CN103751773A - Recombinant BHK cell line for stably expressing classical swine fever virus E0-E1-E2 protein, and applications of the same in preparation of vaccines and diagnosis reagents of classical swine fever - Google Patents

Abstract

The present invention discloses a recombinant cell line for stably expressing classical swine fever virus E0-E1-E2 protein, and applications of the recombinant cell line in preparation of vaccines and diagnosis reagents of classical swine fever, wherein the recombinant cell line is BCSFV-E012, is preserved in the China General Microbiological Culture Collection Center, and has the preservation number of CGMCC No.7720. In addition, the present invention further discloses an establishment method for the cell line for stably expressing classical swine fever virus E0-E1-E2 protein, and a method for preparing a classical swine fever prevention vaccine composition by using the cell line. The present invention further discloses applications of the E0-E1-E2 protein stably expressed by the recombinant cell line in preparation of classical swine fever prevention vaccines and diagnosis reagents. The classical swine fever vaccine prepared by using the recombinant cell line has characteristics of high safety, good immunization effect, easy mass production, less being susceptible to exogenous virus pollution or influence of antibodies, and no classical swine fever virus non-structural protein antibody production so as to identify the vaccinated animal and the virus infected animal.

Description

The recombinant BHK cell system of stably express swine fever virus E0-E1-E2 albumen and in the application of preparing in swine Fever Vaccine and diagnostic reagent

Technical field

The present invention relates to a kind of recombinant BHK cell system of stably express swine fever virus E0-E1-E2 albumen and in the application of preparing in swine Fever Vaccine and diagnostic reagent.More specifically, the present invention relates to a kind of expression swine fever virus E0-E1-E2 Protein reconstitution bhk cell is to be BCSFV-E012.Method and this reconstitution cell of the invention also discloses the described recombinant BHK cell of preparation system tie up to the application in preparation prevention swine Fever Vaccine and swine fever diagnostic reagent.Belong to biological medicine genetic engineering and field of immunology.

Background technology

Swine fever (Classical swine fever, CSF) claim again hog cholera (Hog cholera, HC) be by swine fever virus (Hog Cholera virus, HCV or Classical swine fever virus, CSFV) the acute hot fatal disease of one causing, swine fever has height contagiousness, popular extensive, morbidity is high, very harmful with mortality rate.International Office of Epizootics (OIE) was decided to be category-A infectious disease in the past, now will classify circular epidemic disease the phase as, and China is classified as a class animal epidemic.

In Chinese nationwide, there is swine fever morbidity popular, very harmful to pig industry.To the effective anti-measure of this disease, be vaccine immunity prevention at present.Wherein by Chinese Scientists, study successful swine fever attenuated vaccine (C strain) swine fever prevention and control in world wide have been brought into play to distinguished contribution.This vaccine is still used by multiple countries such as China at present.And on the basis of low virulent strain, developed newborn rabbit Seedling thus, exempt from spleen and drench Seedling, the attenuated vaccine of the various ways such as primary cell Seedling and passage cell Seedling.But a large amount of healthy animal of the need of production of organizing Seedling, in production process, hand labor intensity is large, and cost is high, has these unfavorable factors of Side effects etc. to affect the practical application of this type of vaccine.And produce vaccine with the weak poison of cultured cell breeding, be equally also subject to factors puzzlement, as cell culture can be grown by viral interference with BVDV in serum and antibody, virus antigen titre is difficult to improve, and attenuated vaccine needs omnidistance cold chain preservation etc. all to cause the final result of use of attenuated vaccine to be affected.And all attenuated vaccines are all subject to maternal antibody impact in share process actual, this is also one of factor causing immuning failure.

Along with the development of modern genetic engineering technology and cell biological engineering, many scientific research personnel attempt to develop the New Kind of Vaccine for Classical Swine Fever that can overcome existing vaccine defect with modern molecular biology means so.These novel swine Fever Vaccine have viral live vector vaccine, synthetic peptide vaccine, DNA vaccination, the sub-single vaccine of E2 albumen of baculovirus expression, subunit vaccine of escherichia coli expression albumen etc.Wherein, to the E2 protein subunit vaccine with baculovirus expression that has European scientific research personnel's development being applied at present, this vaccine immunity is not affected by maternal antibody, and can carry out antibody test Differential Diagnosis with viral infection.

CSFV is for there being togavirus, and virion size is about 40-60nm.Viral genome is sub-thread positive chain RNA, is about 12.3kb, containing a large open reading frame (ORF), and the polyprotein containing 3898 amino acid residues of encoding, molecular weight is about 438kDa.Polyprotein is processed into 12 kinds of ripe virus proteins through the protease of virus and host cell in translation and after translation, comprise structural protein and non-structural protein, on its polyprotein, from N epidemic disease to C, the order of end is followed successively by: Npro, C, E0 (Erns), E1, E2, P7, NS2-3, NS4A, NS4B, NS5A, NS5B.NS2-3 can be processed to NS2, NS3 (P80), except C, E0, E1 and E2 are structural protein. and all the other are non-structural protein.The processing of structural protein is to be undertaken by the signal peptidase mediation of host cell, and first protease shear polyprotein between nucleocapsid protein C and E012 precursor, shears and opens subsequently at the C of E2 end, and last E012 is cut into rapidly E01 and E2.After E2 discharges from E012 precursor, E01 is processed to E0 and E1, and finally these 3 kinds of membrane glycoproteins pass through in molecule or intermolecular disulfide bond formation complex, are assembled into virion structure.

The genome structure of swine fever virus and other banzi virus have some similar feature as banzi virus such as encephalitis b viruss.Genome is all through shearing, to form structural protein and non-structural protein after a large polyprotein precursor of coding.Known as the structural protein gene of the banzi virus such as JEV, in mammalian cell, express to assemble form virus-like particle (virus like particle, VLP) structure.VLP antigen has the natural structure conformation close to virion, can induce body to produce humoral immunization and cellular immunization.And not containing viral nucleic acid, not reproducible, no pathogenicity, safe, it is the first-selection of preparing new virus disease vaccine that all these advantages make to express VLP antigen.The structural protein of swine fever virus can form VLP structure after expressing in mammalian cell and also do not report at present so.This research department has carried out in recent years with mammal cell line expresses different banzi virus cell protein genes.This seminar has explored in the present invention different express cell systems and has finally found suitable express cell to be, overcome and expressed the toxic action of banzi virus memebrane protein to cell, and the technology such as Large-scale Screening have been set up, result has successfully been prepared expression swine fever virus structural protein cell line, and expressing protein energy assembling assembly virus-like particle structure.This expression of cell lines VLP antigen can produce good immunoreation to immune swine induction.This antigen presentation amount is high simultaneously, is easy to purification, can be as the antigen of CSFV antibody test.The prepared vaccine of this expression of cell lines antigen is expected to provide novel, efficient preventing preparation for the prevention of swine fever.To China and to the prevention and control of swine fever in world wide, can play a significant role.

Summary of the invention

One of object of the present invention is to provide the recombinant BHK cell system of stably express swine fever virus E0-E1-E2 albumen.

Two of object of the present invention is to provide a kind of aforementioned stable that builds and expresses the method that the recombinant BHK cell of swine fever virus E0-E1-E2 albumen is.

Three of object of the present invention is that the recombinant BHK cell system of described expression swine fever virus E0-E1-E2 albumen and expressed swine fever virus E0-E1-E2 albumen are applied to and prepare swine Fever Vaccine, or uses it for the reagent that is prepared into diagnosis or detects swine fever virus infection.

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

Of the present invention a kind of for preventing the vaccine combination of swine fever, it is characterized in that containing swine fever virus E0-E1-E2 albumen and the adjuvant of through the bhk cell system of stably express swine fever virus E0-E1-E2 albumen, expressing in described vaccine combination, wherein said E0-E1-E2 albumen is virus-like particle antigen.

In the present invention, preferred, the aminoacid sequence of described swine fever virus E0-E1-E2 albumen is as shown in SEQ ID No.1.

In the present invention, preferred, the bhk cell system of described stably express swine fever virus E0-E1-E2 albumen builds and obtains by the following method:

(1) build eukaryon expression plasmid, this eukaryon expression plasmid comprises the cDNA sequence of the coding swine fever virus E0-E1-E2 albumen wherein inserting;

(2) by described eukaryon expression plasmid transfection BHK-21 cell;

(3) through the cell of plasmid transfection, to have added the culture fluid of G418, select to cultivate;

(4) will, through selecting cultured cells to dilute clone, gather in the crops clone cell culture supernatant and cell, detect and compare the expression of CSFV E 2 protein, obtain the recombinant BHK cell system of stably express swine fever virus E0-E1-E2 albumen.

Wherein, preferred, the cDNA sequence of described coding swine fever virus E0-E1-E2 albumen is as shown in SEQ ID No.2.

According to the method described above, the present invention has obtained the recombinant BHK cell system that a strain can stably express swine fever virus E0-E1-E2 albumen, called after BCSFV-E012, Classification And Nomenclature is baby hamster kidney cell (Baby hamster kidney cell), be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, its culture presevation is numbered: CGMCC No.7720, the preservation time is on June 18th, 2013.

The invention allows for described bhk cell is expressed swine fever virus E0-E1-E2 albumen, it is characterized in that described E0-E1-E2 albumen is virus-like particle protein.

The recombinant cell lines of expression swine fever virus E0-E1-E2 albumen prepared by the present invention, easily cultivate, propagation fast, can infinitely expand, stable in properties, expressing quantity is high, and expressing protein can form virus-like particle after cell self processing is modified, after expressing protein immune swine, can produce virucidin by induced animal body, can resist viral infection.Can be with detecting antibody against swine fever virus after expressing protein in cell culture fluid is purified.

Therefore, further, the invention allows for bhk cell system or the application of its culture in preparation prevention swine fever virus disease vaccine medicine of described stably express swine fever virus E0-E1-E2 albumen, the wherein said culture virus-like particle protein that the swine fever virus E0-E1-E2 albumen self assembly expressed forms of serving as reasons.And

The bhk cell system of described stably express swine fever virus E0-E1-E2 albumen or its culture are in preparation diagnosis or detect the application in swine fever virus infection reagent, the wherein said culture virus-like particle protein that the swine fever virus E0-E1-E2 albumen self assembly expressed forms of serving as reasons.

Further, the present invention proposes a kind of method of the vaccine combination of preparing described prevention swine fever, it is characterized in that comprising the following steps:

(1) by bhk cell described above, be after BCSFV-E012 normally goes down to posterity, to grow to 90% when full, changing serum content is 1～2%(v/v) low blood serum medium continue to cultivate 4-6d, harvesting culture supernatant is stored in 4 ℃;

(2) after cell conditioned medium liquid is adjusted to E2 antigen ELISA and tires as 1:32 to 1:64 after molecular cut off is 100kD ultrafiltration and concentration, adding final concentration is 0.02%(w/w) be vaccine antigen liquid after thimerosal;

(3) vaccine antigen liquid and oily adjuvant, by volume for 1:1.5 mixes and fully emulsified, obtain.

Beneficial effect of the present invention is as follows:

1. the culture of the reconstitution cell of expression of the present invention swine fever virus E0-E1-E2 albumen is prepared into vaccine, can produces the neutralizing antibody for CSFV by induced animal body, and can be in vivo or in external and CSFV, viral infection animal body stoped.

2. the selected expression system of the present invention is BHK-21 cell, and the recombinant cell lines antigen presentation amount obtaining is high, can suspension culture or high density fermentation cultivate, be easy to a large amount of production.

3. recombinant expressed cell line of the present invention can utilize serum-free medium or low blood serum medium to carry out culture expression, can reduce antigen or production of vaccine cost.

4. the present invention has carried out gene codon optimization to antigenic protein gene, is conducive to improve antigen presentation amount.

5. the oil-adjuvant vaccine and all virucidins of energy induced animal body generation more efficient valency of water Adjuvanted vaccines that utilize the cell culture of recombinant expressed cell line of the present invention to prepare.

6. vaccine immunity animal prepared by the antigen that utilizes recombinant cell lines of the present invention to express does not produce swine fever virus non-structural protein antibody, therefore, can to vaccine immunity and viral infection animal, differentiate by detecting swine fever virus non-structural protein antibody.

7. the prepared swine Fever Vaccine of the present invention does not contain viral nucleic acid, not reproducible, and no pathogenicity, has high biological safety, and vaccine immunity is not subject to maternal antibody or has had antibody interferes with.

Accompanying drawing explanation

Fig. 1 is restructuring recombinant mammalian expressing vector structure schematic diagram;

Fig. 2 is that after transfection, different clone cells are expressed the detection of E2 protein ELISA;

Fig. 3 is that the different generation cellular expression of screening and cloning cell line E2 protein ELISA detects;

Fig. 4 is restructuring expression of cell lines CSFV E2 protein I FA detection;

A, the 5th generation of clone cell; B, the 25th generation of clone cell; C, normal BHK-21 cell contrast;

Fig. 5 is restructuring expression of cell lines swine fever virus E0-E1-E2 albumen formation virus-like particle electron microscopic observation;

Arrow is depicted as the CSFV virus-like particle that cellular expression E0-E1-E2 albumen forms in cell;

Fig. 6 is immune swine serum swine fever virus ELISA antibody test;

Fig. 7 is restructuring expression of cell lines Protein Detection swine fever serum antibody result.

The specific embodiment

Below with reference to embodiment, describe the present invention in detail, described embodiment is only intended as illustrative explanation the present invention, rather than intention limits the scope of the invention.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these modifications and replacement be all within protection scope of the present invention.

Structure and the detection of the recombinant cell lines of embodiment 1 stably express swine fever virus E0-E1-E2 albumen

1 materials and methods

1.1 plasmids, bacterial strain and cell

Eukaryotic expression plasmid pCAG-neo, DH5 α competent cell and BHK-21 cell are preserved by Vet Biotechnology National Key Laboratory of Harbin Veterinary Medicine Inst., China Academy of Agriculture; in plasmid, extraction reagent kit and RNA extraction test kit are QIAGEN company product; glue reclaims test kit purchased from Shanghai Hua Shun Bioisystech Co., Ltd; G418 is purchased from Gibco company; pancreatin is purchased from Hyclone company, Reverse Transcriptase M-MLV ﹑ PrimeSTAR ^tMhS DNA Polymerase ﹑ Sal I ﹑ Xho I ﹑ BamH I ﹑ T4DNA ligase is purchased from TaKaRa company, the monoclonal antibody of anti-CSFV E2 albumen and anti-CSFV E0 albumen is prepared by this seminar, swine fever virus antigen ELISA detecting kit is Median Diagnostics company product, CSFV E 2 protein antibody ELISA detection kit is IDEXX company product, FITC labelling goat anti-mouse IgG is purchased from biotech firm of Zhong Shan Golden Bridge, FuGENE hD Transfection Reagent transfection reagent box is purchased from Roche company.

The structure of 1.2 recombinant expression plasmids

Opti-CSFV-E012 gene is the gene of the coding CSFV E0-E1-E2 albumen codon optimized through the inclined to one side preferendum of eukaryotic cell.The aminoacid sequence of CSFV E0-E1-E2 albumen is with reference to the protein sequence (GenBank:AAC68902.2) of CSFV Shimen strain, the 258th amino acids V of E2 albumen in this protein sequence is revised as to aminoacid I, at the N of E0 albumen end, add an aminoacid M and 22 amino acid residue sequences as signal peptide, the aminoacid sequence of E0-E1-E2 albumen is as shown in SEQ ID NO.1.When this gene of design, before start codon, be added with Kozak sequence and Sac I restriction enzyme site and protectiveness base; E protein gene coding end be added with terminator and Xho I restriction enzyme site and protectiveness base.Opti-CSFV-E012 gene and this gene specific amplimer are synthesized by Nanjing Genscript Biotechnology Co., Ltd., synthetic gene opti-CSFV-E012 size is 2.475bp, Sac I ﹑ Xho I restriction enzyme site is contained at two ends, sequence is as shown in SEQ ID NO.2, and synthetic gene is cloned in pUC57 plasmid.Sac I ﹑ Xho I double digestion processing for carrier for expression of eukaryon pCAG-neo, then with through the two opti-CSFV-E012 genes that cut back to close of Sac I and Xho I under the effect of T4 DNA ligase, be connected, after transforming DH5 α competent cell, be coated with the LB flat board containing ammonia benzyl, picking list bacterium colony amplification cultivation extract plasmid after incubated overnight, identifies it with Sac I ﹑ Xho I double digestion; Enzyme action is identified positive plasmid called after pCAGneo-opti-CSFV-E012, send biotech firm to carry out sequence verification simultaneously.Fig. 1 is shown in by plasmid construction schematic diagram.

1.3 cell transfectings and screening

Select the BHK-21 cell dissociation that growth conditions is good to be passaged in 24 orifice plates, until BHK-21 cell, grow to 90% when full, according to FuGENE recombiant plasmid pCAGneo-opti-CSFV-E012 transfectional cell for HD Transfection Reagent transfection reagent box operating instruction.After transfection 48h, add containing the cultivation of pressurizeing of G418 (1000 μ g/m L) selective medium, after 4d by cell trypsinization, with limiting dilution assay, go down to posterity and in 96 orifice plates, continue to cultivate, after 7d, under inverted microscope, observe clone's number of every porocyte.Select the hole amplification culture in 24 orifice plates, 6 orifice plates, Tissue Culture Flask in succession that contains 1 cell colony (i.e. 1 cell mass), each cell is carried out IFA evaluation and expressing protein is carried out to ELISA detection simultaneously.The strong and high cell clone of antigen expressed amount of screening IFA signal.

The ELISA that 1.4 clone cells are expressed E2 albumen detects

Clone cell is cultivated after 2d in 24 orifice plates, collect supernatant, take the culture supernatant of untransfected BHK-21 cell as contrast, with E2 albumen in CSFV antigen detection kit detection clone cell supernatant culture fluid, ELISA detects by test kit explanation and is undertaken, measure OD450 value, each cell clone antigen expressed is carried out to relative quantification and relatively screen.

1.5 expression of indirect immunofluorescence assay testing goal albumen in transfectional cell

Clone cell is seeded in 24 holes or 12 orifice plates, after 24h, remove culture fluid, wash 3 times with PBS, 4% paraformaldehyde is fixed 10min, PBS washes 3 times, with the PBS function cells 10min containing 0.1% Triton X100, PBS washes 3 times, with the PBS sealing 2h containing 4%BSA, PBS washes 1 time, add the anti-E2 protein monoclonal antibody of 1:500 dilution or the E0 protein monoclonal antibody of 1:200 dilution, 4 ℃ of overnight incubation, PBS washes 3 times, add by the FITC labelling goat anti-mouse IgG of 1:200 dilution, incubated at room 2h, PBS washes 3 times, observed result under fluorescence microscope.

The RT-PCR of 1.6 clone cells identifies

With RNA, extract test kit and extract cell total rna, get 10 μ L and carry out reverse transcription, add Oligo dT 1 μ L, RNase inhibitor 1 μ L, M-MLV 5 × Buffer 5 μ L, M-MLV transcriptase 1 μ L, dNTP(10m M) 2.5 μ L, add distilled water to 25 μ L, mix, 42 ℃ of heating 60min, 95 ℃ of 5min cessation reactions.Then utilize opti-CSFV-E012 gene-specific primer, pcr amplification genes of interest.

1.7 recombinant cell lines expressing proteins form the electron microscopic observation of virus-like particle

Will through IFA and ELISA detect can stably express destination protein passage cultivate, cultivate again after 4 days after changing cell maintenance medium, cultured cell power transmission mirror cell is fixed to ultra-thin cell section, swine fever virus sample granule in transmission electron microscope observing cell.To filtering out high expressed amount cell clone, cultivate in a large number, results supernatant after after ultrafiltration and concentration again through sucrose density gradient centrifugation purification antigen expressed, purifying antigen is carried out to negative staining electron microscopic observation

CSFV-VLP。

2 results

The structure of 2.1 recombinant expression plasmids

Fig. 1 is shown in by the structure chart of the recombinant expression plasmid pCAGneo-opti-CSFV-E012 building.The recombiant plasmid extracting is after Xho Ι ﹑ Sac Ι double digestion, carry out agarose gel electrophoresis analysis, find that there is two bands, enzyme action product electrophoresis banding pattern is consistent with expected results, and simultaneously sequencing result shows that the gene of the coding E0-E1-E2 albumen of the sequence between Sac Ι ﹑ Xho Ι restriction enzyme site and design in recombiant plasmid is in full accord.

The foundation of 2.2E0-E1-E2 stable gene express cell system

2.2.1 the screening of transfection cell strain

After cell transfecting 48h, add G418 to select culture medium, with limiting dilution assay, go down to posterity and in 96 orifice plates, continue to cultivate, after 7d, under inverted microscope, pick out containing the clone of individual cells colony and through IFA and identify, filter out 5 positive cell clones.

2.2.2 the RT-PCR of clone cell identifies

Utilize RT-PCR to identify that to IFA positive cell clone carries out after the amplification of genes of interest, result shows, the cell of the different generations to filtered out cell clone all can amplify and theory object band of the same size.Show genes of interest stable being blended in cellular genome, and inheritance stability.

2.3.3 the ELISA of express cell system detects screening

5 clone cell culture supernatant are carried out to CSFV E2 proteantigen ELISA to be detected, No. 5 clone's expressing quantities the highest (Fig. 2) in 5 clones that result shows to filter out through IFA, choose No. 5 cell clonies and go down to posterity and cultivate and further specificity analysis.No. 5 clone cell carries out the detection of CSFVE2 protein ELISA to culture supernatant after different generations go down to posterity, and result shows that the clone cell filtering out ties up to after repeatedly going down to posterity and still keeps good protein expression level (Fig. 3).Result shows the recombinant cell lines energy stably express genes of interest building.

By obtain can stably express swine fever virus E0-E1-E2 albumen No. 5 clones, called after BCSFV-E012, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its culture presevation is numbered: CGMCC No.7720, the preservation time is on June 18th, 2012.

2.3.4 indirect immunofluorescence assay detects the expression of E2 albumen in transfectional cell

Indirect immunofluorescence assay demonstration, cell clone first generation after clone can present stronger yellow-green fluorescence signal, and compared with control cells does not present fluorescence signal.And this cell clone, still can stably express destination protein (Fig. 4) when being passaged to for the 20th generation.

2.3.5 indirect immunofluorescence assay detects the expression of E0 albumen in transfectional cell

Indirect immunofluorescence assay shows, detects the cell line cell filtering out can present green fluorescence signal through E2 expression ELISA after E0 monoclonal antibody detects.Show that E0 albumen is expressed in cell.

2.3.6 recombinant cell lines expressing protein forms the electron microscopic observation of virus-like particle

Recombinant cell lines shows through cell section electron microscopic observation, in the endomembrane system endoplasmic reticulum of cell, can observe CSFV virus like particle, and diameter is about 40nm.In cell culture supernatant, the antigen of purification is also observed CSFV virus-like particle structure (Fig. 5) through Electronic Speculum.The albumen that shows constructed expression of cell lines can be assembled into CSFV-VLP.

The immunoprotection test of embodiment 2 expression of cell lines recombiant proteins to pig

Vaccine preparation: by the recombinant cell lines BCSFV-E012(CGMCC No.7720 of embodiment 1 constructed screening) cell grows to 90% when full after normally going down to posterity, change low blood serum medium (serum content is 1～2%) and continue to cultivate 4-6d, harvesting culture supernatant is stored in 4 ℃, and cell conditioned medium liquid is adjusted to after molecular cut off is 100kD ultrafiltration and concentration that after E2 antigen ELISA is tired as 1:32 to 1:64, to add final concentration be after 0.02% thimerosal, to be vaccine antigen liquid.Vaccine antigen liquid and oily adjuvant are by volume for 1:1.5 mixes and fully emulsified.Matched group attenuated vaccine is swine fever cell vaccine (the bull testis primary cell source) product of market sale.

Animal grouping and immunity: experiment is all taken a blood sample before immunity with pig, and to carry out CSFV antibody test negative for separation of serum.Choose 15 of the close wean Chang-Bai piglets of age in days, be divided at random 3 groups: first group of 5 of pig is attenuated vaccine immunity matched group, weak 5 part/pigs of malicious Seedling of immune CSFV, once (attenuated vaccine group) of an immunity; Second group of 5 of pig is oil seepage group prepared by restructuring expression of cell lines E0-E1-E2 antigen, and immune vaccine 2ml, carries out once (recombinant antigen vaccine group) of booster immunization for the first time after immune surrounding; The 3rd group of 5 of pig is blank group, carries out PBS injection contrast (PBS matched group).

Serum neutralizing antibody detects: experiment pig is taken a blood sample once before immunity, and through ELISA, detecting all porcine blood serums is CSFV negative antibody.One exempt from after surrounding oil seepage immune group carry out booster immunization once.2 weeks and the 4 weeks separation of serum of taking a blood sample respectively after immunity for the first time.The 2nd, 4,8,12,16,20,24,28 weeks blood sampling separation of serum after secondary immunity.Laboratory animal porcine blood serum all carries out the detection of CSFV virucidin, and detecting neutralizing antibody method is the NPLA method that OIE handbook is recommended.Concrete grammar is: respectively organize after 56 ℃ of deactivation 30min of porcine blood serum, start to carry out 10 times of dilutions with DMEM in 96 porocyte plates, carry out successively 2 times of dilutions later again.By the serum having diluted 50 μ L and equal-volume CSFV virus (Shimen strain, 200TCID ₅₀/ 0.1ml) mix.Hatch 1h for 37 ℃, then add 100 μ L PK-15 cells, after 37 ℃ of cultivation 3d, cell is fixed with 20% acetone PBS.Take anti-CSFV E2 protein monoclonal antibody as primary antibodie, HRP labelling sheep anti mouse enzymic-labelled antibody is two anti-, develops the color with ACE.Each blood serum sample carries out 3 times to be repeated.Set up blank simultaneously.Cell plates through colour developing after in light Microscopic observation result of determination.To reduce more than 50% the maximum serum diluting multiple that infects, tire as the neutralization of serum.

Serum ELISA antibody test: immunity before and after institute the porcine blood serum that gathers with IDEXX hog cholera antibody detection kit, detect, detection method by test kit illustrate carry out.By specification method calculating antibody blocking-up rate respectively.Serum is carried out detecting serum antibody titer (the highly diluted multiple of serum that antibody blocking rate is greater than 40%) after doubling dilution.

Result: serum neutralizing antibody testing result shows, the expression swine fever virus E0-E1-E2 Protein reconstitution bhk cell of the constructed preparation of the present invention is can induce pig to produce CSFV virucidin after the expressed antigen vaccine immunity pig of preparing.Two exempt from after the high energy of two weeks (just exempt from after 6 weeks) neutralizing antibodies reach 5120, reach neutralizing antibody peak, it is worth apparently higher than attenuated vaccine immunity group neutralizing antibody level.Two exempt from rear surrounding (just exempt from after 8 weeks) recombinant subunit vaccine, and to induce NAT be 2560.Neutralizing antibody is slow decreasing trend later, but still keeps high-level neutralizing antibody.To latter 7 months (28 weeks) of immunity, vaccine immunity papova neutralizing antibody level prepared by the present invention still reaches 320, far away higher than providing immunoprotection required virucidin's level (40～50) to pig.And in experiment immunity contrast pig in its virucidin's level of whole experimental session all for being less than 10.Specific experiment data are as shown in table 1.

Blocking-up ELISA detects Antibody Results and shows, within after recombinant subunit vaccine immune swine 2 weeks, can induce and produce swine fever specific antibody (antibody blocking rate is greater than 40%), antibody horizontal raises gradually subsequently, 6-8 week peaking after immunity.The duration of antibody, recombinant subunit vaccine immune group antibody horizontal is all higher than attenuated vaccine group antibody horizontal.All the time there is (Fig. 6) in this high-level antibody 32 cycles of observing after immunity.

Table 1 recombinant subunit vaccine immune swine serum-virus neutralizing antibody testing result

^arecombinant antigen vaccine group is carried out booster immunization once in 4 weeks after initial immunity.

Embodiment 3 use recombinant cell lines antigen expressed indirect elisa methods detect antibody against swine fever virus

By BCSFV-E012 cell amplification culture, harvesting culture supernatant.Cell conditioned medium liquid is clarified through 0.45 μ m membrane filtration remove the impurity such as cell debris through low-speed centrifugal after again.The filter membrane ultrafiltration and concentration that the supernatant of clarification is 100kD through molecular cut off again.After approximately 40 times of volumes are concentrated, more centrifugal through 12000rpm, remove insoluble impurity.Supernatant is 10% to 50% continuous sucrose density gradient centrifugation through concentration.Collect 20%-30% concentration sucrose layer liquid, then measure protein content through ultrafiltration desaccharide and after concentrating, purifying antigen saves backup in-70 ℃.With purification CSFV-E012 albumen be envelope antigen, coated 96 hole polystyrene ELISA Plate.With pH9.6,0.1M carbonate buffer solution dilution antigen to final concentration is 3 μ g/ml, adds in ELISA Plate 4 ℃ of coated spending the night by 100 μ l/ holes.Then use PBST(PBS+0.05%Tween) detersive enzyme target 3 times; Containing the PBST sealase target of 1%BSA, 37 ℃ of sealing 2h, wash plate 3 times with washing liquid PBST after sealing, immediately for detection of or-20 ℃ deposit standby.

Antibody test operation sequence: add porcine blood serum to be detected (100 times of dilutions of qualitative detection serum, antibody titer detects serum and carries out doubling dilution, set up positive serum contrast to contrast with negative serum and the blank of increase serum not) simultaneously, hatch 1h for 37 ℃, PBST washing liquid washing 3 times, each 3 minutes; Add horseradish peroxidase-labeled goat-anti pig IgG (Goat-anti-pig IgG-HRP), hatch 1h for 37 ℃, PBST washing liquid washing 4 times, each 3 minutes; Add HRP chromogenic substrate (TMB developer), within incubated at room 5-15 minute, observe chromogenic reaction; Fully, after colour developing, add the reaction of 2M sulphuric acid color development stopping; By microplate reader, measure the light absorption value of 450nm wavelength; Result of determination.

During result of determination: blank and negative serum hole light absorption value are less than or equal to 0.3, and it is effective that positive serum control wells light absorption value is greater than 0.4 o'clock result; Calculate P/N value=(detection hole OD value-blank hole OD value)/(negative serum OD value-blank hole OD value), P/N value be equal to or greater than 2 o'clock positive; Antibody titer take the maximum dilution multiple of reacting positive serum as this sample serum.

Choose each 20 parts of the positive porcine blood serum of known antibody against swine fever virus and negative porcine blood serum, take purification CSFV-E012 albumen as envelope antigen, carry out indirect ELISA detection respectively, with the detection effect of detectable antigens.As shown in Figure 7, all positive serum OD450 values are all greater than 0.5 to testing result, and all negative serum OD450 values are all less than 0.3.Show that this antigen has good specificity and sensitivity.

Sequence table

Claims (10)

1. one kind for preventing the vaccine combination of swine fever, it is characterized in that containing swine fever virus E0-E1-E2 albumen and the adjuvant of through the bhk cell system of stably express swine fever virus E0-E1-E2 albumen, expressing in described vaccine combination, wherein said E0-E1-E2 albumen is virus-like particle antigen.

2. vaccine combination as claimed in claim 1, is characterized in that the aminoacid sequence of described swine fever virus E0-E1-E2 albumen is as shown in SEQ ID No.1.

3. vaccine combination as claimed in claim 1 or 2, is characterized in that the bhk cell system of described stably express swine fever virus E0-E1-E2 albumen builds and obtains by the following method:

(1) build eukaryon expression plasmid, this eukaryon expression plasmid comprises the cDNA sequence of the coding swine fever virus E0-E1-E2 albumen wherein inserting;

(2) by described eukaryon expression plasmid transfection BHK-21 cell;

(3) through the cell of plasmid transfection, to have added the culture fluid of G418, select to cultivate;

(4) will, through selecting cultured cells to dilute clone, gather in the crops clone cell culture supernatant and cell, detect and compare the expression of CSFV E 2 protein, obtain the recombinant BHK cell system of stably express swine fever virus E0-E1-E2 albumen.

4. vaccine combination as claimed in claim 3, is characterized in that the cDNA sequence of described coding swine fever virus E0-E1-E2 albumen is as shown in SEQ ID No.2.

5. according to any one vaccine combination in claim 1-4, the bhk cell that it is characterized in that stably express swine fever virus E0-E1-E2 albumen is BCSFV-E012, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its culture presevation is numbered: CGMCC No.7720.

6. the bhk cell of stably express swine fever virus E0-E1-E2 albumen system, called after BCSFV-E012, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and its culture presevation is numbered: CGMCCNo.7720.

7. by bhk cell claimed in claim 6, be expressed swine fever virus E0-E1-E2 albumen, it is characterized in that described E0-E1-E2 albumen can be self-assembled into as virus-like particle protein at cell inner expression.

8. the bhk cell of stably express swine fever virus E0-E1-E2 albumen claimed in claim 6 system or its culture application in preparation prevention swine fever virus disease vaccine medicine, the wherein said culture virus-like particle protein that the swine fever virus E0-E1-E2 albumen self assembly expressed forms of serving as reasons.

9. the bhk cell of stably express swine fever virus E0-E1-E2 albumen claimed in claim 6 system or its culture be in preparation diagnosis or detect the application in swine fever virus infection reagent, the wherein said culture virus-like particle protein that the swine fever virus E0-E1-E2 albumen self assembly expressed forms of serving as reasons.

10. a method of preparing the vaccine combination of the prevention swine fever described in claim 1-5 any one, is characterized in that comprising the following steps:

(1) by bhk cell claimed in claim 6, be after BCSFV-E012 normally goes down to posterity, to grow to 90% when full, changing serum content is 1～2%(v/v) low blood serum medium continue to cultivate 4-6d, harvesting culture supernatant is stored in 4 ℃;

(2) after cell conditioned medium liquid is adjusted to E2 antigen ELISA and tires as 1:32 to 1:64 after molecular cut off is 100kD ultrafiltration and concentration, adding final concentration is 0.02%(w/w) be vaccine antigen liquid after thimerosal;

(3) vaccine antigen liquid and oily adjuvant, by volume for 1:1.5 mixes and fully emulsified, obtain.

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