in cell, screening might develop into the recombinant HBsAg gene of hepatitis B subunit vaccine of new generation.We also express a series of HBsAg gene clones of band preS district's deletion mutation in expression plasmid of yeast pVT102U in yeast S78 XV700-6B.Yeast cells is after glass strain concussion fragmentation, the gained cell extract dyes colour developing with anti-HBsAg antibody mediated immunity post precipitation through separation of SDS-protein electrophoresis and silver, perhaps with " radioimmunity the detects the hepatitis B surface antigen test kit " quantitative assay of the dilution of the extract behind broken yeast back.

Content of the present invention is specifically narration in following embodiment:

The structure of embodiment 1, S311 gene and the expression in mammalian cell thereof.

Materials and methods:

A.DNA polymerase I Klenow fragment, restricted enzyme, T4 dna ligase are the products of New England Biolab, the Taq archaeal dna polymerase is the Promega product.The primed DNA of PCR is synthesized by U.S.'s Wisconsin university biotechnology center and Chinese Academy of Sciences's Shanghai biochemistry.

B. monkey kidney cell line COSM6 and CV-1 and human hepatoma cell strain HepG2 and Huh7 are provided by the J.Mertz of U.S. Wisconsin university.The Vero cell is provided by the biochemical Wang Yuan of institute, and above-mentioned cell is all cultivated in containing the DMEM culture fluid of 5% hyclone.

C. the transfection of cell.COSM6 cell DEAE-dextran method transfection (Yu, X.M., " The C-terminal half of the preS1 region is essential for the secretion of human hepatitis B virus large protein devoid of the N-terminal retention sequence ", Virology, (1991), 181,386).The DNA transfection of coprecipitation of calcium phosphate of other cells.Cell is incubated in 37 ℃ CO2 incubator and changes culture fluid after 5 hours after the transfection, cultivates harvesting and culture fluid confession radioimmunity and euzymelinked immunosorbent assay (ELISA) (ELISA) mensuration after 48 hours again.Perhaps be used for isotopic labeling.

The antigenic ELISA of d.HBsAg radioimmunoassay and preS1 measures

Cell after freeze thawing repeatedly in the supernatant HBsAg albumen detect hepatitis B surface antigen test kit (Beijing Biological Product Inst.'s product) with AUSRIA medicine box (Abbott company product) or radioimmunity and measure.ELISA measures the preS1(a.a.12-32 that adopts OrganonTeknick company) medicine box mensuration.

E. isotopic labeling and immunoassay

³⁵The S methionine is the product of Amersham company.The antibody of anti-HBsAg is DAKO Corp company product.Anti-preS1(a.a.12-47) antibody is provided by Neurath.Cell pulse labeling and tracking and immunoprecipitation and SDS-protein electrophoresis method are seen document (Yu, X.M., " The C-terminal half of the preS1 region is essential for the secretion of human hepatitis B virus large protein devoid of the N-terminal retention sequence ", Virology, (1991), 181,386).

We describe the concrete steps of enforcement step by step:

(1) expression plasmid pS311 makes up

The SV40 expression plasmid is to derive from plasmid pSP65, and its structure is seen document (Chinese science B collects (1991) 6,633 for Yu Xianming etc., " the HBV surface antigen large protein that can secrete mammalian cell ").The S311 gene is that hepatocyte receptor binding domain (a.a.21-47) coding region and the fusion of S coding region in preS1 district forms.Its structure adopts two step PCR methods.Pcr template is pS301 gene (Chinese science B collects (1991) 6,633 for Yu Xianming etc., " the HBV surface antigen large protein that can secrete mammalian cell ").The coding region of first couple of primer amplification preS1 a.a.21-47, and the point of contact of holding a restricted enzyme NcoI of introducing at the 3' of the dna fragmentation that increases.A restricted enzyme NcoI point of contact is also introduced at the 5' end of the dna fragmentation of amplification in the coding region in second pair of primer amplification S district (a.a.1-32).Two dna fragmentations are connected with carrying out three fragments with plasmid pS301 through BamHI and XbaI enzyme cutting behind BamHI, NcoI or NcoI, the XbaI enzyme cutting respectively, obtain the pS311 expression plasmid.The structure of plasmid has also been carried out determined dna sequence with restriction map spectrometry probatio inspectionem pecuoarem to the zone of pcr amplification, does not find that nucleotide sequence has any change.Proteic deletion mutant of LS of such S311 gene code, 29 aminoacid of its preS head of district add 226 aminoacid in carboxylic end S district, and 255 aminoacid of length overall are more bigger than S molecular weight of albumen.

The expression plasmid pS311 that has this recombination deposits in escherichia coli, Escherichia coli TG-1/pS311, and stored table is designated as CCTCC 94024.Extract plasmid by escherichia coli and carry out people's " molecular cloning " such as () J.Sambrook by usual way.

(2) the proteic construction features of S311

After we use pS311 plasmid DNA transfection monkey kidney COSM6 cell and pulse labeling and tracking.Find in the cell ³⁵The S311 albumen of S methionine labelling can be respectively by anti-HBsAg and anti-preS1 antibody precipitation (see figure 3).Because the antibody of anti-HBsAg is mainly discerned the configuration type antigenic determinant in S district.S311 albumen has been pointed out its S district to keep the proteic basic structure of S by the immunoprecipitation of this antibody, and promptly the peptide chain folding in S district is identical with S albumen with the disulfide bond generation type.Be exposed to the surface of protein molecular simultaneously with the immunoprecipitation explanation preS1 district of anti-preS1 antibody, be easy to be discerned by anti-preS1 antibody.Fig. 3 also shows saccharifying and the proteic molecular weight of non-saccharifying S311 and from conforming to that nucleotide sequence is inferred, and there is no that other HBsAg albumen such as S are proteic to express simultaneously.

(3) proteic secreted of S311 and stability

³⁵The comparison (see figure 4) of the S311 albumen of S labelling content in cell and in the culture fluid shows that the S311 albumen of most labellings is present in the culture fluid in pulse labeling and after following the trail of 24 hours, and this explanation S311 protein excretion is good.With radioimmunology measure respectively different time S311 in cell and the content in the culture fluid show that the S311 total amount of labelling is constant pulse labeling 3 hours or after following the trail of 24 hours.S311 albumen stable existence in cell and culture fluid is described.

According to the result of quantitative radioimmunoassay, the secretion level of S311 albumen in monkey kidney COSM6 cell is identical with S albumen.At Vero, in HepG2 and the Huh7 cell the proteic secretion of S311 good, but total expression is more lower slightly than S albumen.

(4) the proteic antigenicity of S311

³⁵The S311 of S methionine labelling can be precipitated by the antibody of anti-chemical synthesising peptide section (S district a.a.122-145).PreS district exists the zone that does not have the major antigen determinant in S district that tangible coverage effect is arranged in this explanation S311 albumen, and it is possible having pointed out the preS district that presents exposure on same protein molecular simultaneously and the epitope cluster in S district.And scholar in the past thinks that to the serious coverage effect in S district hepatitis vaccine should be the HBsAg albumen and the proteic mixture of the S (Alberti in band preS district in view of the proteic long preS district (163 or 174 aminoacid) of LS, A., et al., " Nature and display of hepatitis Bvirus envelope proteins and the humoral immune response ", inIn Immunopathology Springer-Verlag, (1990), 5).

The S interval of hepatocyte receptor binding domain (a.a.21-47) and carboxylic end is every 116 or 127 amino acid residues in LS albumen, but directly continuous in this two zone of S311 albumen.With the ELISA method the antigenic mensuration of preS1 is shown that the antigenicity of S311 albumen and S301 albumen (the proteic deletion mutant of LS, it has lacked the a.a.2-19 of preS, but distance becomes between above-mentioned these two zones) is identical.Even the most of disappearance in this explanation preS district still is easy to be discerned by corresponding antibody with the preS district antigenic determinant that the S district directly links to each other.

(5) S311 albumen can form the HBsAg granule

The measuring principle of the radioimmunology that we are used is two sandwich assays, and this method only can be measured the HBsAg albumen that exists with particle form.Proteic expression of S311 in mammalian cell and yeast cells and S albumen with measured by radioimmunoassay are approaching, and the result of protein electrophoresis is consistent behind this result and the immunoprecipitation.This shows at the S311 of these two system expressions albumen and can form the HBsAg granule separately.

The expression of embodiment 2.S311 albumen in Yeast system

Identical among the proteic immunoprecipitation of yeast HBsAg and methods such as SDS-protein electrophoresis and the embodiment 1.

Yeast strains S78XV700-6B and the expression plasmid of yeast pVT102U that contains the ADH1 promoter are provided by T.Vernet.The HBsAg gene in band preS district is built into a series of HBsAg expression plasmids after inserting expression plasmid pVT102U behind BamHI and the HindIII enzyme action.The conversion of yeast cells and transformant screening are seen document (Vernet, T., et al., " A family of yeast expression vectors containing the phage fl intergenic region ", Gene, (1987), 52,225).Yeast Cultivation is in the YPD culture fluid, and 30 ℃ of shaken cultivation 48 hours reach 10.0 left and right sides collecting cells to 0.D.650.Cell suspension contains 5mMEDTA, 1mM PMSF, 1% NP-40 at the long-pending PBS(of triploid) in, adding the long-pending bead (diameter 0.45mm) of monoploid again, concuss 8 times each 30 seconds, carries out immunoprecipitation after getting supernatant in centrifugal 30 minutes.

The S311 albumen of expressing in yeast cells is the same with S albumen, though can not secrete, is present among the endochylema with soluble protein, after the bead fragmentation, can extract with the buffer that contains detergent.The S311 protein expression level is suitable with S albumen in yeast.

Claims (8)

1, class blocking hepatitis B virus infection or be used for the new generation vaccine of chronic hepatitis treatment effectively, it is characterized in that the proteic ammonia end of hbs antigen (HBsAg) as main constituent has the preS district (being the preS1 district or the preS2 district of excalation) of excalation, the carboxylic end is complete S district; This class HBsAg albumen is expressed with secernent particle form in mammalian cell; In yeast cells, can form granule, and can after fragmentation, use the conventional method purification at cell.

2,, it is characterized in that and to form by preS1 district, complete preS2 district and the complete S district of excalation according to the HBsAg protein described in the claim 1.

3,, it is characterized in that and to form by the preS1 district of excalation and complete S district according to the HBsAg protein described in the claim 1.

4, according to the HBsAg protein described in the claim 1,3, it is characterized in that can be by preS(a.a.21-47) and complete S district form.

5, according to the HBsAg albumen described in the claim 1,2,3,4 be surface antigen large protein (LS albumen) from HBsAg gene code HBsAg gene expression or complete of the preS coding region of band portion disappearance and complete S coding region through biochemistry or chemical method degradation treatment, disappearance part preS district obtains.

6, make up with preS district in the vitro recombination method excalation HBsAg gene according to the HBsAg gene described in the claim 1,2,3,4, perhaps earlier with chemical method or the synthetic dna fragmentation of biochemical process corresponding to the preS district, then and link corresponding to the dna fragmentation of S coding region, the expression plasmid pS311 that has this recombination deposits in escherichia coli, Escherichia coli TG-1/pS311, stored table is designated as CCTCC 94024.

7, comprise zooblast-plasmid expression system, zooblast-expression of recombinant virus system, yeast expression system and prokaryotic expression system (for example escherichia coli and bacillus subtilis etc.) according to the HBsAg expression of gene system that makes up described in the claim 5.

8, also can directly with dna form be used for genetic immunization or gene therapy except that being used for the HBsAg expression albumen according to the HBsAg gene that makes up described in the claim 5.

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