CN104826100A - Preparation method and application of classical swine fever virus recombinant subunit vaccine - Google Patents

Abstract

The invention discloses a preparation method and application of a classical swine fever virus recombinant subunit vaccine with the amino acid sequence shown as SEQ ID No.1. The preparation method of the classical swine fever virus recombinant subunit vaccine typically includes the following steps: classical swine fever E2 truncated protein (TE2) coding gene is cloned into baculovirus vector pFastBacTM1, and is then transfected into Sf9 insect cells to obtain recombinant baculovirus capable of expressing protein TE2. The high five insect cells in logarithmic growth phase are infected by the recombinant baculovirus, so that a large amount of the protein TE2 can be expressed in a cell culture supernatant. Finally, the cell culture supernatant is recovered and purified to obtain a large amount of the recombinant protein TE2 with the purity more than 90%. According to the method, the target protein can be harvested from the cell culture supernatant, the time of protein purification is reduced, consumption of a large amount of time can be avoided, and the vaccine production process can be simplified. Under the premise of simplification of the vaccine production process, the recombinant protein TE2 has the advantages of strong immunogenicity and high safety, and the animal experiments prove that the recombinant protein can effectively stimulate the body to produce a highly effective humoral immune response.

Description

A kind of preparation method of swine fever virus recombinant subunit vaccine and application

Technical field

The present invention relates to a kind of preparation method and application of swine fever virus recombinant subunit vaccine, belong to biovaccine preparing technical field.

Background technology

Swine fever is called that in Europe classic swine fever (Classical Swine Fever, CSF) is caused by swine fever virus (ClassicalSwine Fever Virus, CSFV) that the one of pig is acute, hot, contagious disease.With urgency of falling ill, high heat is delaied and to be caused with the degeneration of thin vessels wall the extensively pathological changes such as hemorrhage, infraction and necrosis to be feature, and domestic and wild pig is its unique natural host.OIE (OIE) is decided to be category-A infectious disease, and China's " animal epidemic prevention method " is classified as a class infectious disease.Swine fever is one of main epidemic disease endangering China's pig industry development at present.

Swine fever virus belongs to flaviviridae, pestivirus member, is strand linear RNA virus.Common antigen is had between this virus and the bovine viral diarrhea virus belonged to together.Virion is slightly rounded, has lipoprotein envelope, and virion surface has fragile fine lug structure.Think that swine fever virus still only has One serotype at present, but the virulence of Strain has by force, in, weak point.CSFV genome is about 12.5kb, only containing 1 large open reading frame (ORF), this ORF coding about 4000 amino acid residue molecular amounts are about the polyprotein of 438ku, be processed as 12 kinds of maturation proteins, be followed successively by N under the effect of virus and host leukoprotease ^pro, C, E ^ms, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B.Wherein C, E ^ms, E1 and E2 is structural protein, and all the other are non-structural protein.E2 is the topmost immunogenic protein of CSFV, body can be induced to produce neutralizing antibody and pig can be protected to resist the attack of CSFV virulent strain.

Vaccine immunity controls the popular important means of swine fever.Through applying for many years, the effective attenuated vaccine strain of generally recognized as safe has three kinds: 1. Chinese rabbitization attenuated vaccine; 2. Japanese GPE (-) cell weak-toxic vaccine; 3. France's " Thiveosal " cold variation low virulent strain.The China that wherein Chinese scholar succeeds in developing is (C system) hog cholera lapinised virus vaccine, from nineteen fifty-seven, except except China's extensive use, has been generalized to Eurasian a lot of country, and has helped these state controls or eliminated swine fever.This vaccine is acknowledged as swine Fever Vaccine more satisfactory in the world at present.Swine Fever Vaccine conventional in current China market has 3 kinds, and namely swine fever cell live vaccine (cell vaccine), swine fever breast rabbit organize live vaccine (Tissue vaccine), swine fever spleen to drench live vaccine (spleen pouring Seedling).

Live vaccines of hog cholera (cell source) is prepared from by In vitro culture bovine testicle cell virus of proliferation.The advantage of live vaccines of hog cholera (cell source) is antigenic content relatively high (high efficiency cell Seedling in the market reaches 7500RID/ head part); Higher antibody horizontal can be produced; Can be mass-produced, Quality Control is easy, easy to operate, confession amount is sufficient.But owing to employing primary cell in vaccine preparation process, therefore there is many defects in this vaccine, as: there is batch wise differences; Acellular pathological changes, wayward viral yield; The raw and auxiliary material of live vaccines of hog cholera (cell source) relates to bovine testicle cell and Ox blood serum, causes the risk that there is BVDV pollution in its preparation process.BVDV infected pigs can cause the symptom of doubtful swine fever.In recent years, just the swine diseases reporting that the swine Fever Vaccine polluted by BVDV causes was had in China.Although breast rabbit Tissue vaccine and becomes the immunogenicity of rabbit spleen pouring Seedling relatively good, and without BVDV pollution risk, owing to needing use rabbit in vaccine preparation process, therefore this kind of vaccine has the following disadvantages, as: be not easy large-scale production; Different cultivars, batch, the sensitivity of rabbit differs in environment; The outer derived bacterium of easy pollution; Anaphylaxis can be there is after individual animal injection.

Summary of the invention

CSFV E2 subunit vaccine advantage of the present invention: 1. abundance is measured in large-scale production, confession, Quality Control is easy; 2. safety is high; 3. stable batch; 4. production cost is low; 5. without BVDV pollution risk.

The problem to be solved in the present invention is to provide a kind of preparation method and application of swine fever virus recombinant subunit vaccine.

A preparation method for swine fever virus recombinant subunit vaccine, comprises the steps:

1) the truncate albumen TE2 encoding gene of swine fever virus E2 is cloned in baculovirus expression system obtains recombinant baculovirus plasmid Bacmid-KSPTE2.

2) transfection sf9 cell obtains recombinant baculovirus Bv-KSPTE2, obtains TE2 albumen with reclaiming purification after insect cell amplification culture.

3) swine fever virus recombinant subunit vaccine is obtained after swine fever virus TE2 albumen and pharmaceutically acceptable adjuvant fully being mixed.

Particularly, 1) construction method of described recombinant baculovirus plasmid Bacmid-KSPTE2 is for be first cloned into carrier pFastBac by Kozak sequence gene and GP67 signal peptide gene ^tMmiddle interstitial granules pFastBac1-KSP is obtained on 1, again TE2 protein coding gene (containing His label) is cloned in middle interstitial granules pFastBac1-KSP and obtains recombiant plasmid pFastBac1-KSPTE2, finally recombiant plasmid pFastBac1-KSPTE2 is converted in escherichia coli DH 10Bac competent cell, utilizes Tn7 Site-specific recombinase method finally to obtain recombinant baculovirus plasmid bacmid-KSPTE2.

2) described amplification culture is: (cell density is 1.5-2.5 × 10 to recombinant baculovirus plasmid bacmid-KSPTE2 transfection logarithmic (log) phase sf9 cell ⁶/ ml) obtain P1 poison; Amplification culture is carried out with P3 for viral infection logarithmic (log) phase insect cell High five after the amplification of two generations.

2) described purification is recovered as: collected by centrifugation 2) amplification culture cell conditioned medium, successively obtain destination protein by affinity chromatograph and cation exchange chromatography.

3) described recombinant subunit vaccine adjuvant used is ISA 201VG.

Described TE2 protein coding gene nucleotide sequence is as shown in SEQ ID NO.2, and GP67 signal peptide nucleotide sequence is as shown in SEQ ID NO.4, and Kozak sequence is as shown in SEQ ID NO.5.

Described swine fever virus E2 truncate albumen (TE2) is applied to subunit vaccine preparation.

Accompanying drawing explanation

Fig. 1, pFastBac ^tM1 plasmid map.

Fig. 2, pFastBac1-KSPTE2 double digestion qualification result.M:DNA Marker DL5,000; 1: recombiant plasmid pFastBac1-KSPTE2 double digestion electrophoresis result.

Fig. 3, bacmid-KSPTE2PCR qualification result.M:DNA Marker DL5,000; 1.M13 primer PCR qualification recombinant baculovirus plasmid bacmid-KSPTE2 electrophoresis result.

Fig. 4, Western blot (anti-His) identifies TE2 protein expression.Sun: positive control; M:PrestainedProtein Ladder; Cloudy: negative control; TE2:TE2 culture supernatant.

Fig. 5, affinity chromatography chromatogram and SDS-PAGE result.

Fig. 6, Source 15Q column chromatography figure and SDS-PAGE result.

Antibody titer result figure after Fig. 7, IDEXX test kit detection TE2 immunity.

Detailed description of the invention

The structure of embodiment 1 expression vector

1.1 swine fever virus RNA extract

From swine fever virus culture supernatant, the template of RNA as reverse transcription is extracted with Trizol.

1.2RNA reverse transcription becomes cDNA

The Primescript RT-PCR kit of takara is used to test.

1.3PCR amplimer (restriction enzyme site underscore marks):

Forward primer: 5 '-CG gAATTCcTAGCCTGCAAGGAAGATTAC-3 '

Downstream primer: 5 '-CCC aAGCTTtTA GTGATGGTGATGGTGATGAACAAATTCTGCGAAGTAATC-3 '

Application of sample system is (50 μ l):

Pcr amplification program:

1.4 glue reclaim DNA fragmentation:

(1) 50 μ l system reactant liquors of step 1.3 are carried out 1% agarose gel electrophoresis (98V 45min);

(2) under uviol lamp, cut glue and reclaim DNA fragmentation in 1.5ml EP pipe; Utilize the DNA extraction kit of sky root biology to reclaim DNA fragmentation, concrete operation step is as follows:

(3) in the 1.5ml EP pipe in step (2), 500 μ l PC buffer are added, 50 DEG C, water-bath 10min;

(4) solution in step (3) is moved to adsorption column center, leave standstill 2min, centrifugal 12,000rpm 30sec;

(5) abandon waste liquid, add 600 μ l PW buffer to adsorption column center, leave standstill 3min, centrifugal 12,000rpm, 30sec;

(6) step (5) is repeated;

(7) centrifugal 12, the 000rpm 1min of suction attached column;

(8) 30 μ l ddH are added to adsorption column center ₂o, leaves standstill 3min, centrifugal (12,000rpm, 2min);

(9) collect step (8) DNA sample and carry out electrophoresis.

1.5 double digestions reaction (40 μ l system):

In 1.5ml EP pipe, carry out application of sample, mixing according to step 1.5, then these two 40 μ l reactant liquors are placed in 37 DEG C of thermostat water baths, water-bath 2h.

1.6 glue reclaim double digestion product:

With step 1.4

1.7 coupled reactions (10 μ l system):

In 1.5ml EP pipe, carry out application of sample, mixing according to above-mentioned system, take out after then coupled reaction liquid being placed in 16 DEG C of water-bath 16h, after 65 DEG C of water-bath 15min, carry out deactivation, by the preservation of 4 DEG C, sample.

1.8 transformation experiments:

(1) take out 10 μ l coupled reaction liquid of step 1.7, add 100 μ l E.coli DH5 α competent cells wherein, mixing;

(2) ice bath 30min;

(3) 42 DEG C of heat shock 90sec;

(4) ice bath 2min;

(5) in EP pipe, 600 μ l LB fluid mediums are added, 37 DEG C of water-bath 1h;

(6) centrifugal (8,000rpm, 2min), remove 600 μ l liquid, remain the resuspended thalline of 100 μ l LB;

(7) get bacterium liquid and be plated on (Amp concentration is 100 μ g/ml) in LB flat board, LB flat board is placed in biochemical constant incubator 37 DEG C and cultivates 12h.

1.9 recombiant plasmid extract and enzyme action qualification:

(1) from transformation plate, picking monoclonal bacterium colony is in 3ml LB fluid medium, and 37 DEG C, 260rpm shakes bacterium and spends the night;

(2) get 1ml bacterium liquid in 1.5ml EP pipe, centrifugal (12,000rpm, 2min), abandon supernatant;

(3) in the EP pipe in step (2), 250 μ l P1buffer are added, resuspended thalline;

(4) in step (3) solution, add 250 μ l P2buffer, gentle mixing, leaves standstill 2min;

(5) in step (4) solution, 350 μ l P3buffer are added, gentle mixing;

(6) by step (5) solution centrifugal (12,000rpm, 10min);

(7) supernatant solution in step (6) is moved to adsorption column center, centrifugal (8,000g, 30sec);

(8) abandon waste liquid, add 500 μ l wash buffer to adsorption column center, centrifugal (9,000g, 30sec);

(9) step (8) is repeated;

(10) suction attached column centrifugal (9,000g, 1min);

(11) add 30 μ l Elution buffer to adsorption column center, leave standstill 2min, centrifugal (12,000rpm, 2min);

(12) collect step (11) DNA sample and carry out electrophoresis;

(13) as shown in step 1.5, enzyme action qualification is carried out to the plasmid extracted, then carry out 1% agarose gel electrophoresis.

Recombiant plasmid pFastBac1-KSPTE2 enzyme action the results are shown in Figure 2.As shown in the figure, after BamH I and HindIII double digestion, obtain carrier segments and about 1, the 200bp object fragment of about about 4,800bp respectively, in the same size with expection.

Embodiment 2 is recombinated pFastBac1-KSPTE2 plasmid transformation escherichia coli DH 10Bac

(1) the DH10Bac competent cell getting 100 μ l is placed on ice;

(2) adding at least 1ng recombinates in pFastBac1-KSPTE2 plasmid to 100 μ l DH10Bac competent cell, ice bath 30min;

(3) 42 DEG C of water-bath heat shock 45sec, EP pipe turns back on ice, leaves standstill 2min;

(4) in super-clean bench, add the nonresistant LB culture fluid of 900 μ l;

4h is cultivated in (5) 37 DEG C of 220rpm concussions;

(6) get 10 μ l bacterium liquid after 4h and be coated with the anti-flat board of KGT tri-(50 μ g/ml Kanamycin, 7 μ g/mlGentamicin and 10 μ g/ml Tetracycline, 100 μ g/ml Bluo-gal, and 40 μ g/ml IPTG), cultivate 48h in 37 DEG C of lucifuges;

(7) select white macula and be forwarded to the anti-flat board continuation of KGT tri-37 DEG C of cultivation about 24h.After select uniform white macula, each monoclonal adds in the anti-LB fluid medium of 50 μ l KGT tri-, and 37 DEG C, 220rpm shakes cultivation;

According to step 1.3, PCR qualification is carried out to cultivation bacterium liquid, the results are shown in Figure 3.As seen from Figure 3, M13 primer can amplify the fragment of 3,500bp.As can be seen here, genes of interest is successfully inserted in Baculovirus Gene group by Tn7 transposition.

Embodiment 3bacmid-KSPTE2 extracts

(1) get bacterium liquid PCR to be accredited as positive white macula bacterium liquid and to be transferred in the anti-LB fluid medium of 5ml KGT tri-and to continue 37 DEG C of 225rpm incubated overnight;

(2) the 1ml bacterium liquid getting incubated overnight is inoculated into and in the anti-LB fluid medium of 100ml KGT tri-, continues 37 DEG C of 225rpm incubated overnight, according to alkaline lysis extracting Bacmid plasmid;

(3) the bacterium liquid of cultivation is dispensed in the centrifuge tube of 2 50ml, 4 DEG C of 12,000rpm centrifugal 10min;

(4) supernatant is abandoned, blot culture fluid as far as possible, reclaim thalline, add 10ml ice cooled solution I (50mmol/L glucose, 25mmol/L TrisHCl, 10mmol/L EDTA, pH 8.0,40 μ g/ml RNase A are added during use) blow and beat gently with pipettor, thalline is suspended completely;

(5) add the solution II 20ml (1%SDS, 0.2M NaOH) of fresh configuration, cover tightly the mouth of pipe, put upside down centrifuge tube gently for several times, with contents were mixed, be sure not, with forced oscillation, then centrifuge tube to be placed in 5min on ice;

(6) solution III (the 5M potassium acetate of 15ml ice pre-cooling is added, 2M glacial acetic acid), cover tightly the mouth of pipe, put upside down mixing gently, solution III is uniformly dispersed in sticky bacterial lysate, be placed in 5-10min on ice, now can be observed to occur white flock precipitate (protein and genome of E.coli DNA);

(7) 4 DEG C, the centrifugal 30min of 12,000rpm, careful to draw after supernatant centrifugal 10min again, and in supernatant, add the cold isopropanol of 2/3 times of volume, put upside down mixing gently several times, more than ice bath 30min, 4 DEG C centrifugal, 12,000rpm 30min;

(8) after discarding supernatant, add 10ml 75% washing with alcohol precipitation, 4 DEG C of 12,000rpm centrifugal 10min, discards supernatant, drying at room temperature 5-10min will be precipitated to transparent, with appropriate 10mM Tris-HCl, pH8.0 dissolution precipitation and subpackage, be restructuring Bacmid plasmid, be placed in 4 DEG C or-20 DEG C of preservations, with nano spectrophotometric determination bacmid sample concentration.

The acquisition of embodiment 4 recombinant baculovirus Bv-KSPTE2

(1) get out the sf9 cell of suspension culture, cell is in exponential phase, and density is about 1.5-2.5 × 10 ⁶cell/ml, spreads six orifice plates, 8-9 × 10, every hole ⁵individual cell;

(2) in Biohazard Safety Equipment, get Cellfectin II6-8 μ l and join in 100 μ l unsupplementedGrace ' s Medium, mix gently, leave standstill 5min; Separately get bacmid 1-2 μ g to join in 100 μ lunsupplemented Grace ' s Medium, mix gently, then Cellfectin II and bacmid is mixed gently, incubated at room 15-45min.

(3) after having hatched, add 0.8ml unsupplemented Grace ' s Medium, after gently mixing, slowly add in six orifice plates implanting cell along wooden partition, put into 27 DEG C of incubators and hatch 5h.

(4) discard the mixed liquor in six orifice plates, every hole adds 2ml Sf-900 ^tMiII SFM culture medium, hatches 72h in 27 DEG C of incubators.Observe transfection phenomenon.

Get after cultured cell liquid supernatant mixes with 5 × SDS-PAGE loading buffer, boil 10min, can polyacrylate hydrogel electrophoresis be carried out after suitably centrifugal, then carry out western blot detection.As shown in Figure 4, TE2 is great expression in cell culture fluid, and molecular weight of albumen is consistent with expection.

Embodiment 5 protein purification

5.1 affinity chromatography

Collecting cell culture supernatant: 4 DEG C, the centrifugal 1h of 8,000rpm, abandons precipitation, supernatant 0.45 μm of filter membrane sucking filtration, prevents blocking Histrap FF protein chromatographic post to remove impurity;

Prepacked column process: balance 5CV (column volume) with ultra-pure water, discharges ethanol conserving liquid;

In conjunction with (4 DEG C of operations): use peristaltic pump, sample is pumped into pillar, and flow velocity is 1ml/min, collect stream and wear liquid, get 20 μ l and detect for SDS-PAGE;

Eluting: with 20CV with Binding buffer (the 20mM NaH of upper volume ₂pO ₄, 500mM NaCl, pH7.4) and wash post, flow velocity is 2ml/min, and the albumen in conjunction with upper prop is rinsed well, to OD ₂₈₀till nm baseline is steady, use 1% successively, 2%, 3%, 4%, Elution buffer (the 20mM NaH of 20% ₂pO ₄, 150mM NaCl, 500mM imidazoles, pH 7.4) and gradient elution;

Protein electrophoresis: prepare 12% polyacrylamide gel, concentrated glue 80V constant voltage electrophoresis, separation gel 120V constant voltage electrophoresis, SDS-PAGE the results are shown in Figure 5.

5.2 ion-exchange chromatography

Source 15Q column equilibration: with A buffer (the 10mM NaH for 20CV ₂pO ₄, pH 8.0) and equilibrium ion exchange column, flow velocity 2ml/min;

Sample treatment: by the A buffer dialysis sample liquid of 2L, dialyse for 4 DEG C, 1h/ time, dialyse 3 times altogether, rear sample liquid of having dialysed crosses 0.45 μm of filter;

Loading and eluting: loading flow velocity is 1ml/min, elution flow rate is 2ml/min, with B Buffer (10mMNaH ₂pO ₄, 500mM NaCl, pH 8.0) and gradient elution: 0-15%B, 8CV eluting;

Protein electrophoresis: prepare 12% polyacrylamide gel, concentrated glue 80V constant voltage electrophoresis, separation gel 120V constant voltage electrophoresis, SDS-PAGE the results are shown in Figure 6.

5.3 determination of protein concentration: adopt BCA method to measure protein concentration;

Albumen yield is 21mg/L.

The preparation of embodiment 6 vaccine and immunity test

Appropriate TE2 albumen is joined (volume ratio is 46: 54) in ISA 201VG adjuvant, make final concentration of protein be 30 μ g/ml, ultrasonic emulsification.

Select 3-4 Landrace in age in week to test, each immune 1ml TE2 protein vaccine (30 μ g/ml), just exempts from booster immunization after three weeks and once, gets weekly blood 1 time, separation of serum after immunity, measure antibody titer by stop band restrain method.

As can be seen from Fig. 7 result, TE2 subunit vaccine blocking-up rate drenches Seedling apparently higher than the one-tenth rabbit spleen of certain brand on market.The antibody (blocking-up rate > 70%) produced when one exempts from 21 days has been enough to the infection protecting swinery from swine fever virus in theory.Exempt within 7 days and two, to exempt from 14 days blocking-up rates two and be about 90%, reached the upper limit of detection of test kit, therefore do not have to embody the rising of tiring.

Claims (8)

1. a preparation method for swine fever virus recombinant subunit vaccine, its basic feature comprises the steps:

1) the truncate albumen TE2 of swine fever virus E2 is characterized in that the protein as (a) or (b):

A protein that () is made up of the aminoacid shown in SEQ ID NO.1;

(b) aminoacid sequence in (a) through replacing, disappearance or add an aminoacid or several aminoacid and there is the antigenic protein derivative by (a) of CSFV E 2 protein.

2) TE2 protein coding gene is cloned in baculovirus expression system obtains recombinant baculovirus plasmid Bacmid-KSPTE2.

3) transfection sf9 cell obtains recombinant baculovirus Bv-KSPTE2, obtains TE2 albumen with reclaiming purification after insect cell amplification culture.

4) swine fever virus recombinant subunit vaccine is obtained after TE2 albumen and pharmaceutically acceptable adjuvant fully being mixed.

2. vaccine preparation method according to claim 1, is characterized in that 2) construction method of described recombinant baculovirus plasmid Bacmid-KSPTE2 for first by Kozak sequence gene and GP67 signal peptide sequence gene clone to carrier pFastBac ^tMmiddle interstitial granules pFastBac1-KSP is obtained on 1, again TE2 protein coding gene (containing His label) is cloned in middle interstitial granules pFastBac1-KSP and obtains recombiant plasmid pFastBac1-KSPTE2, finally recombiant plasmid pFastBac1-KSPTE2 is converted into escherichia coli DH10Bac competent cell, utilizes Tn7 Site-specific recombinase method finally to obtain recombinant baculovirus plasmid bacmid-KSPTE2.

3. vaccine antigen preparation method according to claim 1, is characterized in that 3) described amplification culture is: (cell density is 1.5-2.5 × 10 to recombinant baculovirus plasmid bacmid-KSPTE2 transfection logarithmic (log) phase sf9 cell ⁶/ rnl) obtain P1 poison; Amplification culture is carried out with P3 for viral infection logarithmic (log) phase insect cell High five after the amplification of two generations.Preferably, described baculovirus infection plural number (MOI) is 0.5-10, is optimized for 1 further; Preferably, described incubation time is 56-63h.

4. vaccine preparation method according to claim 1, is characterized in that 3) described recovery purification is: collected by centrifugation 3) amplification culture cell conditioned medium, successively obtain destination protein by affinity chromatograph and anion-exchange chromatography purification.Preferably but be not limited to, described affinity chromatograph is Histrap FF chromatographic column; Preferably but be not limited to, described anion-exchange chromatography is source 15Q chromatographic column; Preferably but be not limited to, the buffer that described anion-exchange chromatography uses comprises: buffer A: 10mMNaH ₂pO ₃, pH8.0; Buffer B: 10mM NaH ₂pO ₃, 500mM NaCI, pH8.0; Preferably but be not limited to, the elution program of described elution buffer is with 0-100% elution buffer B, and 5-10 column volume carries out gradient elution.

5. vaccine preparation method according to claim 1, is characterized in that 4) described recombinant subunit vaccine adjuvant used is ISA201VG.Preferably, described TE2 albumen and adjuvant ISA201VG 46: 54 mixing and emulsifyings by volume.

6. TE2 protein coding gene nucleotide sequence according to claim 1 is as shown in SEQ ID NO.2, and GP67 signal peptide nucleotide sequence is as shown in SEQ ID NO.4, and Kozak sequence is as shown in SEQ ID NO.5.

7. described in claim 1, TE2 protein expression system includes but not limited to insect cell, escherichia coli, yeast and mammalian cell etc.

8. the CSFV E 2 protein (TE2) of truncate described in claim 1 is applied to subunit vaccine preparation.