CN103751773B - The recombinant BHK cell system of stably express CSFV E0-E1-E2 albumen and in the application of preparing in hog cholera vaccine and diagnostic reagent - Google Patents

Abstract

The invention discloses a kind of recombinant cell lines of stably express CSFV E0-E1-E2 albumen and in the application of preparing in hog cholera vaccine and diagnostic reagent. Wherein, the recombinant cell of described expression CSFV E0-E1-E2 albumen is BCSFV-E012, and this clone is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, does is its deposit number: CGMCC? No.7720. In addition, the invention also discloses the method for building up of stably express CSFV E0-E1-E2 albuminous cell system and use the method for the vaccine combination of described clone preparation prevention swine fever, further, the E0-E1-E2 albumen that the invention also discloses described recombinant cell lines expression is in the application of preparing in vaccine and the diagnostic reagent that prevents swine fever. The hog cholera vaccine that adopts recombinant cell lines of the present invention to prepare is safe, good immune effect, easily large-scale production, be not subject to exogenous virus pollution or antibody impact, and do not produce CSFV non-structural protein antibody, therefore can differentiate vaccine immunity and virus infections animal.

Description

The recombinant BHK cell system of stably express CSFV E0-E1-E2 albumen and in the application of preparing in hog cholera vaccine and diagnostic reagent

Technical field

The recombinant BHK cell that the present invention relates to a kind of stably express CSFV E0-E1-E2 albumen is and is makingApplication in standby hog cholera vaccine and diagnostic reagent. More specifically, the present invention relates to a kind of CSFV of expressingE0-E1-E2 Protein reconstitution bhk cell system is BCSFV-E012. The invention also discloses the described restructuring BHK of preparationThe method of clone and this recombinant cell tie up to the application in preparation prevention hog cholera vaccine and swine fever diagnostic reagent. Belong toIn biological medicine genetic engineering and field of immunology.

Background technology

Swine fever (Classicalswinefever, CSF) claims that again hog cholera (Hogcholera, HC) is by hog choleraA kind of acute heat that poison (HogCholeravirus, HCV or Classicalswinefevervirus, CSFV) causesProperty fatal disease, swine fever has height contagiousness, popular extensively, morbidity is high, very harmful with the death 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 asOne class animal epidemic.

There is swine fever morbidity popular in Chinese nationwide, very harmful to pig industry. At present to this disease effectivelyAnti-measure is vaccine immunity prevention. Wherein study successful swine fever attenuated vaccine (C strain) to generation by Chinese ScientistsWithin the scope of boundary, distinguished contribution has been brought into play in swine fever prevention and control. This vaccine is still used by multiple countries such as China at present. And byOn the basis of this low virulent strain, develop newborn rabbit seedling, exempted from spleen and drench seedling, the multiple shapes such as primary cell seedling and passage cell seedlingThe attenuated vaccine of formula. But a large amount of healthy animal of the need of production of organizing seedling, in production process, hand labor intensity is large,Cost is high, has these unfavorable factors of Side effects etc. to affect the practical application of this type of vaccine. And it is thin to cultivateBorn of the same parents breed weak poison production vaccine and are equally also subject to factors puzzlement, as cell is cultivated with BVDV and antibody in serumCan grow by viral interference, viral antigen titre is difficult to improve, and attenuated vaccine needs omnidistance cold chain preservation etc. all to causeThe final result of use of attenuated vaccine is affected. And all attenuated vaccines are all subject to mother actual in share processSource antibody impact, this is also one of factor causing immuning failure.

So along with the development of modern genetic engineering technology and cell biological engineering, many scientific research personnel attempt withModern molecular biology means are developed the New Kind of Vaccine for Classical Swine Fever that can overcome existing vaccine defect. The pig that these are novelPestilence seedling has viral live vector vaccine, synthetic peptide vaccine, DNA vaccination, the E2 albumen of baculovirus expressionSub-single vaccine, subunit vaccine of Bacillus coli expression albumen etc. Wherein to the European Section that has being applied at presentGrind the E2 protein subunit vaccine with baculovirus expression of personnel's development, this vaccine immunity is not subject to source of parents anti-Body impact, and can carry out antibody test antidiastole with virus infections.

CSFV is for there being togavirus, and virion size is about 40-60nm. Viral genome be sub-thread justChain RNA, is about 12.3kb, containing a large open reading frame (ORF), encodes one containing 3898The polyprotein of amino acid residue, molecular weight is about 438kDa. Polyprotein translation in and translationBe processed into 12 kinds of ripe virus proteins by virus and the protease of host cell, comprise structural proteins withNon-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 proteins. all the other are non-structural protein. Structure eggWhite processing is to be undertaken by the signal peptidase mediation of host cell, protease first at nucleocapsid protein C andBetween E012 precursor, shear polyprotein, shear and open subsequently at the C of E2 end, last E012 is cut rapidlyBe cut into E01 and E2. After E2 discharges from E012 precursor, E01 is processed to E0 and E1, lastThese 3 kinds of membrane glycoproteins are interior or intermolecular disulfide bond formation compound by molecule, is assembled into virion knotStructure.

The genome structure of CSFV and other flavivirus have some as flavivirus such as japanese encephalitis virusesSimilar feature. Genome is all to form structure egg through shearing after a large polyprotein precursor of codingWhite and non-structural protein. The known structural protein gene as flavivirus such as JEV is expressed in mammalian cellCan assembling form virus-like particle (viruslikeparticle, VLP) structure. VLP antigen has close to diseaseThe natural structure conformation of virion, can induce body to produce humoral immunity and cellular immunity. And not containing viral coreAcid, not reproducible, no pathogenicity, safe, it is preparation that all these advantages make to express VLP antigenThe first-selection of new virus disease vaccine. So the structural proteins of CSFV in mammalian cell, express after energyCan not form VLP structure does not also report at present. This research department has carried out in recent years with mammal cell lineExpress different flavivirus cell protein genes. Different expression clone has been explored also in the present invention by this seminarFinally find suitable expression clone, overcome and expressed the toxic action of flavivirus memebrane protein to cell, and builtFound the technology such as Large-scale Screening, result has successfully been prepared expression CSFV structural proteins clone, andExpressing protein energy assembling assembly virus-like particle structure. This expression of cell lines VLP antigen can produce immune swine inductionRaw good immune response. This antigen presentation amount is high simultaneously, is easy to purifying, can be as the inspection of CSFV antibodyThe antigen of surveying. The prepared vaccine of this expression of cell lines antigen is expected to provide novel, efficient for the prevention of swine feverPreventing preparation. Can play a significant role to China and to the prevention and control of swine fever in world wide.

Summary of the invention

One of object of the present invention is to provide the recombinant BHK cell of stably express CSFV E0-E1-E2 albumenSystem.

Two of object of the present invention is to provide a kind of weight that builds aforementioned stable expression CSFV E0-E1-E2 albumenThe method of group bhk cell system.

Three of object of the present invention is by the recombinant BHK cell system of described expression CSFV E0-E1-E2 albumenAnd expressed CSFV E0-E1-E2 albumen is applied to and prepares hog cholera vaccine, or use it for to be prepared into and examineDisconnected or detect the reagent of 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 in described vaccine combinationThere is the CSFV E0-E1-E2 egg of expressing through the bhk cell system of stably express CSFV E0-E1-E2 albumenBletilla adjuvant, wherein said E0-E1-E2 albumen is virus-like particle antigen.

In the present invention, preferred, the amino acid sequence of described CSFV E0-E1-E2 albumen is as SEQIDShown in No.1.

In the present invention, preferred, the bhk cell system of described stably express CSFV E0-E1-E2 albumenBuild and obtain by the following method:

(1) build eukaryon expression plasmid, this eukaryon expression plasmid comprises the coding CSFV wherein insertingThe cDNA sequence of E0-E1-E2 albumen;

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

(3) select to cultivate to have added the nutrient solution of G418 through the cell of plasmid transfection;

(4) by through selecting the cell of cultivating to dilute clone, gather in the crops clone cell culture supernatant and cell, inspectionSurvey and compare the expression of CSFV E 2 protein, obtain the restructuring of stably express CSFV E0-E1-E2 albumenBhk cell system.

Wherein, preferred, the cDNA sequence of described coding CSFV E0-E1-E2 albumen is as SEQIDNo.2Shown in.

According to the method described above, the present invention has obtained the weight that a strain can stably express CSFV E0-E1-E2 albumenGroup bhk cell system, called after BCSFV-E012, Classification And Nomenclature is baby hamster kidney cell (BabyhamsterkidneyCell), be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is exposed to the sun in BeijingNo. 1, North Star West Road, district institute of microbiology of the Chinese Academy of Sciences of institute, its culture presevation is numbered: CGMCCNo.7720,The preservation time is on June 18th, 2013.

The invention allows for described bhk cell is expressed CSFV E0-E1-E2 albumen, its featureBe that described E0-E1-E2 albumen is virus-like particle protein.

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

Therefore, further, the stably express CSFV E0-E1-E2 albumen described in the invention allows forThe application in preparation prevention CSFV disease vaccine medicine of bhk cell system or its culture, wherein said cultivationThing serve as reasons express the self assembly of CSFV E0-E1-E2 albumen form virus-like particle protein. And

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

Further, the present invention proposes a kind of method of the vaccine combination of preparing described prevention swine fever, itsBe characterised in that and comprise the following steps:

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

(2) cell conditioned medium liquid is adjusted to E2 antigen ELISA effect after molecular cut off is 100kD ultrafiltration concentrationAfter valency is 1:32 to 1:64, 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, to obtain final product.

Beneficial effect of the present invention is as follows:

1. the culture of the recombinant cell of expression CSFV E0-E1-E2 albumen of the present invention is prepared into vaccine,Can induced animal body produce the neutralizing antibody for CSFV, and can be in vivo or in external and CSFV,Stop virus infections animal body.

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

3. recombinant expressed clone of the present invention can utilize serum free medium or low blood serum medium to cultivateExpress, 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 the water adjuvant that utilize the cell culture of recombinant expressed clone of the present invention to prepareVaccine is the virucidin of energy induced animal body generation more efficient valency all.

6. it is non-that vaccine immunity animal prepared by the antigen that utilizes recombinant cell lines of the present invention to express does not produce CSFVStructural proteins antibody, therefore, can come vaccine immunity and virus by detecting CSFV non-structural protein antibodyInfection animal is differentiated.

7. the prepared hog cholera vaccine of the present invention does not contain viral nucleic acid, not reproducible, and no pathogenicity, has highBiological safety, vaccine immunity is not subject to maternal antibody or has existed antibody to disturb.

Brief description of the drawings

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 clone E2 protein ELISA detects;

Fig. 4 is restructuring expression of cell lines CSFVE2 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 CSFV 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 CSFV ELISA antibody test;

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

Detailed description of the invention

Below describe the present invention in detail with reference to embodiment, described embodiment is only intended as illustrative explanation the present invention, andNot that intention limits the scope of the invention. It will be understood by those skilled in the art that and do not departing from essence of the present inventionUnder god and scope, can the details of technical solution of the present invention and form be modified or be replaced, but these amendments and replacingChange all within protection scope of the present invention.

Structure and the detection of the recombinant cell lines of embodiment 1 stably express CSFV 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 by Chinese agricultureHarbin veterinary institute Vet Biotechnology National Key Laboratory of the academy of sciences preserve, in plasmid extraction reagent kit andIt is QIAGEN company product that RNA extracts kit, and it is limited purchased from Shanghai China Shun biotechnology that glue reclaims kitCompany, G418 is purchased from Gibco company, and pancreatin is purchased from Hyclone company, ReverseTranscriptaseM-MLV﹑PrimeSTAR^TMHSDNAPolymerase ﹑ SalI ﹑ XhoI ﹑ BamHI ﹑ T4DNA connectsEnzyme is purchased from TaKaRa company, and the monoclonal antibody of anti-CSFVE2 albumen and anti-CSFVE0 albumen is studied by thisGroup preparation, CSFV antigen ELISA detecting kit is MedianDiagnostics company product, hog choleraPoison E2 protein antibodies ELISA detection kit is IDEXX company product, FITC mark goat anti-mouse IgGPurchased from biotech firm of Zhong Shan Golden Bridge,HDTransfectionReagent transfection reagent box is purchased from RocheCompany.

The structure of 1.2 recombinant expression plasmids

Opti-CSFV-E012 gene is the coding CSFVE0-E1-E2 egg codon optimized through the inclined to one side preferendum of eukaryoticWhite gene. The amino acid sequence of CSFVE0-E1-E2 albumen is with reference to the protein sequence of CSFVShimen strain(GenBank:AAC68902.2), the 258th amino acids V of E2 albumen in this protein sequence is revised as to amino acid I,Add an amino acid M and 22 amino acid residue sequences as signal peptide at the N of E0 albumen end, E0-E1-E2 albumenAmino acid sequence as shown in SEQIDNO.1. In the time of this gene of design, before initiation codon, be added with KozakSequence and SacI restriction enzyme site and protectiveness base; E protein gene coding end be added with terminator and XhoI enzymeCut site and protectiveness base. Opti-CSFV-E012 gene and this gene specific amplimer by Nanjing gold thisAuspicious bio tech ltd is synthetic, and synthetic gene opti-CSFV-E012 size is 2.475bp, and two ends are containedSacI ﹑ XhoI restriction enzyme site, sequence is as shown in SEQIDNO.2, and synthetic gene is cloned in pUC57 plasmid.SacI ﹑ XhoI double digestion processing for carrier for expression of eukaryon pCAG-neo, then with through SacI cuts with XhoI pairThe opti-CSFV-E012 gene reclaiming connects under the effect of T4DNA ligase, transforms DH5 α competence thinAfter born of the same parents, coating is containing the LB flat board of ammonia benzyl, and picking list bacterium colony amplification cultivation extract plasmid after incubated overnight, uses SacI﹑ XhoI double digestion is identified it; Enzyme is cut qualification 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, treat that BHK-21 cell grows to90% when full, according toRestructuring matter for HDTransfectionReagent transfection reagent box operating instructionGrain pCAGneo-opti-CSFV-E012 transfectional cell. After transfection 48h, add containing G418 (1000 μ g/mL)The selective medium cultivation of pressurizeing, by cell trypsinization, goes down to posterity in 96 holes with limiting dilution assay after 4dIn plate, continue to cultivate, after 7d, under inverted microscope, observe clone's number of every porocyte. Select and contain 1 carefullyThe hole of born of the same parents' colony (i.e. 1 cell mass) in succession expands and cultivates in 24 orifice plates, 6 orifice plates, Tissue Culture Flask,Each cell is carried out IFA qualification and expressing protein is carried out to ELISA detection simultaneously. The strong and table of screening IFA signalReach the cell clone that antigen amount is high.

The ELISA that 1.4 clone cells are expressed E2 albumen detects

Clone cell is cultivated after 2d in 24 orifice plates, collects supernatant, with the culture supernatant of untransfected BHK-21 cellLiquid is contrast, and with E2 albumen in CSFV antigen detection kit detection clone cell supernatant nutrient solution, ELISA detectsUndertaken by kit explanation, measure OD450 value, each cell clone expression antigen 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, removes nutrient solution, wash 3 times with PBS,4% paraformaldehyde is fixed 10min, and PBS washes 3 times, thin with the PBS effect containing 0.1%TritonX100Born of the same parents 10min, PBS washes 3 times, and with the PBS sealing 2h containing 4%BSA, PBS washes 1 time, addsThe E0 protein monoclonal antibody of the anti-E2 protein monoclonal antibody of 1:500 dilution or 1:200 dilution, incubates for 4 DEG CEducate and spend the night, PBS washes 3 times, adds by the FITC mark goat anti-mouse IgG of 1:200 dilution, and room temperature is incubatedEducate 2h, PBS washes 3 times, observed result under fluorescence microscope.

The RT-PCR qualification of 1.6 clone cells

Extract kit with RNA and extract cell total rna, get 10 μ L and carry out reverse transcription, add OligodT1 μ L, RNase inhibitor 1 μ L, M-MLV5 × Buffer5 μ L, M-MLV transcriptase 1 μ L, dNTP(10MM) 2.5 μ L, add distilled water to 25 μ L, mix, and 42 DEG C of heating 60min, 95 DEG C of 5min stop anti-Should. 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, change after cell maintenance medium againCultivate after 4 days, cultured cell power transmission mirror cell is fixed, ultra-thin cell section, in transmission electron microscope observing cellCSFV sample particle. Cultivate in a large number filtering out high expressed amount cell clone, results supernatant after through ultrafiltrationAfter concentrated, express antigen through SDGC purifying again, 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 restructuring of extractingPlasmid, after Xho Ι ﹑ Sac Ι double digestion, carries out agarose gel electrophoresis analysis, finds that there is two bands, and enzyme is cut productThing electrophoresis banding pattern is consistent with expected results, and simultaneously sequencing result shows in recombinant plasmid between Sac Ι ﹑ Xho Ι restriction enzyme siteSequence and the gene of the coding E0-E1-E2 albumen of design in full accord.

2.2E0-E1-E2 stable gene is expressed the foundation of clone

2.2.1 the screening of transfection cell strain

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

2.2.2 the RT-PCR of clone cell qualification

Utilize RT-PCR to carry out after the amplification of genes of interest IFA qualification positive cell clone, result shows, to instituteThe cell of the different generations of the cell clone filtering out all can amplify and theory object band of the same size. ShowGenes of interest is stable being blended in cellular genome, and inheritance stability.

2.3.3 the ELISA that expresses clone detects screening

To 5 clone cell culture supernatant carry out CSFVE2 proteantigen ELISA detect, result show throughIn 5 clones that IFA filters out, No. 5 clone's expressing quantities the highest (Fig. 2), choose No. 5 cell clones and go down to posterityCultivate and further specificity analysis. No. 5 clone cell carries out CSFV to culture supernatant after different generations go down to posterityE2 protein ELISA detects, and result shows that the clone cell filtering out ties up to after repeatedly going down to posterity and still keeps good albumenExpression (Fig. 3). Result shows the recombinant cell lines energy stably express genes of interest building.

By obtain can stably express CSFV E0-E1-E2 albumen No. 5 clones, called afterBCSFV-E012, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and its bacterial classification is protectedTibetan is numbered: CGMCCNo.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 fluorescenceSignal, and control cells does not present fluorescence signal. And this cell clone, in the time being passaged to for the 20th generation, still can be stablizedExpress destination protein (Fig. 4).

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 through E0 through E2 expression ELISAMonoclonal antibody can present green fluorescence signal after detecting. 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 observeCSFV virus like particle, diameter is about 40nm. In cell culture supernatant, the antigen of purifying is also observed through Electronic SpeculumCSFV virus-like particle structure (Fig. 5). 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 recombinant proteins to pig

Vaccine preparation: by the recombinant cell lines BCSFV-E012(CGMCCNo. of embodiment 1 constructed screening7720) cell grows to 90% when full after normally going down to posterity, and changes low blood serum medium (serum content is 1～2%) and continuesCultivate 4-6d, harvesting culture supernatant is stored in 4 DEG C, and cell conditioned medium liquid is 100kD ultrafiltration through molecular cut offAfter concentrated, being adjusted to E2 antigen ELISA, to tire as adding final concentration after 1:32 to 1:64 be 0.02% thimerosalBe vaccine antigen liquid afterwards. Vaccine antigen liquid and oily adjuvant are by volume for 1:1.5 mixes and fully emulsified. RightSwine fever cell vaccine (the bull testis primary cell source) product that is market sale according to group attenuated vaccine.

Animal grouping and immunity: test the separation of serum of all taking a blood sample with pig and carry out CSFV antibody test before immunityNegative. 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 weak poisonVaccine immunity control group, weak 5 part/pigs of malicious seedling of immune CSFV, an immunity is (attenuated vaccine group) once; Second5 of group pigs are oil seepage group prepared by restructuring expression of cell lines E0-E1-E2 antigen, and immune vaccine 2ml, exempts from for the first timeAfter epidemic disease surrounding, carry out once (recombinant antigen vaccine group) of booster immunization; The 3rd group of 5 of pig is blank group, entersRow PBS injection contrast (PBS control group).

Serum neutralizing antibody detects: experiment pig is taken a blood sample once before immunity, detects all pig serum be through ELISACSFV negative antibody. One exempt from after surrounding oil seepage immune group carry out booster immunization once. In immunity for the first time rear 2Week and the separation of serum of taking a blood sample respectively for 4 weeks. Within the 2nd, 4,8,12,16,20,24,28 weeks after secondary immunity, adoptBlood separation of serum. Animal used as test pig serum all carries out the detection of CSFV virucidin, detects neutralizing antibody methodFor the NPLA method of OIE handbook recommendation. Concrete grammar is: respectively organize after 56 DEG C of deactivation 30min of pig serum, 96In porocyte plate, start to carry out 10 times of dilutions with DMEM, carry out successively again 2 times of dilutions later. By what dilutedSerum 50 μ L and equal-volume CSFV virus (Shimen strain, 200TCID₅₀/ 0.1ml) mix. Hatch 1h for 37 DEG C,Then add 100 μ LPK-15 cells, after 37 DEG C of cultivation 3d, cell is fixed with 20% acetone PBS. With anti-CSFVE2 protein monoclonal antibody is primary antibodie, and HRP mark 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 are sentenced in light Microscopic observation after colour developingDetermine result. Tire as the neutralization of serum to reduce more than 50% the maximum serum diluting multiple that infects.

Serum ELISA antibody test: immunity before and after the pig serum that gathers with IDEXX hog cholera antibody detection kitDetect, detection method is undertaken by kit explanation. By specification method calculating antibody blocking-up rate respectively. By bloodCarry out clearly 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 CSFV of the constructed preparation of the present inventionE0-E1-E2 Protein reconstitution bhk cell is can induce pig to produce after the expressed antigen vaccine immunity pig of preparingCSFV virucidin. Two exempt from after the high energy of two weeks (just exempt from after 6 weeks) neutralizing antibodies reach 5120, reach neutralizationAntibody peak, it is worth apparently higher than attenuated vaccine immunity group neutralizing antibody level. Two exempt from rear surrounding (just exempting from latter 8 weeks)It is 2560 that recombinant subunit vaccine can be induced NAT. Neutralizing antibody is slow decreasing trend later, but complies withSo keep high-level neutralizing antibody. To latter 7 months (28 weeks) of immunity, vaccine immunity papova prepared by the present inventionNeutralizing antibody level still reaches 320, far away higher than providing immunoprotection required virucidin's level to pig(40～50). And in experiment immunity contrast pig in its virucidin's level of whole experimental session all for being less than10. 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 generation swine feverSpecific antibody (antibody blocking rate is greater than 40%), antibody horizontal raises gradually subsequently, and after immunity, 6-8 week reachesPeak value. The duration of antibody, recombinant subunit vaccine immune group antibody horizontal is all higher than attenuated vaccine group antibody waterFlat. 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 are expressed antigen indirect ELISA method and are detected antibody against swine fever virus

BCSFV-E012 cell is expanded and cultivated, harvesting culture supernatant. Cell conditioned medium liquid is through low-speed centrifugalAfter removing the impurity such as cell fragment, clarify through 0.45 μ m membrane filtration again. The supernatant of clarification is again through molecular cut offFor the filter membrane ultrafiltration concentration of 100kD. After approximately 40 times of volumes are concentrated, more centrifugal through 12000rpm, remove insolubleImpurity. Supernatant is 10% to 50% continuous SDGC through concentration. Collect 20%-30% concentrationSucrose layer liquid, then measure protein content through ultrafiltration desugar and after concentrating, purifying antigen saves backup in-70 DEG C. With pureChanging CSFV-E012 albumen is envelope antigen, coated 96 hole polystyrene ELISA Plates. With pH9.6,0.1M carbonic acidSalt buffer dilution antigen to final concentration is 3 μ g/ml, adds in ELISA Plate 4 DEG C 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 of 1%BSATarget, 37 DEG C of sealing 2h, wash plate 3 times with washing lotion PBST after sealing, immediately for detection of or-20 DEG C deposit standbyWith.

Antibody test operation sequence: add pig serum to be detected (100 times of dilutions of qualitative detection serum, antibody titerDetect serum and carry out doubling dilution, set up positive serum contrast to contrast with negative serum and the sky of increase serum not simultaneouslyWhite contrast), hatch 1h for 37 DEG C, PBST washing lotion washing 3 times, each 3 minutes; Add horseradish peroxidaseMark goat-anti pig IgG (Goat-anti-pigIgG-HRP), hatches 1h for 37 DEG C, and PBST washing lotion washing 4 times is everyInferior 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 sulfuric acid color development stopping; Measure the light absorption value of 450nm wavelength with ELIASA; SentenceDetermine result.

When result of determination: blank and negative serum hole light absorption value are less than or equal to 0.3, positive serum control wellsIt is effective that light absorption value is greater than 0.4 o'clock result; Calculate P/N value=(detecting hole OD value-blank hole OD value)/(the moonProperty serum OD value-blank hole OD value), P/N value be equal to or greater than 2 o'clock positive; With reacting positive bloodClear maximum dilution multiple is the antibody titer of this sample serum.

Choose each 20 parts of the positive pig serum of known antibody against swine fever virus and negative pig serum, respectively with purifyingCSFV-E012 albumen is that envelope antigen carries out indirect ELISA detection, with the detection effect of detectable antigens. Detect knotAs shown in Figure 7, all positive serum OD450 values are all greater than 0.5 to fruit, and all negative serum OD450 values are all less than0.3. Show that this antigen has good specificity and sensitiveness.

Claims (8)

1. for preventing a vaccine combination for swine fever, it is characterized in that containing in described vaccine combination throughThe CSFV E0-E1-E2 albumen that the bhk cell system of stably express CSFV E0-E1-E2 albumen expresses andAdjuvant, wherein said E0-E1-E2 albumen is virus-like particle antigen, described CSFV E0-E1-E2 eggWhite amino acid sequence is as shown in SEQIDNo.1;

The vaccine combination of described prevention swine fever prepares by following steps:

(1) be after BCSFV-E012 normally goes down to posterity, to grow to 90% when full by bhk cell, clear volume percentage exchanges blood transfusionThe low blood serum medium that is 1～2% than content continues to cultivate 4-6d, and harvesting culture supernatant is stored in 4 DEG C;

(2) cell conditioned medium liquid is adjusted to E2 antigen ELISA effect after molecular cut off is 100kD ultrafiltration concentrationAfter valency is 1:32 to 1:64, according to mass percent meter, adding final concentration is after 0.02% thimerosal, to be vaccineAntigen liquid;

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

Wherein, described bhk cell is BCSFV-E012, is deposited in Chinese microorganism strain preservation conservatorMeeting common micro-organisms center, its culture presevation is numbered: CGMCCNo.7720.

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

(1) build eukaryon expression plasmid, this eukaryon expression plasmid comprises the coding CSFV wherein insertingThe cDNA sequence of E0-E1-E2 albumen;

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

(3) select to cultivate to have added the nutrient solution of G418 through the cell of plasmid transfection;

(4) by through selecting the cell of cultivating to dilute clone, gather in the crops clone cell culture supernatant and cell, inspectionSurvey and compare the expression of CSFV E 2 protein, obtain the restructuring of stably express CSFV E0-E1-E2 albumenBhk cell system.

3. vaccine combination as claimed in claim 2, is characterized in that described coding CSFV E0-E1-E2The cDNA sequence of albumen is as shown in SEQIDNo.2.

4. the bhk cell of stably express CSFV E0-E1-E2 albumen system, called after BCSFV-E012, protectsEnsconce China Committee for Culture Collection of Microorganisms's common micro-organisms center, its culture presevation is numbered: CGMCCNo.7720, the amino acid sequence of described CSFV E0-E1-E2 albumen is as shown in SEQIDNo.1.

5. be expressed CSFV E0-E1-E2 albumen by bhk cell claimed in claim 4, its spyLevy and be that described E0-E1-E2 albumen can be self-assembled into as virus-like particle protein at cell inner expression, describedThe amino acid sequence of CSFV E0-E1-E2 albumen is as shown in SEQIDNo.1.

6. the bhk cell of stably express CSFV E0-E1-E2 albumen claimed in claim 4 system or its trainingSupport the application of thing in preparation prevention CSFV disease vaccine medicine, the wherein said culture swine fever of expressing of serving as reasonsThe virus-like particle protein that the self assembly of virus E0-E1-E2 albumen forms, described CSFV E0-E1-E2 albumenAmino acid sequence as shown in SEQIDNo.1.

7. the bhk cell of stably express CSFV E0-E1-E2 albumen claimed in claim 4 system or its trainingSupport thing in preparation diagnosis or detect the application in swine fever virus infection reagent, wherein said culture is served as reasons and is expressedThe virus-like particle protein that the self assembly of CSFV E0-E1-E2 albumen forms, described CSFV E0-E1-E2The amino acid sequence of albumen is as shown in SEQIDNo.1.

8. prepare a method for the vaccine combination of the prevention swine fever described in claim 1-3 any one, its featureBe to comprise the following steps:

(1) be after BCSFV-E012 normally goes down to posterity, to grow to 90% when full by bhk cell claimed in claim 4,The clear volume percent content that exchanges blood transfusion is 1～2% low blood serum medium continuation cultivation 4-6d, harvesting culture supernatantLiquid is stored in 4 DEG C;

(2) cell conditioned medium liquid is adjusted to E2 antigen ELISA effect after molecular cut off is 100kD ultrafiltration concentrationAfter valency is 1:32 to 1:64, according to mass percent meter, adding final concentration is after 0.02% thimerosal, to be vaccineAntigen liquid;

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