CN107674883A

CN107674883A - Preparation method and application of recombinant classical swine fever E2 protein and subunit vaccine thereof

Info

Publication number: CN107674883A
Application number: CN201710409930.6A
Authority: CN
Inventors: 钱泓; 吴有强; 车影; 吴素芳; 查银河
Original assignee: Novo Biotech Corp
Current assignee: Novo Biotech Corp
Priority date: 2016-08-01
Filing date: 2017-06-02
Publication date: 2018-02-09

Abstract

The invention discloses a preparation method and application of a recombinant swine fever E2 protein and subunit vaccine thereof, wherein the preparation method of the recombinant swine fever E2 protein comprises the following steps of 1) cloning a encoding gene of the swine fever E2 protein into a eukaryotic expression vector to obtain a recombinant plasmid containing a encoding gene of the swine fever E2 protein, 2) transfecting the recombinant plasmid containing the encoding gene of the swine fever E2 protein into a CHO cell strain, 3) obtaining a highly expressed cell strain by culturing, screening and domesticating the CHO cell strain in the 2), and 4) fermenting and culturing the cell strain in the 3) to obtain the recombinant swine fever E2 protein after purification.

Description

Recombinate the preparation method and application of swine fever E2 albumen and its subunit vaccine

Technical field

The present invention relates to a kind of Chinese hamster ovary celI strain of suspending stabilized high efficient expression swine fever E2 albumen and structure, screen the cell The method of strain and the preparation method and application of swine fever E2 protein subunit vaccines, belong to animal vaccine and veterinary biologicses technology Field.

Background technology

It is by CSFV that swine fever is referred to as classic swine fever (Classical Swine Fever, CSF) in Europe A kind of acute, hot, fatal disease caused by (Classical Swine Fever Virus, CSFV).Swine fever has height Contagiousness, anxious, high fever of falling ill are delaied causes the characteristics of lesion such as extensive bleeding, infraction and necrosis with the denaturation of thin vessels wall.Family Support and wild pig is its unique natural host.OIE (OIE) is set to A class infectious diseases, China《Animal Assanation》It is classified as a kind of infectious disease.Swine fever is one of main epidemic disease for endangering China's pig industry development at present.

CSFV belongs to flaviviridae, pestivirus member, is single-stranded linear RNA virus.Virion is slightly rounded, With lipoprotein envelope, virion surface has the fine lug structure of fragility.CSFV genomes are about 12.5kb, only big containing 1 Open reading frame (ORF), this ORF encode about 3898 amino acid residues, molecular weight about 438kDa polyprotein.This is more Polyprotein is processed into 12 kinds of maturation proteins while translation and upon translation through virus with host cell proteins enzyme, is followed successively by N^pro,C,E^rns, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B, wherein C, E^rns, E1 and E2 are structural proteins, its Remaining is non-structural protein.E2 is the most important immunogenic proteins of CSFV, and body can be induced to produce neutralizing antibody and can Protect the attack of pig resistance CSFV velogen strains, and the important target protein of research hog cholera genetic engineering bacterin.

Swine fever worldwide has prevalence, very harmful to pig industry, is to effective precautionary measures of the disease at present Vaccine immunity.By applying for many years, it is recognized that safely and effectively attenuated vaccine strain has three kinds：1. Chinese rabbitization attenuated vaccine；2. day This GPE (-) cell weak-toxic vaccine；3. French " Thiveosal " cold variation low virulent strain.The China that wherein Chinese scholar succeeds in developing It is (C systems) hog cholera lapinised virus vaccine, it is Eurasian many national except in addition to, being generalized in China from nineteen fifty-seven, and Help these state controls or eliminate swine fever.The vaccine is acknowledged as at present more satisfactory hog cholera vaccine in the world.At present The hog cholera vaccine commonly used in China market has 3 kinds, i.e. swine fever cell live vaccine (cell vaccine), swine fever breast rabbit tissue live vaccine (group Knit seedling), swine fever spleen leaching live vaccine (spleen leaching seedling).The production of Tissue vaccine, spleen leaching seedling needs a large amount of healthy animals, people in production process The extensive use that work labor intensity is big, cost is high, has the unfavorable factors such as Side effects to have impact on such seedling.Cell vaccine is equally Perplexed by factors, batch wise differences such as be present；Acellular lesion, it is difficult to control viral yield；Cell vaccine raw and auxiliary material relates to And bovine testicle cell and cow's serum, cause the risk that BVDV pollutions in its preparation process be present.BVDV infected pigs can cause doubtful Like the symptom of swine fever.In recent years, swine disease caused by the hog cholera vaccine polluted as BVDV is just had been reported that in China.It is further, since long Phase uses live vaccine, leads to not fundamentally purify swine fever.

In view of many disadvantages and deficiency of existing hog cholera vaccine, and with modern genetic engineering technology and cell technology Development, many scientific research personnel attempt to develop the new swine fever that can overcome existing vaccine defect with modern molecular biology means Vaccine.These new hog cholera vaccines have viral live vector vaccine, synthetic peptide vaccine, DNA vaccination, Bacillus coli expression albumen Subunit vaccine, the sub- single vaccine of the E2 albumen of baculovirus expression.Wherein there is European scientific research people to what is be applied at present The E2 protein subunit vaccines with baculovirus expression that member develops, the vaccine immunity are not influenceed by maternal antibody, and It can be infected with virus and carry out antibody test antidiastole.But the vaccine is expressed by insect baculovirus expression system, Post translational processing can be carried out for the expression system is with respect to prokaryotic expression system, after protein expression to a certain extent with repairing Decorations, but still have different with viral native antigen protein structure, folded after protein expression and be not so good as mammalian cell with modification Expression system.And the system production, when preparing antigen, cell can be cracked with dead, to downstream purification through cells following viral infection Increase difficulty etc. production technology.In addition, the yield that the system prepares destination protein will not be very high, typically only 200-300mg/L, It is extremely difficult to 500mg/L yield.

Chinese hamster ovary celI is nineteen fifty-seven Univ Colorado-Boulder USA Theodore doctors T.Puck from an Adult female Hamster ova Nest separation obtains, and is the adherent type cell of epithelium.The cell has immortality, and it is more than generation can to pass on hundred, is current biological work Widely used cell in journey.Relative to other expression systems, it has following advantage：(1) there is accurate posttranscriptional modification Function, the albumen of expression is in terms of molecular structure, physicochemical property and biological function closest to native protein molecule；(2) both Can adherent growth, can suspend culture again, and have higher tolerance shearing force and osmotic pressure ability；(3) with recombination Efficient amplification and ability to express, the integration of foreign protein are stable；(4) there is product exocytosis function, and seldom secrete certainly The intrinsic protein of body, is easy to downstream product to isolate and purify；(5) it can reach high with suspension training method or in serum free medium Density culture, and volume of culture can reach 1, more than 000L, can mass produce.

Chinese hamster ovary celI type is relatively more, such as：DG44, DXB11, CHO K1 and CHO-S etc..Opened from the 80-90 ages in 20th century Begin, industrially relatively early to use DHFR (dihyrofolate reductase deficiency) gene magnification screening system, host cell strain is DG44.When containing methotrexate (MTX) (methotrexate, MTX) in cell culture medium, dihyrofolate reductase is suppressed, and is passed through Feedback regulation so that the gene is expanded, and the gene in the range of its upstream and downstream 100-1,000kb can all expand therewith, therefore Target gene is inserted into this site scope can be expanded.The system of many monoclonal antibody productions is still DG44 DHFR bodies now System.GS (glutamine synthelase) amplification system, it is that a kind of novel gene developed in recent years expands using CHO-K1 as host cell Increase screening system, have obvious superiority compared with DHFR systems, have been widely recognized in the world at present.Its principle is that GS exists While ATP hydrolysis provides energy, glutamine is synthesized using intracellular ammonia and glutamic acid.Lacking external source glutamine GS inhibitor methionine sulfoxide imonium (L-methioninesulfoximine, MSX) is added in culture medium, GS genes can be made And the target gene being attached thereto effectively is expanded, the horizontal purpose of destination gene expression is improved so as to reach.The system Advantage mainly exists：The CHO-K1 cell lines of gene defection type are not needed as host cell；CHO-K1 cells are easy to cultivate stronger It is strong；Glutamine need not be added in the medium, glutamine can be avoided to decompose and cause the problem of ammonia level is high in cultivating system, The difficulty of technology controlling and process is reduced, and cell fermentation density can be effectively improved and extend the cells survival time.

But the present inventor has found the gene of swine fever E2 albumen not when using expressing cho cell swine fever E2 albumen at the beginning During through codon optimization, Chinese hamster ovary celI does not express swine fever E2 albumen substantially, therefore, is using expressing cho cell swine fever E2 albumen When, codon optimization is an extremely important step.

The content of the invention

The problem to be solved in the present invention is to provide one kind can large-scale industrial production CSFV recombinant subunit vaccine Preparation method and application.

According to an aspect of the present invention, the invention provides a kind of preparation method for recombinating swine fever E2 albumen, the system Preparation Method contains the following steps：1) the swine fever E2 protein coding genes after codon optimization are cloned into carrier for expression of eukaryon and obtained To the recombinant plasmid containing swine fever E2 protein coding genes；2) again by the Transfected Recombinant Plasmid containing swine fever E2 protein coding genes Into Chinese hamster ovary celI strain；3) by cultivate, screen, tame 2) described in Chinese hamster ovary celI strain obtain the cell line that altimeter reaches；With And 4) fermented and cultured 3) described in cell line, obtain recombinating swine fever E2 albumen after purification.

In the preferred technical solution of the present invention, it is preferable that swine fever E2 protein coding genes after the codon optimization Nucleotide sequence is as shown in SEQ ID NO.2.

In the preferred technical solution of the present invention, it is preferable that the carrier for expression of eukaryon be pEE6.4, pEE12.4, pGL4.13、pcDNA3.1.It is highly preferred that the carrier for expression of eukaryon is pEE12.4.

In the preferred technical solution of the present invention, it is preferable that the Chinese hamster ovary celI strain is DG44, DXB11, CHO-K1, CHO-S Cell line.It is highly preferred that the Chinese hamster ovary celI strain is CHO-K1.

According to another aspect of the present invention, present invention also offers one kind to contain restructuring swine fever E2 protein subunit vaccines Preparation method, restructuring swine fever E2 albumen and pharmaceutically acceptable adjuvant that above-mentioned preparation method obtains fully are mixed, obtained To swine fever E2 recombinant subunit vaccines.

In the preferred technical solution of the present invention, it is preferable that adjuvant used in the recombinant subunit vaccine is the VG of ISA 201.

In the preferred technical solution of the present invention, it is preferable that the restructuring swine fever E2 albumen presses body with the VG of adjuvant ISA 201 Product ratio 46:54 mixing and emulsifyings.

In accordance with a further aspect of the present invention, present invention also offers one kind restructuring swine fever E2 albumen and CSFV restructuring are sub- Application of the subunit vaccine in dependent diagnostic reagent is prepared.

The present invention builds and has screened the Chinese hamster ovary celI strain of suspending stabilized high efficient expression swine fever E2 albumen, cell line expression E2 protein yields are high, are easy to purify, are easy to mass produce, and the swine fever E2 proteantigens expressed can be induced immune swine and produced Good immune response is simultaneously resistant to swine fever Strain Shimen strong virus attack；By restructuring E2 albumen prepare subunit vaccine have with Lower advantage：1. large-scale production, for measuring, sufficient, Quality Control is easy；It is 2. safe, immunogenicity is good；It is 3. stable between batch；4. life It is low to produce cost；5. without BVDV pollution risks.

The system of present invention selection expression swine fever E2 albumen is CHO expression systems, and the system research is clearer, and this hair The cell line of person of good sense's expression used is to be adapted to the culture that suspends, and yield is up to 1g/L, therefore is industrially very easy to amplification, special Not Shi He large-scale industrial production, relative to other eukaryotic expression systems, production finished product is very low；In addition, the present inventor makes With the swine fever E2 albumen of expressing cho cell be secretion cell conditioned medium in because foreign protein content is less in cell conditioned medium, because This purifying is got up more convenient quick, and the present inventor, which studies, to be found, expresses the swine fever E2 albumen in cell conditioned medium in SDS- Purity on PAGE can reach more than 80% (see the SDS-PAGE testing results of stoste in Fig. 6), therefore only need simple A ni-sepharose purification purity is crossed with regard to more than 95% can be reached, is much met needed for the preparation of swine fever E2 subunit vaccines.

Brief description of the drawings

Fig. 1 represents pEE12.4-OPTI-E2 plasmid maps.

Fig. 2 represents pCDNA3.1-OPTI-E2 plasmid maps.

Fig. 3 represents pEE12.4-OPTI-E2 double digestion qualification results：3 be DNA Marker：1kb ladder；1 is The non-restriction enzyme digestion and electrophoresis results of pEE12.4-OPTI-E2；2 be pEE12.4-OPTI-E2 double digestion electrophoresis results.

Fig. 4 represents pCDNA3.1-OPTI-E2 double digestion qualification results：3 be DNA Marker：DL10000；1st, 2 are all PEE12.4-OPTI-E2 double digestion electrophoresis results.

Fig. 5 represents cell shake flask fermentation result.

Fig. 6 represents affinity chromatography chromatogram and SDS-PAGE results.

Fig. 7 represents the immune rear antibody titer result of IDEXX kits detection restructuring swine fever E2 protein subunit vaccines.

Fig. 8 represents the nucleotide sequence comparison before and after codon optimization：E2 represents the sequence before codon optimization；OPTI- E2 represents the sequence after codon optimization.

Embodiment

Below with reference to drawings and examples, the present invention will be further described, and embodiments of the invention are merely to illustrate this The technical scheme of invention, and the non-limiting present invention.

Bacterial strain, plasmid and reagent used in the embodiment of the present invention are commercially available prod.

The source list of reagent and medicine of the present invention is as follows：

CHO-K1 cell deriveds are in the Shanghai life of the cell bank Chinese Academy of Sciences of the American Type Culture Collection committee of the Chinese Academy of Sciences Order Science Institute's cell bank；

Dihyrofolate reductase deficiency Chinese hamster ovary cell (CHO/dhfr^-) it is purchased from American Type Culture Collection (ATCC) Products (SCO610)；

Cell culture medium is purchased from Gibco companies of the U.S., and hyclone is purchased from Hyclone companies；

Carrier for expression of eukaryon pEE12.4 is purchased from upper Hailin deep pool bio tech ltd；

Carrier for expression of eukaryon pCDNA3.1 (+) is purchased from Invitrogen companies of the U.S.；

Lipofectamine LTX are purchased from Thermo Fisher companies of the U.S.；

Aminomethyl petrin (methotrexate, MTX) is purchased from Sigma companies；

Methionine sulfoxide imonium ((L-methioninesulfoximine, MSX)) is purchased from Sigma companies；

BCA quantification of protein kit is purchased from Thermo Fisher companies of the U.S.；

The VG of ISA 201 are purchased from match BIC Corp of France.

Embodiment 1：Swine fever E2 albumen codon optimizations

By carrying out codon optimization to swine fever E2 protein nucleotide sequences, OPTI-E2 sequences are obtained, such as SEQ ID Shown in NO.2, the working delegation Suzhou Jin Weizhi bio tech ltd completes.

SEQ ID NO.2 are compared with SEQ ID NO.3 sequence, find the swine fever E2 albumen after codon optimization Nucleotide sequence has 21.3% difference, i.e. 1044 nucleotides with the swine fever E2 protein nucleotide sequences without codon optimization In have 222 nucleotides differences.Specifically as shown in Figure 8.

Embodiment 2：PEE12.4-OPTI-E2 construction of recombinant plasmid

2.1 PCR amplification purpose fragments OPTI-E2

2.1.1 PCR reacts

(1) design of primers and synthesis

Sense primer:5’-CCAAGCTTGCCGCCACCATGAAAGTGCTGAGGGGCCAG-3’

Anti-sense primer:5’-CCGGAATTCTTAGTGATGGTGATGGTGATGAGC-3’

(2) system 50 μ L are loaded, it is as shown in the table：

PCR amplification programs：

2.1.2 PCR primer carries out glue reclaim

(1) sample collection EP pipes, adsorption column and collecting pipe have been marked；

(2) the empty EP pipe weight marked is weighed, and records numerical value；

(3) single target DNA band is carefully cut with scalpel from Ago-Gel on bale cutting instrument be put into it is dry In net 1.5mL centrifuge tubes；

(4) 600 μ L PC buffer are added in the 1.5mL centrifuge tubes into step (3), it is left that 5min is placed in 50 DEG C of water-baths The right side, centrifuge tube is gently constantly spun upside down therebetween, to ensure that blob of viscose fully dissolves；

(5) column equilibration：Into adsorption column CB2, (adsorption column is placed in advance in collecting pipe) adds 500 μ L equilibrium liquid BL, centrifugation 12,000rpm/min, 1min, the waste liquid in collecting pipe is outwelled, adsorption column is placed back in collecting pipe；

(6) step (5) resulting solution is added in adsorption column CB2, stands 2min, 10,000rpm/min, centrifugation 30s, Fall the waste liquid in collecting pipe, then adsorption column CB2 is put into collecting pipe；

(7) 600 μ L rinsing liquid PW buffer are added into adsorption column, stand 3min, centrifuge 10,000rpm/min, 30s, The waste liquid in collecting pipe is outwelled, adsorption column CB2 is put into collecting pipe；

(8) repeat step (7)；

(9) suction attached column centrifuges, and 12,000rpm/min, 2min, removes rinsing liquid as far as possible, adsorption column is placed in into room temperature puts 10min is put, is thoroughly dried；

(10) adsorption column CB2 is put into collecting pipe, 50 μ L Elution is vacantly added dropwise to adsorbed film centre position Buffer (65 DEG C of preheatings), stands 3min, centrifuges 12,000rpm/min, 2min；

(11) centrifuge tube in step (10) is taken out from centrifuge, the adsorption column CB2 of centre is abandoned, covers centrifugation lid Son, retain the DNA sample in centrifuge tube；

(12) DNA sample in step 11 is placed in 4 DEG C of preservations, prepares agarose gel electrophoresis identification glue reclaim DNA pieces Section.

2.2 PCR primers and the reaction of carrier double digestion

(1) mark needs well the 1.5mL EP used to manage, and sample-adding is carried out according to the following table in 1.5mL EP pipes, mixes：50μ L reaction systems

(2) the 1.5mL EP pipes in step (1) are placed in corresponding enzyme optimum temperature thermostat water bath, water-bath 2-3h.

Double digestion product glue reclaim：Above-mentioned double digestion system is taken out, enters row agarose gel electrophoresis to reclaim DNA therein Fragment, method is the same as PCR primer glue reclaim in 1.2.1.

2.3 coupled reaction

(1) it is some to prepare clean 1.5mL EP pipes, carries out mark, is placed in stand-by on EP pipe supports.

(2) sample-adding, mixing is carried out according to the following table in 1.5mL EP pipes.

(3) after completing sample-adding according to form in step (2), each 10 μ l reaction systems is placed in 16 DEG C of cryogenic liquids and followed In ring machine, water-bath 10-16h；

(4) take out EP in step (3) to manage, be placed in 65 DEG C of water-baths, water-bath 15min；

(5) the EP pipes in step (4) are taken out, are placed in 4 DEG C of preservations.

1.2.4 conversion reaction

(1) 10 μ l coupled reaction liquid are rapidly joined in 100 μ l competent cells, and blows and beats mixing, ice bath 30min；

(2) sample cell is taken out, is placed in 42 DEG C of water-bath 100s, immediately after ice bath 2min；

(3) sample cell is taken out, in superclean bench, 600 μ L LB liquid mediums are added into sample cell, then by sample QC is placed in 37 DEG C of constant-temperature tables, 220rpm/min, cultivates 1h；

(4) coated plate：Sample cell in step (3) is taken out, room temperature centrifuges 8,000rpm/min, 2min, removes 600 μ l supernatants The thalline of bottom of the tube is resuspended in body, remaining supernatant, and the bacterium solution of resuspension is put into corresponding conversion plate center, will be turned with bacteria stick is applied The bacterium solution for changing plate center is uniformly spread out.

(5) step of converting (4) flat board is just being placed in biochemical constant incubator, after 37 DEG C of culture 1h, flat-plate inverted will be being converted Put and carry out culture 15h；

(6) conversion results are observed.

2.5 plasmid extractions are identified with double digestion

2.5.1 plasmid extraction

(1) with 10 μ L liquid transfer gun heads from conversion flat board in picking monoclonal to 5ml benzyls containing ammonia resistance LB fluid nutrient mediums In, 37 DEG C, 220rpm/min shakes bacterium and stayed overnight；

(2) bacterium solution is moved in 1.5ml EP pipes, room temperature centrifugation, 12,000rpm/min, 2min, abandons supernatant；

(3) 250 μ L plasmid extraction reagent P1 buffer, thorough suspension thalline are added into the EP pipes of step (2)；

(4) 250 μ L P2 buffer are added into step (3) solution, immediately gently reverse 5-10 mixing of centrifuge tube, room Temperature stands 2-4min；

(5) 350 μ L P3 buffer are added into step (4) solution, immediately gently reverse 5-10 mixing of centrifuge tube；Room Temperature stands 2-4min；

(6) by step (5) solution, room temperature centrifugation, 14,000rpm/min, 10min；

(7) supernatant solution in step (6) is moved into adsorption column center, room temperature centrifugation, 12,000rpm/min, 30s, outwelled Liquid in collecting pipe；

(8) 500 μ L Buffer DW1 are added to adsorption column center, room temperature centrifugation, 12,000rpm/min, 30s, outwells receipts Liquid in collector；

(9) 500 μ L wash solution are added to adsorption column center, room temperature centrifugation, 12,000rpm/min, 30s, fallen Fall liquid in collecting pipe, be repeated once；

(10) suction attached column, room temperature centrifugation, 12,000rpm, 2min.

(11) adsorption column is put into a clean 1.5ml centrifuge tube, 30 μ L Elution is added to absorption center membrane Buffer, it is stored at room temperature 5min, room temperature centrifugation, 12,000rpm, 2min.Preserve DNA solution in pipe.

2.5.2 double digestion is identified

(1) mark needs well the 1.5mL EP used to manage, and sample-adding is carried out according to the following table：20 μ L reaction systems

(2) the μ L reaction systems of EP pipes 20 in step (1) are placed in 37 DEG C of thermostat water baths, water-bath 2h.

(3) the double digestion system sample in step (2) is entered into row agarose gel electrophoresis, whether checks Insert Fragment size Correctly；Experimental result is shown in Fig. 3.

(4) selecting Insert Fragment, correctly clone send sequencing company to be sequenced.

2.6 endotoxin-free plasmids carry greatly

2.6.1 endotoxin-free plasmid extraction

(1) correctly clone is seeded in the culture medium of 100ml benzyls containing ammonia resistance for sequencing, in 37 DEG C of constant-temperature tables, 220rpm/min, cultivate 15h；

(2) bacterium solution by culture in step (1) is transferred in 50mL centrifuge tubes, room temperature, 8,000rpm/min, centrifugation 5min, thalline is collected, discards supernatant culture medium；

(3) 8mL solution P1 are added into the centrifuge tube of step (2), thalline is fully resuspended with pipettor；

(4) 8mL solution P2 are added into the centrifuge tube of step (3), gently overturn centrifuge tube immediately 6-8 times, are stored at room temperature 5min；

(5) 8mL solution P4 are added into the centrifuge tube of step (4), are turned upside down 6-8 times immediately, are fully mixed to solution There is white flock precipitate, room temperature places 10min or so.8,000rpm/min room temperatures centrifuge 5-10min, make white precipitate from extremely Ttom of pipe；

(6) by supernatant in step (5) all careful immigration filter CS1, slowly push handle filter, filtrate collection exists In clean 50mL centrifuge tubes；

(7) column equilibration：Into adsorption column CP6, (adsorption column is put into 50mL collecting pipes) adds 2.5mL equilibrium liquid BL, room 8,000rpm/min of temperature centrifuges 2min, outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe；

(8) isopropanol of 0.3 times of filtrate volume is added into step (6) filtrate, absorption is transferred to after mixing of turning upside down In post CP6.Room temperature 8,000rpm/min centrifugation 2min, outwells liquid in collecting pipe, adsorption column CP6 is reentered into same receipts In collector；

(9) 10mL rinsing liquid PW are added into step (8) adsorption column CP6, room temperature 8,000rpm/min centrifugation 2min, abandon receipts Waste liquid in collector, adsorption column is placed back in collecting pipe；

(10) step (9) is repeated once；

(11) 3mL absolute ethyl alcohols are added into step (10) adsorption column CP6, room temperature 8,000rpm/min centrifugation 2min, are fallen Fall waste liquid；

(12) step (11) adsorption column CP6 is placed back in collecting pipe, room temperature 8,000rpm/min centrifugations 5min.It will inhale Attached column CP6 uncaps, and is placed in room temperature and places several minutes and dries；

(13) adsorption column in step (12) is put into clean 50mL centrifuge tubes, adding 1-2mL in adsorbed film center delays Fliud flushing TB, 5min is stored at room temperature, room temperature 8,000rpm/min centrifugation 2min, the eluent in 50mL centrifuge tubes is all moved into one Individual clean 1.5mL centrifuge tubes, survey concentration, -20 DEG C of preservations.

(14) plasmid DNA solution obtained by 1-2 μ L is taken to enter row agarose gel electrophoresis and preserve electrophoresis result data.

Embodiment 3：PCDNA3.1-OPTI-E2 construction of recombinant plasmid

Operated according to the step in embodiment 2, double digestion identification experiment result is shown in Fig. 4.

Embodiment 4：PEE12.4-OPTI-E2 Transfected Recombinant Plasmid CHO-K1 cells and the foundation of monoclonal screening

4.1 CHO-K1 cell transfectings

(1) prepare：Biohazard Safety Equipment ultraviolet sterilization 30min；DMEM/F12 (containing 10% serum, 1% is dual anti-), DMEM/F12 37 DEG C of water-baths, which are placed in, with PBS is preheated to 37 DEG C.

(2) cell (10cm Tissue Culture Dish), supernatant discarding culture medium, with the 8ml of pre-temperature are taken out from 37 DEG C of incubators PBS washes cell once, and discards PBS.

(3) each 10cm Tissue Culture Dish adds 1-2ml 0.25%trypsin-EDTA, room temperature digestion 2min or so, shows Micro- Microscopic observation cell shrinkage is rounded, and is in individual cells.

(4) 4ml DMEM/F12 (contain 10% serum, 1% is dual anti-) termination digestion reactions are added, and with pipettor by cell Dispel.

(5) cell digested is transferred in 15ml centrifuge tubes, normal temperature centrifugation, 200g, 5min.

(6) with DMEM/F12 (containing 10% serum, 1% is dual anti-) again suspension cell, count.

(7) diluting cells are to 2 × 10⁵Individual/ml, the cell for taking 2ml to mix are added to six orifice plates, and six orifice plates are placed into 37 DEG C, 5%CO₂It is incubated overnight in cell culture incubator.

(8) step (7) Tissue Culture Dish is taken out, observes cell state：When cell degree of crossing reaches 80%-90% Start to transfect, culture medium is changed into the DMEM/F12 of antibiotic-free serum-free, 2mL/ holes before transfection.

(9) plasmid is diluted：Plasmid is diluted with OPTI-MEM, 2.5 μ g plasmids is added in 125 μ l OPTI-MEM, then adds 2.5 μ l plus, mix, be stored at room temperature 5min.

(10) Lipofectamine LTX are diluted：9 μ l Lipofectamine LTX are added in 125 μ l OPTI-MEM, Then 2.5 μ l plus are added, gently mixes, is stored at room temperature 5min.

(11) step (10) and step (11) mixture are gently mixed.Room temperature places 5min, and six holes are then added dropwise It is uniformly distributed in plate.

(12) six orifice plates are placed in 37 DEG C, 5%CO₂4-6h is cultivated in cell culture incubator.

(13) liquid is changed：Supernatant culture medium is discarded, 2ml DMEM/F12 (dual anti-containing 10% serum 1%) are added, by six orifice plates 37 DEG C are placed in, 5%CO₂Cultivated in cell culture incubator.

4.2 pressurization screenings

24h starts to pressurize after transfection：Six orifice plate cells are taken out from 37 DEG C of incubators, supernatant discarding culture medium, add 2ml DMEM/F12 (+25 μM of MSX containing 10% serum), pressurize 7days, and centre observation cell, dead cell changes liquid more.

4.3 monoclonals screen

(1) when death ray basic to negative control cell is screened in pressurization, about 7day, monoclonal screening is started.

(2) six orifice plates are taken out, discard culture medium, PBS is washed once, then adds 300 μ l 0.25%trypsin-EDTA, Room temperature digestion 2min or so, adds 2ml DMEM/F12 (+25 μM of MSX containing 10% serum) and terminates digestion reaction, and use pipettor Cell is dispelled.

(3) cell digested is transferred in 15ml centrifuge tubes, normal temperature centrifugation, 200g, 5min.

(4) with DMEM/F12 (+25 μM of MSX containing 10% serum) again suspension cell, count.

(5) bed board：To 5/ml, the cell for taking 200 μ L to mix is added in 96 orifice plates diluting cells, is placed into 37 DEG C, 5%CO₂4-6h is incubated in cell culture incubator.

(6) hole of individual cells is recorded.

(7) when the hole length of individual cells in 96 orifice plates is got up, culture medium is discarded, PBS is washed once, adds 100 μ l 0.25%trypsin-EDTA, room temperature digestion 2min or so, adds 2ml DMEM/F12 (+25 μM of MSX containing 10% serum) and terminates Digestion reaction, and dispelled cell with pipettor.Cell liquid is transferred to 12 orifice plates, when 12 orifice plates cover with, takes supernatant, Whether ELISA detections clone is positive, and the positive colony of high efficient expression continues to expand culture, frozen.

(8) by screening, 2 plants of cell lines are harvested altogether, numbering is 133 plants, 54 plants.

Embodiment 5：PCDNA3.1-OPTI-E2 Transfected Recombinant Plasmids CHO/dhfr^-Cell and the foundation of monoclonal screening

5.1 CHO/dhfr^-Cell transfecting

By the plasmid pCDNA3.1-OPTI-E2 and pSV2-dhfr built according to mol ratio 5:1 mixing, is used Lipofectamine LTX cotransfections CHO/dhfr^-Cell.The CHO-K1 cell transfectings of embodiment 4 are shown in concrete operations.

5.2 pressurization screenings

24h starts to pressurize after transfection：Six orifice plate cells are taken out from 37 DEG C of incubators, supernatant discarding culture medium, add 2ml DMEM/F12 (contains 10% serum+25nM MTX), and pressurize 7d, and centre observation cell, dead cell changes liquid more.

5.3 monoclonals screen

25 μM of MSX in monoclonal screening step in embodiment 4 are replaced with into 25nM MTX, other operations are the same.

By screening, 1 plant of cell line is harvested altogether, numbering is 251 plants.

Embodiment 6：The domestication of CHO-K1 cell lines is cultivated into suspending

(1) prepare：Biohazard Safety Equipment ultraviolet sterilization 30min；DMEM/F12 (containing 10% serum, 25 μM of MSX) is placed in 37 DEG C 37 DEG C are preheated in water-bath.

(4) 4ml DMEM/F12 (contain 10% serum, 25 μM of MSX) termination digestion reactions are added, and with liquid-transfering gun by cell Dispel.

(6) 100%DMEM/F12 (containing 10% serum, 25 μM of MSX) suspension cell is used, is counted.

(7) diluting cells are to 5 × 10⁵Individual cell/ml inoculation 30ml cultures are based in a 125ml shaking flask.Cell culture Bottle is placed into 37 DEG C, 5%CO₂120rpm/min is incubated overnight on rail mounted oscillator in cell culture incubator.

(8) bio-safety counter top is sterilized with 75% alcohol wipe, ultraviolet irradiation 30min.

(9) cell density and vigor are counted every 24h.

(10) second generation culture is carried out when cell survival rate reaches 94-97% after first generation cell culture once.

(11) prepare：Biohazard Safety Equipment ultraviolet sterilization 30min；100%DMEM/F12 (contains 10% serum, 25 μM of MSX), EX-CELL 302 is placed in CO₂37 DEG C are preheated in cell culture incubator.

(12) take out cell from 37 DEG C of incubators to be transferred in 50ml centrifuge tubes, normal temperature 200g centrifugations 5min.

(13) DMEM/F12 (containing 10% serum, 25 μM of MSX) and EX-CELL 302 is pressed 1：1 mixing mixes, and hangs again Floating cell, is counted.

(14) diluting cells are to 5 × 10⁵Individual cell/ml inoculation 30ml cultures are based in a 125ml shaking flask.Cell culture Bottle is placed into 37 DEG C, 5%CO₂120rpm/min is incubated overnight on rail mounted oscillator in cell culture incubator.

(15) bio-safety counter top is sterilized with 75% alcohol wipe, ultraviolet irradiation 30min.

(16) cell density and vigor are counted every 24h.

(17) cell survival rate that second generation culture obtains afterwards twice is more than 95%；Three to six be commissioned to train support obtain afterwards three times Cell survival rate be more than 95%.After 7 weeks, cell breeds three generations after being inoculated with 3 days, and density reaches 1 × 10⁶Individual cell/ml, simultaneously Cell survival rate reaches 95%, and the cell is considered as being already adapted to the culture that suspends.Inoculum density is reduced to 3 × 10⁵Individual/ml.

(18) through domestication, 133 plants, 54 plants all meet to require, this shows that 133 plants, 54 plants are all tamed successfully.

Embodiment 7：CHO/dhfr^-Cell line domestication is cultivated into suspending

25 μM of MSX in embodiment 6 are replaced with into 25nM MTX, other operations are the same.

Through domestication, 251 plants meet to require, this shows that 251 plants are tamed successfully.

Embodiment 8：Cell shake flask fermentation

(1) preparation of Secondary Culture base：60% CD-CHO+40% Ex-cell 302 is placed in 37 DEG C of water-baths in advance Heat is to 37 DEG C.

(2) from CO₂Constant-temperature table takes out shaking flask cell, is counted.

(3) diluting cells are to 2.5-3.5 × 10⁵Individual cell/ml inoculation 30ml cultures are based in a 125ml shaking flask.Carefully Born of the same parents' blake bottle is placed into 37 DEG C, 5%CO₂100rpm/min is incubated overnight in constant-temperature table.

(4) cell density and vigor are counted every 24h, surveys glucose, when less than 2g/L, addition glucose arrives 4g/L；1ml samples are taken daily, and supernatant is used to detect protein expression situation.

(5) feed supplement (the about the 4th day)：70g/L CB5 are supplemented, add the 10% of basal medium.

Start within (6) the 5th days, by CO₂Incubator temperature is adjusted to 32 DEG C.

(7) the 9th days, 70g/L CB5 are supplemented, add the 10% of basal medium.

(8) the 12nd days, harvesting supernatant.

(9) as shown in figure 4, being detected through werstern, 133 plants of expression yield highests, it is adapted to needed for large-scale production.

Embodiment 9：Protein purification

Cells and supernatant in embodiment 8 is collected, 4 DEG C, 8,000g centrifugation 30min, supernatant is taken, crosses 0.8 μm of filter membrane, on Sample, 5 × SDS- sample buffers that 80 μ l samples add 20 μ l are reserved, are detected for SDS-PAGE.

Column equilibration：With ultrapure 2~3CV of water balance (column volume column volumes), discharge ethanol preserves liquid；Then use Buffer A(20mM NaH₂PO₄(pH 7.4), 500mM NaCl) balance 2~3CV, 4~7ml/min.

Loading：If 5ml prepacked columns one, 1ml/min carries out loading and (according to prepackage column volume regulation loading flow velocity, retained Time 5min), Flow through (FT) are collected, take 80 μ l samples to add 20 μ l 5 × SDS- sample buffers, for SDS- PAGE is detected.

Washing：With 4%buffer B (20mM NaH₂PO₄(pH 7.4), 500mM NaCl, 20mM imidazole) wash Post, flow velocity 4ml/min, the weaker foreign protein of the albumen and binding ability of uncombined upper prop is rinsed well, to OD280nm bases Untill line is steady.

Elution：50%buffer B (20mM NaH₂PO₄(pH 7.4), 500mM NaCl, 500mM imidazole) elution Destination protein, flat, 2ml/min is washed to baseline, collected：10ml/ is managed；(Elutethrough-ET) takes 80 μ after collecting sample mixing L samples add 20 μ l 5 × SDS- sample buffers, are detected for SDS-PAGE.As shown in Figure 5.

Washing：100%buffer B (20mM NaH₂PO₄(pH 7.4), 500mM NaCl, 500mM imidazole), 4ml/min, do not collect, rinse 2-3 column volume, washed to UV baselines flat.Ultrapure 2~3CV of water balance.Preserve HisTrap Excel posts can preserve liquid with 20% ethanol and balance 2~3CV.

Liquid is changed in ultrafiltration：Millipore 30KD milipore filter bag carries out ultrafiltration and changes liquid, using being concentrated after 10 times of dilutions of PBS Method to original volume carries out changing liquid, in triplicate, dilutes 1000 times altogether and changes liquid.

Aseptic filtration：In Biohazard Safety Equipment, 0.22 μm of low protein binding syringe needle filter is crossed, or a large amount of protein solutions are crossed and gone out The Nalgene of 0.22 μm of filter membrane of bacterium filter, the protein solution sample filtered deposit in -80 DEG C of refrigerators.

Protein concentration and purity testing：Protein concentration after purification is determined using BCA methods, it is overall according to gained albumen after purification Product calculates gained Tot Prot after purification, and albumen yield is calculated further according to the cell conditioned medium volume taken, by calculating, 133 plants Albumen yield is 1g/L, and other are less than 500mg/L；Purity is detected using HPLC methods, purity is attained by more than 95%.

Embodiment 10：Prepared by vaccine tests with Immunization

10.1 prepared by vaccine

The swine fever E2 albumen of appropriate expressing cho cell is added to (volume ratio 46 in the VG adjuvants of ISA 201:54), make Final concentration of protein is 30 μ g/ml, and 4 DEG C of preservations are placed in after emulsification, quality inspection are qualified.

The swine fever E2 albumen of appropriate baculovirus expression is added to (volume ratio 46 in the VG adjuvants of ISA 201: 54) it is 140 μ g/ml, to make final concentration of protein, and 4 DEG C of preservations are placed in after emulsification, quality inspection are qualified.

10.2 Immunizations experiment (Jin Yu Bao Ling Biotechnology Ltd. live vaccine national project test into OK)

Screening 3-4 week old Landrace (hog cholera antibody antigen feminine gender, annulus antigen negative, blue ear antigen negative, every group 5 Head) tested, the swine fever E2 protein subunit vaccines (30 μ g/ml) of immune 1ml expressing cho cells and insect are shaft-like every time The swine fever E2 protein subunit vaccines (140 μ g/ml) of expressing viral, booster immunization once, takes weekly after immune after just exempting from three weeks Blood 1 time, serum is separated, with IDXEE kit measurement antibody titers.

Two exempt to carry out attacking poison, every pig muscle with swine fever Strain Shimen blood poison (being purchased from China Veterinery Drug Inspection Office) within 3 weeks afterwards Inject swine fever Strain Shimen blood poison 2ml (1/2), Continuous Observation 16 days.Set up with dead more than 4/5 experiment of control group pig.

Fig. 7 results are shown：Blocking rate is always 70% or so after 2 groups of immune groups are exempted from two, but expressing cho cell E2 eggs E2 protein contents only 30 μ g/ml in white vaccine, well below the E2 protein contents in baculovirus expression E2 protein vaccines, this says After the expressing cho cell E2 protein immunizations of bright low dosage with caused antibody after the baculovirus expression E2 protein immunizations of high dose Potency is suitable.

In addition, after attacking poison, control group pig is all dead in 8 days after poison is attacked, and illustrates that challenge viral dosage is set up；Chinese hamster ovary celI E2 protein immunization groups are expressed during poison is attacked without death, and 5 pigs are also without any clinical symptoms (such as body temperature liter High, spiritual depressed, anorexia etc.), therefore integrated protection rate is 100%；Baculovirus expression E2 protein immunizations group is attacking poison Without death during although, there is pig body temperature during poison is attacked to be increased to more than 40.5 DEG C, and spirit it is depressed, Appetite substantially declines, and can determine whether to attack swine fever disease symptom after poison, therefore comprehensive descision protective rate is 80%.So from attacking poison From the point of view of protecting result, expressing cho cell E2 protein immunizations group will get well compared with baculovirus expression E2 protein immunization groups protecting effect.

The present invention is illustrated by above embodiment, it is understood, however, that the present invention is not limited to institute here The particular example and embodiment of description.Purpose herein comprising these particular examples and embodiment is to help this area In technical staff put into practice the present invention.Any those of skill in the art are easy to do not departing from spirit and scope of the invention In the case of be further improved and perfect, therefore the present invention is only by the content of the claims in the present invention and limiting for scope System, its intention cover the alternative in all spirit and scope of the invention for being included in and being limited by appendix claim and waited Same scheme.

Sequence table

<110>Zhejiang oceanic rise bio tech ltd

<120>Recombinate the preparation method and application of swine fever E2 albumen and its subunit vaccine

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 345

<212> PRT

<213>CSFV E 2 protein amino acid sequence

<400> 1

Leu Ala Cys Lys Glu Asp Tyr Arg Tyr Ala Ile Ser Ser Thr Asp Glu Ile Gly Leu Leu

25 30 35 40

Gly Ala Gly Gly Leu Thr Thr Thr Trp Lys Glu Tyr Asn His Asp Leu Gln Leu Asn Asp Gly

45 50 55 60 65

Thr Val Lys Ala Ser Cys Val Ala Gly Ser Phe Lys Val Thr Ala Leu Asn Val Val Ser Arg Arg

70 75 80 85

Tyr Leu Ala Ser Leu His Lys Lys Ala Leu Pro Thr Ser Val Thr Phe Glu Leu Leu Phe Asp

90 95 100 105

Gly Thr Asn Pro Ser Thr Glu Glu Met Gly Asp Asp Phe Arg Ser Gly Leu Cys Pro Phe Asp

110 115 120 125

Thr Ser Pro Val Val Lys Gly Lys Tyr Asn Thr Thr Leu Leu Asn Gly Ser Ala Phe Tyr Leu Val

130 135 140 145 150

Cys Pro Ile Gly Trp Thr Gly Val Ile Glu Cys Thr Ala Val Ser Pro Thr Thr Leu Arg Thr Glu Val

155 160 165 170

Val Lys Thr Phe Arg Arg Asp Lys Pro Phe Pro His Arg Met Asp Cys Val Thr Thr Thr Val Glu

175 180 185 190 195

Asn Glu Asp Leu Phe Tyr Cys Lys Leu Gly Gly Asn Trp Thr Cys Val Lys Gly Glu Pro Val

200 205 210 215

Val Tyr Thr Gly Gly Val Val Lys Gln Cys Arg Trp Cys Gly Phe Asp Phe Asp Gly Pro Asp

220 225 230 235

Gly Leu Pro His Tyr Pro Ile Gly Lys Cys Ile Leu Ala Asn Glu Thr Gly Tyr Arg Ile Val Asp Ser

240 245 250 255 260

Thr Asp Cys Asn Arg Asp Gly Val Val Ile Ser Thr Glu Gly Ser His Glu Cys Leu Ile Gly Asn

265 270 275 280

Thr Thr Val Lys Val His Ala Ser Asp Glu Arg Leu Gly Pro Met Pro Cys Arg Pro Lys Glu Ile

285 290 295 300 305

Val Ser Ser Ala Gly Pro Val Met Lys Thr Ser Cys Thr Phe Asn Tyr Thr Lys Thr Leu Lys Asn

310 315 320 325

Arg Tyr Tyr Glu Pro Arg Asp Ser Tyr Phe Gln Gln Tyr Met Leu Lys Gly Glu Tyr Gln Tyr Trp

330 335 340 345

Phe Asp Leu Asp Ala Thr Asp Arg His Ser Asp Tyr Phe Ala His His His His His His

350 355 360 365

<210> 2

<211> 1044

<212> DNA

<213>CSFV E 2 protein nucleotide sequence after codon optimization

<400> 2

ctg gcttgcaagg aagactacag gtacgccatc 120

tcctccacag atgagattgg actgctgggc gctggaggac tgaccaccac ctggaaggag 180

tacaaccatg acctgcagct gaacgacggc accgtgaagg cctcctgtgt ggctggctcc 240

tttaaggtga ccgccctgaa tgtggtgagc cggaggtacc tggcctccct gcacaagaag 300

gccctgccta cctccgtgac cttcgaactg ctgttcgacg gcaccaaccc ctccaccgag 360

gagatgggcg atgatttccg gagcggcctg tgtcccttcg acacctcccc cgtcgtgaag 420

ggcaagtaca acacaaccct gctgaacggc agcgcctttt acctggtgtg ccctatcggc 480

tggaccggcg tcatcgagtg cacagccgtc tcccctacca ccctgcggac agaagtggtg 540

aagaccttcc ggagggacaa gcctttcccc caccggatgg actgtgtcac caccaccgtg 600

gagaacgagg atctgttcta ctgcaagctg ggcggcaact ggacctgcgt gaagggcgag 660

cctgtggtgt acaccggagg cgtggtgaag cagtgcaggt ggtgcggctt cgacttcgat 720

ggacctgacg gcctgcctca ctatcccatc ggcaagtgca ttctggccaa cgagaccggc 780

taccggatcg tggattccac cgactgcaac cgggacggcg tggtcatctc caccgaaggc 840

agccacgagt gcctgatcgg caacaccaca gtgaaggtgc atgctagcga tgaaaggctg 900

ggacccatgc cctgcaggcc taaggagatt gtctcctccg ccggccccgt gatgaagacc 960

agctgcacct tcaattacac caagaccctg aagaaccggt actacgagcc tcgggactcc 1020

tacttccagc agtacatgct gaagggcgag taccagtact ggttcgacct ggatgccacc 1080

gacaggcaca gcgactattt cgctcatcac catcaccatc actaagaatt c 1131

<210> 2

<211> 1044

<212> DNA

<213>CSFV E 2 protein nucleotide sequence before codon optimization

<400> 3

ctagcctgca aggaagatta caggtacgca atatcgtcaa ccgatgagat agggctactt 60

ggggccggag gtctcaccac cacctggaag gaatacaacc acgatttgca actgaatgac 120

gggaccgtca aggccagttg cgtggcaggt tcctttaaag tcacagcact taatgtggtc 180

agtaggaggt atttggcgtc attgcataag aaggctttac ccacttccgt gacattcgag 240

ctcctgttcg acgggaccaa cccatcaact gaggaaatgg gagatgactt caggtccggg 300

ctgtgcccgt ttgatacgag tcctgttgtt aagggaaagt acaatacgac cttgttgaac 360

ggtagtgctt tctatcttgt ctgcccaata gggtggacgg gtgtcataga gtgcacagca 420

gtgagcccaa caactctgag gacagaagtg gtaaagacct tcaggagaga caagcccttt 480

ccgcacagaa tggattgtgt gaccaccaca gtggaaaatg aagatttatt ctattgtaag 540

ttggggggca actggacatg tgtgaaaggc gagccagtgg tctacacagg gggggtagta 600

aaacaatgta gatggtgtgg cttcgacttc gatgggcctg acggactccc gcattacccc 660

ataggtaagt gcattttggc aaatgagaca ggttacagaa tagtagattc aacggactgt 720

aacagagatg gcgttgtaat cagcacagag gggagtcatg agtgcttgat cggtaacacg 780

actgtcaagg tgcatgcatc agatgaaaga ctgggcccta tgccatgcag acctaaagag 840

attgtctcta gtgctggtcc tgtaatgaaa acctcctgta cattcaacta cacaaaaact 900

ttgaagaaca ggtactatga gcccagggac agctacttcc agcaatatat gcttaagggt 960

gagtatcagt actggtttga cctggatgcg actgaccgcc actcagatta cttcgcacat 1020

caccatcacc atcactaaga attc 1044

Claims

1. a kind of preparation method for recombinating swine fever E2 albumen, it is characterised in that the preparation method comprises the following steps：

1) the swine fever E2 protein coding genes after codon optimization are cloned into carrier for expression of eukaryon and obtained containing swine fever E2 eggs The recombinant plasmid of white encoding gene；

2) again by the Transfected Recombinant Plasmid containing swine fever E2 protein coding genes into Chinese hamster ovary celI；

3) by cultivate, screen, tame 2) described in Chinese hamster ovary celI strain obtain the cell line that altimeter reaches；

4) fermented and cultured 3) described in cell line, obtain after purification recombinate swine fever E2 albumen.

2. according to the method for claim 1, it is characterised in that the swine fever E2 protein coding genes after the codon optimization Nucleotide sequence as shown in SEQ ID NO.2.

3. according to the method for claim 1, it is characterised in that the carrier for expression of eukaryon be pEE6.4, pEE12.4, pGL4.13、pcDNA3.1。

4. according to the method for claim 3, it is characterised in that the carrier for expression of eukaryon is pEE12.4.

5. method according to claim 1 or 2, it is characterised in that the Chinese hamster ovary celI strain be DG44, DXB11, CHO-K1, CHO-S cell lines.

6. according to the method for claim 5, it is characterised in that the Chinese hamster ovary celI strain is CHO-K1.

7. a kind of preparation method containing restructuring swine fever E2 protein subunit vaccines, it is characterised in that will be according to claim 1 institute The restructuring swine fever E2 albumen that the preparation method stated obtains fully mixes with pharmaceutically acceptable adjuvant, obtains swine fever E2 albumen weights Group subunit vaccine.

8. according to the method for claim 7, it is characterised in that adjuvant used in the recombinant subunit vaccine is ISA 201 VG。

9. preparation method according to claim 7, it is characterised in that the restructuring swine fever E2 albumen and adjuvant ISA 201 VG by volume 46:54 mixing and emulsifyings.

10. a kind of exist according to any described restructuring swine fever E2 albumen of claim 1~7 and CSFV recombinant subunit vaccine Prepare the application in dependent diagnostic reagent.