CN104388453B - Porcine circovirus (PCV) cap protein inserted swine fever virus B cell epitope recombinant virus and application thereof - Google Patents

Abstract

The invention discloses a porcine circovirus (PCV) cap protein inserted swine fever virus B cell epitope recombinant virus and application thereof. A synthetic cap-24 gene is expressed in a baculovirus transfer vector pH promoter to obtain the recombinant baculovirus (CCTCC NO:V201432). When being used for immunizing a mouse, the subunit vaccine prepared from the inserted PCV2 VLP protein expressed by the recombinant baculovirus can induce the organism to generate the specific humoral immunity and can generate the specific antibody level against CSFV and PCV2. The invention provides a favorable candidate vaccine for simultaneously controlling the two diseases in clinic, and also provides feasibility test data for researching expression of other exogenous antigens by using circovirus as a vector.

Description

A kind of recombinant virus of the chimeric CSFV B cell epi-position of pig circular ring virus cap albumen And application

Technical field

The invention belongs to technical field of veterinary biology, and in particular to a kind of chimeric CSFV B of pig circular ring virus cap albumen The recombinant virus of cell epitope and application.

Background technology

Pig circular ring virus (PCV) be PCV-II section (Circoviridae) Circovirus (Circovirus) into Member, including P CV1 and PCV2, is the vertebrate viruse minimum in the world having now been found that.PCV1 no pathogenicities, and PCV2 has It is pathogenic.PCV2 can clinically infect the pig of 5-12 week old and wean pig multisystem exhaustion syndrome (postweaning occurs Mul tisystemic wastingsyndrome, PMWS), can also cause pigskin inflammation nephrotic syndrome (porcine Dermatitis and nephropathy, PDNS), porcine respiratory disease is combined disease (porcine respiratory Diease complex, PRDC), Hypertrophic necrotizing pneumonia (proliferative and necrotizing Pneumonia, PNP) etc. disease.At present, these diseases are referred to as Porcine circovirus desease for the convenience of research in the world (postweaning multisystemic wasting syn drome associated diease,PCVAD).PCV's (nt 1,735-1,034), the Cap protein of its coding is made up of ORF2 full length gene 702bp 234 amino acid, and size is 27.8kDa.Cap is main immunogenic protein and can induce PCV2 neutralizing antibodies (Pogranichnyy et specially Al., 2000), thus ORF2 genes be design PCV2 new generation vaccines preferred object gene.Liu etc. (2001) passes through large intestine bar Bacterium expresses ORF2 genes, it was demonstrated which has very strong immunogenicity；Annulus can be formed after baculovirus expression ORF2 genes Virus-like particle (Virus like particles, VLP) (Nawagitgul, 2000).The N sections of Cap protein are highly conserved And arginic sequence is rich in, before N sections, 41 amino acid fragments are Cap protein nuclear localization signal sequence (NLS), and research shows to lack Losing N sections nuclear localization sequence does not affect the characteristic of its assembling assembly virus-like particle.

Swine fever (Classical swine fever, CSF) is by flaviviridae pestivirus CSFV (Classical Swine f ever virus, CSFV) a kind of high degree in contact, hemorrhagic and the lethal infectious diseases that cause.World animal is defended Raw tissue (O IE) is classified as the animal epidemic that must be reported, China is classified as a class animal epidemic.Current swine fever is at me The many pig farms of state are popular, and clinical signs are premature labor, miscarriage, stillborn foetus, mummy tire and pig production capacity decline etc..While the disease With the multiple infection such as pseudoabies, circovirus infection relevant disease and mixed infection it occur frequently that.E2 albumen is induction body Produce the main protection antigen main component of neutralizing antibody, can resist strong poison attack malicious Protection (Dong et al., 2006；Che n et al.,2011；Llilianne Gangesa et al.,2012).E2 albumen exist 4 it is relatively independent Antigenic region：A, B, C and D, and the amino acid sequence (CKEDYRYAISSTNEIGLLGAGGLT) between B areas 693-716 to belong to B thin Born of the same parents' epi-position, can produce neutralizing antibody and provide completely protective rate (Dong XN, et al., 2006,2007).

In recent years, rhabdovirus expression vector has simple to operate, safe because of which, can accommodate big genes of interest, Expression foreign protein efficiency high, plays the role of posttranslational modification, the immunogenicity of expressing protein, biologically active and natural albumen Similar the advantages of, account on expression vector leading status (Anderson et al., 1995；Wang et al., 2001；Ri beiro et al.,2001).And virus sample particle vaccines developed in recent years, containing virulent one or Multiple antigen proteins, with the particle with virion analog structure, without virulent inhereditary material, it is impossible to autonomous replication, There is no infectivity, can produce good with native conformation and pattern present antigen, effective stimulus humoral and cellular immune response, induction body Immunoprotection.The present invention lacks its 39 amino acid sequence of N sections nuclear location on the ORF2 gene basises of PCV2, inserts The gene order of CSFV B cells epi-position or coupling T cell amino acid, with the chimeric B cell epi-position of baculovirus expression system expression PCV2 Cap protein, study B cell epi-position PCV2 virus sample particle vaccines formed ability.Simultaneously using formation Hybrid virus like particles prepare subunit's bivalent vaccine, by immunity of the animal experiment evaluation to CSFV and pig circular ring virus Originality effect, provides preferable candidate vaccine strain for clinically prevention and control swine fever and Porcine circovirus desease.

The content of the invention

It is an object of the present invention to provide a kind of pig circular ring virus cap GFPs of manually modified synthesis and swine fever Chimeric cDNA gene orders cap-24B of viral B cell epitope gene sequence, its sequence are respectively shown in SEQ ID NO.1, are compiled The protein of code is shown in SEQ ID NO.2.

It is a further object to provide a kind of transfer vector pFBD-cap-24B, is containing the chimeric of 3 copies The baculovirus transfer vector pFBDPHmHNM1P10eGFP of cap-24B cDNA gene orders.

Another object of the present invention is just to provide a kind of expression pig circular ring virus cap albumen and B cell epitope gene Recombinant baculovirus, the strain deliver China typical culture collection center (CCTCC) preservation on October 19th, 2014, its guarantor Tibetan number is CCTCC NO:V201432, Classification And Nomenclature：Recombinant baculovirus Recominant Autographa californi Ca multiple Nucleopolyhedrovirus Ac-cap-24B, address：Wuhan, China Wuhan University.

It is a still further object of the present invention to provide recombinant baculovirus Ac-cap-24B is in subunit vaccine is prepared Using body will be can induce after the vaccine for chimeric virus-like particles immune mouse produce specific swine fever virus resistant and pig circular ring virus 2 The immune response of poison, and the antibody response of the specificity for being directed to swine fever and pig circular ring virus can be produced.

More detailed technical scheme is shown in《Specific embodiment》It is described.

The invention has the beneficial effects as follows：

1. preferences of the present invention according to baculoviral codon, manually modified first to have synthesized pig circular ring virus Cap bases Because N section nuclear localization sequences (NLS) is replaced by gene order Cap-24B of the cDNA gene orders of CSFV B cell epi-position.

2. the present invention is that artificial synthesized genes of interest Cap-24B is placed under baculoviral pH promoters to express, and is improve The expression of foreign gene.It is inserted on baculovirus transfer vector to be placed under baculoviral pH promoters and expresses, because insertion is 3 Copy form can improve the expression of foreign gene.

3. after the chimeric CSFV B cell epi-position of pig circular ring virus Cap genes expressed by the recombinant baculovirus of the present invention, PCV2 sample virions can be equally successfully formed, after showing that pig circular ring virus Cap genes N section nuclear localization sequences (NLS) are replaced The ability for not affecting which to form virus-like particle, provides feasibility foundation for further developing Chimeric porcine circovirus.

4. VLP is fitted together to expressed by the recombinant baculovirus of the present invention and prepares subunit vaccine, can induce machine after immune mouse Body produces the specific immune response for CSFV and pig circular ring virus.

Description of the drawings

The artificial synthesized schematic diagram built with baculovirus transfer vector of Fig. 1 cap-24B genes

A：The artificial synthesized schematic diagram of cap-24B genes；

B：PFBD-cap-24B baculovirus transfer vectors build schematic diagram.

Digestion identification collection of illustrative plates of the Fig. 2 for baculovirus transfer vector pFBD-cap-24B.

M：15000bp DNA Marker；1:PFBD-cap-24B/SpeI+NotI digestions

Fig. 3 restructuring rod granules rBac-cap-24B PCR identifies schematic diagram

Correct baculovirus transfer vector pFBD-cap-24B conversions DH10Bac is identified, blue hickie screening obtains positive Bacterium colony, extracts the electrophoresis picture that rod granule enters performing PCR identification.

M：2000bp；1：Expanded using Cap-24B 4F and Cap-R primer；2：Expanded using Cap-24B 4F and M13R primer Increase；3：Negative control.

Fig. 4 is the schematic diagram that a kind of Western blot analyze recombinant baculovirus Ac-cap-24B protein expressions

M：Albumen marker；1：SF9 cell controls；2：Ac-cap infection sf9 cells (positive control)；3：Ac-cap-24B Infection sf9 cells；

Fig. 5 is a kind of schematic diagram of indirect immunofluorescence analysis recombinant baculovirus Ac-cap-24B protein expressions

Anti-Cap：With PCV2 cap monoclonal antibody testing goal protein expressions, red fluorescence is observed；EGFP：Baculoviral carries EGFP is marked, and observes green fluorescence；DAPI：Observation of cell core；Merge：The picture that red fluorescence is combined with rate fluorescence

Ac-cap:The sf9 cells of Ac-cap infection；Ac-cap-24B：The sf9 cells of Ac-cap-24B infection

Fig. 6 is the Electronic Speculum schematic diagram that baculoviral Ac-cap-24B infects that sf9 cell conditioned mediums form virus-like particle

Recombinant baculovirus Ac-cap-24B is inoculated with sf9 cells, collect after 84 hours sick cell ultrasonic treatment and Ultracentrifugation carries out electron microscopic observation, it is seen that a large amount of sizes be about 17-20nm without togavirus, form and typical PCV2 phases Together, show assembling assembly virus-like particle VLP.

Indirect ELISA antibody and neutralizing antibody schematic diagram of the Fig. 7 for the anti-PCV2 of different time after immune mouse immunity

A：After mouse immune, different time points produce the ELISA antibody horizontals for PCV2 cap；B：14 after mouse immune The neutralizing antibody of anti-PCV2 is produced when it was with 40 days；

Fig. 8 is the anti-CSFV antibody schematic diagram of different time after immune mouse immunity

After mouse immune, different time produces the B cell epi-position IgG content for CSFV.

Specific embodiment

According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real Apply the content described by example and be merely to illustrate the present invention, and should not also without limitation on sheet described in detail in claims Invention.Technical scheme of the present invention, if not otherwise specified, is the conventional scheme of this area, and the reality is not such as especially said It is bright, it is commercialization or published reagent material.

Embodiment 1：

A kind of expression expression pig circular ring virus cap albumen is fitted together to the recombinant baculovirus of CSFV B cell epitope gene Ac-cap-2

The structure of 4B

(1) structure of recombinant baculovirus Ac-cap-24B

1st, the genes of interest of the chimeric CSFV B cell epi-positions of pig circular ring virus cap is obtained

With reference to PCV2 WH separation strains ORF2 gene order (Hua Zhong Agriculture University's Master's thesis：Porcine circovirus 2 type (PCV2) separation identification and the research and application of inactivated vaccine, Master degree candidate：Yan Weidong；Tutor：What opening；The graduation time： In June, 2009), by 117 sequential nucleotide deletions of N sections 39 amino acid of nuclear localization signal, it is replaced by CSFV B cell 72 nucleotide sequence sequences (as shown in Figure 1) of epi-position (CKEDYRYAISSTNEIGLLGAGGLT), according to baculoviral password The cap-24B gene orders of the artificial synthesized 666bp of preferences of son, its nucleotides sequence are classified as shown in SEQ ID NO.1, its coding Amino acid sequence be SEQ ID NO.2 shown in.This sequence obtains recombinant plasmid pUC-cap- in being subcloned into carrier pUC 24B (by Shanghai bio-engineering corporation into).

2nd, the structure of baculovirus transfer vector pFBD-cap-24B

The structure schematic diagram of baculovirus transfer vector pFBD-cap-24B is as shown in Figure 1.

With plasmid pUC-57cap-24B as template, using three pairs of different primer (being shown in Table 1) amplification cap-24B gene pieces Section, is cloned into baculovirus transfer vector pFBDPHmHNM1P10eGFP plasmids (abbreviation pFBD respectively.Money equality, a kind of expression The recombinant baculovirus of the influenza A H 1 N 1 virus HA-NA-M 1 gene of manually modified synthesis, patent publication No. CN101624580A, patent No. ZL200910063217.6).And then pass through BamHI and EcoRI, SpeI and NotI, SalI and Cap-24B genetic fragments are cloned into HindIII the corresponding site of pFFBD respectively, obtain transferring plasmid pFBD-cap-24B. PFBD-cap-24B carriers identify there is 1129bp after as a result showing digestion by BamHI and EcoRI double digestions, 750bp fragments, Size is consistent with expected results, shows that transferring plasmid pFBD-cap-24B is successfully constructed, and digestion qualification result is as shown in Figure 2.Should Transfer vector contains the chimeric cap-24B cDNA gene orders of 3 copies.

Table 1 clones genes of interest the primer

Note：Black matrix is restriction enzyme site sequence.

3rd, the structure of recombinant virus baculoviral Ac-cap-24B

(1) acquisition and identification of restructuring rod granule Ac-cap-24B

Take 1.0 μ g pFBD-cap-24B of transferring plasmid and be added to 100 μ L DH10Bac competent escherichia coli cells (GiBCO BRL Products) mix, and after ice bath 30min, carry out heat shock in 42 DEG C of 45s water-baths, then ice bath 2min, Jin Erjia Enter 900 μ L SOC fluid nutrient mediums, 37 DEG C of shaking culture 4h, by 10^-1、10^-2、10^-3After dilution, respectively take 100 μ L coat containing The high salt LB plate of kanamycins, gentamicin and tetracycline, concentration press Bac-to-Bac B aculovirus Expression System operational manuals (Invitrogen companies), 37 DEG C culture 24-48h, by blue hickie screening, it is pure Change positive bacterium colony, extract restructuring rod granule, and then enter the amplification identification of performing PCR special primer：VBac-cap-24B rod granules then lead to respectively Cross Cap-24B 1-F and Cap-1R；Cap-24B 1-F and M13R two carries out identification detection to primer, its estimated amplification size point It is not 750bp and 1500bp.

Cap-24B 4-F 5’-TTGGATCCGCCACCATGTGCAAGGAAGATTACAGGT-3’；

Cap-1R 5’-TTGAATTCTTACTTTGGGTTCAGTGGAGGGTCCT-3’；

M13R：5’-AGCGGATAACAATTTCACACAGG-3’；

Its PCR amplification system is as follows：

Act on 5 minutes by 95 DEG C；Then 94 DEG C of 45 second minutes of effect, 56 DEG C are annealed 30 seconds, and 72 DEG C extend 1 minute, and totally 35 Individual circulation, the last 72 DEG C reaction conditions for extending 10 minutes are expanded in ABI PCR instruments.By PCR primer Jing of amplification The Ago-Gel of 1.0% (weight/volume, w/v) carries out electrophoresis, as a result shows to amplify the two of 1500bp and 750bp The special purpose band of bar, size are consistent with expection, show that genes of interest cap-24B fragments have successfully been recombined into Baculovirus Gene In group (as shown in Figure 3).

(2) recombinant baculovirus Ac-cap-24B is obtained

Positive bacteria of the aseptic picking containing restructuring rod granule rBac-cap-24B is fallen within high salt LB fluid nutrient medium, is trained at 37 DEG C After supporting 16 hours, with 0.3mL solution Is (50mmol/L glucose, 10mmol/L EDTA, 25mmol/L Tris-Cl (pH8.0)) It is resuspended, add 0.3mL solution IIs (0.2mol/L NaOH, 1%SDS, matching while using) slightly to mix, be stored at room temperature 5min, slowly 0.3mL solution IIIs (potassium acetate of 3mol/L, pH5.0) are added to mix, ice bath 5-10min, 12000r/min centrifugation 10min, on Reset and add in 0.5mL isopropanols, mix 5～10min of ice bath, room temperature 12000r/min centrifugation 20min, the washing of 75% ethanol are heavy Form sediment, be dissolved in 100 μ L TE after being dried, be sub-packed in -80 DEG C of preservations.

Using liposome mediated transfection method (according to Invitrogen companies Bac-to-Bac Baculovirus Expression System operational manuals are operated), by screening restructuring rod granule rBac-cap-24B Jing after purificationIn Reagent transfections sf9 cells (purchased from Wuhan University's Chinese Typical Representative thing collection), cultivate in 28 DEG C, transfection 72h places fluorescence microscope whether there is green fluorescence appearance afterwards, collects cell conditioned medium and obtains recombinant baculovirus Ac-cap- 24B, is placed in -80 DEG C of preservations.Restructuring bar virus Ac-cap-24B embeds caryogram with many with parent's strain autogra-phacalifornica Polyhedrosis virus (AcMNPV) identical morphological characteristic and cultural character, but the recombinant baculovirus have green fluorescent label base Because of eGFP, can be in fluorescence microscopy Microscopic observation virus amplification situation.The virus delivers to Chinese Typical Representative on October 19th, 2014 Organism Depositary carries out preservation, Classification And Nomenclature：Recombinant baculovirus Recominant Autographa californica multiple Nucleopoly hedrovirus Ac-cap-24B；Deposit number：CCTCC NO：V201432.Address：China Wuhan Wuhan University.

(2), the detection of recombinant baculovirus Ac-cap-24B exogenous gene expressions

1st, the expression of Westerning blot analyses detection foreign gene

Sf9 cells are inoculated with into 6 porocyte culture plates, when cell grows up to 80-90% individual layers, 1.0m.o.i Ac-cap- is used 24B and Ac-cap (positive control, Master's thesis：Peng Bo；PCV2 Cap proteins are expressed in Pichia pastoris and baculoviral and sub- The preparation of subunit vaccine.Tutor：Qian Ping) inoculation sf9 cells, connect poison and collect cell after 84 hours and cracked with NP-40 lysates Cell sample.Transferring film after SDS-PAGE, resists (Master's thesis with Cap protein monoclonal antibody prepared by what opening professor of this laboratory as one：Chen Donghuan；The structure of expression 2 type PCV-II Cap protein recombinant salmonella of pig and identification.Tutor：He QiLid), HRP marks Sheep anti-mouse igg (Sigma) be two anti-to carry out the expression that Western-blotting detects Cap protein.As a result show restructuring disease Occur the specific band of a 27KDa or so size, size in the cell sample of malicious Ac-cap and Ac-cap-24B infection respectively It is consistent with expection, and sf9 cell controls are without this specific band, it was demonstrated that Cap protein is expressed, as a result as shown in Figure 4.

2nd, indirect immunofluorescence identifies the expression of foreign gene

Just growing up on the sf9 cells of individual layer, with the dose inoculation recombinant baculovirus Ac-cap-24B of 1.0m.o.i and Cell is fixed after Ac-cap, 48h and carries out the expression of indirect immunofluorescene assay Cap protein.With what opening professor of this laboratory The Cap protein monoclonal antibody of preparation is an anti-(Master's thesis：Chen Donghuan；Expression 2 type PCV-II Cap protein recombinant salmonella of pig Structure and identification.Tutor：What opening), two resist and carry out indirect immunofluorescence for CY3- sheep anti-mouse iggs (Sigma), most rearmounted common Observe under focusing microscope.As a result there is a very strong red fluorescent in showing the sf9 cells for infect Ac-cap-24B, and mainly Expression is in nucleus, and normal sf9 cells do not have any red fluorescent, show recombinant baculovirus Ac-cap and Ac- Cap-24B can effective expression cap albumen, do not change albumen positioning scenarios.As a result it is as shown in Figure 5.

3rd, whether the insertion of foreign gene affects the electron microscopic observation of the external assembling assembly virus-like particle of PCV2 cap albumen

F4 is infected into the sf9 cells of the culture that suspends for Ac-cap-24B with 1.0m.o.i dosage, after infection, collects thin in 84h Born of the same parents, add lysate simultaneously freeze thawing 3 times.Under the conditions of 4 DEG C, 8000g centrifugations 30min, is then concentrated to the 1/ of original volume with sucrose 10, the VLP particles in supernatant i.e. containing expression.By supernatant 27,000rpm/min, ultracentrifugation 3 hours, precipitation is resuspended in In the PBS of 2.0ml, and blown and beaten with the syringe of 1.0ml repeatedly, then viral suspension is carried out with 20-60% sucrose density gradients 27,00rpm/min ultracentrifugations 16 hours, the protein band for collecting the superiors carry out electron microscopic observation, are as a result displayed in electron microscopic Under mirror (H-7000FA), cap-24B albumen can form virus liquid particle (Virus-like particles, VLPs), and diameter is about Size is 17 25nm, and its form and size are all similar to PCV2 totivirus particles (Fig. 6).Show us in PCV2 cap albumen N Section disappearance NLS sequences and replace with CSFV B epitope sequence do not affect cap albumen formed VLP assembling.Embodiment 2：

Applications of the recombinant baculovirus Ac-cap-24B of the present invention in subunit vaccine is prepared, process are as follows：

(1), the preparation of the chimeric VLPs subunit vaccines of recombinant baculovirus Ac-cap-24B

With the recombinant baculovirus Ac-cap-24B that embodiment 1 is obtained, suspend according to the dose inoculation of 1.0m.o.i and cultivate Insect cell High Five^TM(purchased from Invitrogen companies), collects cell and supernatant, at cell crushing instrument after 84h Cell fragment is centrifuged off after reason, and the expressing quantity for calculating cap-24B by quantitative SDS-PAGE parts glue is 52 micrograms Per milliliter, and then sub- vaccine is prepared into ISA-206 oil adjuvant (purchased from SEPPIC companies) emulsifications so as to cap- in per milliliter 24B protein contents be 40 micrograms, be stored in 4 DEG C it is standby, for following examples.

(1) physical form

Outward appearance this product is the uniform emulsion of milky.

Formulation is two-way formulation (W/O/W), takes a cleaning suction pipe and draws a small amount of vaccine and drips in cold water, the water surface and Have in water.

Viscosity exit inside diameter is that 1.2mm suction pipes draw vaccine 1mL under 25 DEG C or so of room temperature, makes its vertical outflow, 0.5mL is flowed out in 8 seconds, be qualified.

(2) steriling test

According to《Chinese veterinary pharmacopoeia》169-171 page of annex is carried out, and as a result shows no bacterial growth.

(2), the security examination of chimeric VLPs subunit vaccines prepared by recombinant baculovirus Ac-cap-24B of the invention Test

(1) newborn piglet safety testing

Chimeric VLPs subunit vaccines 4 batches prepared by laboratory, take 3 bottles, after being well mixed immediately per batch, select health 1 age in days newborn piglet, 10 pigs of every batch of vaccine inoculation, the chimeric VLPs subunit vaccines of 2 multiple dose 4mL of each intramuscular injection separately set 4 Head pig is blank.1 week before vaccinating and after vaccinating, 2 body temperature of measurement simultaneously observe pig to all piglets daily Upgrowth situation, spirit and appetite, Continuous Observation 14 days.Vaccine inoculation same day body temperature, spirit are normal with appetite, without bad anti- Should, show that vaccine is safe to newborn piglet.

(2) weanling pig safety testing

Chimeric VLPs subunit vaccines 4 batches prepared by laboratory, take 3 bottles, after being well mixed immediately per batch, select health 30 age in days weanling pigs, 10 pigs of every batch of vaccine inoculation, the chimeric VLPs subunit vaccines of 2 multiple dose 4mL of each intramuscular injection separately set 4 pigs are blank, are ibid observed 14 days.Vaccine inoculation same day body temperature, spirit and appetite normally, have no adverse reaction, show Vaccine is safe to weanling pig.

(3) in-pig safety testing

Chimeric VLPs subunit vaccines 4 batches prepared by laboratory, take 3 bottles, after being well mixed immediately per batch, select health 80 age in days in-pigs, 10 pigs of every batch of vaccine inoculation, the chimeric VLPs subunit vaccines of 2 multiple dose 4mL of each intramuscular injection separately set 2 pigs are blank.Raise under the same terms to farrowing, observe impact that vaccine produces to reproduction performance of gestation sow and newly Cub's pig developmental state, after vaccine inoculation, body temperature, spirit and appetite are normal, without phenomenons such as miscarriage, stillborn foetus, mummies, show epidemic disease Seedling is safe to in-pig.

(3), chimeric immunogenicity experiments of the VLP subunit vaccines in mouse of recombinant baculovirus Ac-cap-24B

In order to evaluate the chimeric VLPs subunit vaccines of above-mentioned preparation for pig circular ring virus and the immunogene of CSFV Property, 6 week old female Ba/bc mouse 18 are bought, 2 groups are randomly divided into, 6 per group.PBS is feminine gender, Ac-cap-24B groups.Immunity Dosage is every mouse immune 200ul of Ac-cap-24B groups, makes cap-24B protein contents be every mouse of 40 microgram.Immunization route Inject for hind leg muscle, after 3 weeks with same dosage booster immunization once.Head carries out tail vein blood in 0,14,28,40 days after exempting from, Evaluate its immune effect.

(1) body produces the antibody horizontal for PCV2 cap albumen

With the PCV2 cap albumen pronucleus expressions product coating enzyme-linked reaction plate of 0.1 microgram, examined by indirect ELISA method Survey the antibody horizontal of anti-cap in mice serum.As a result show that the chimeric VLP subunit vaccines of recombinant baculovirus Ac-cap-24B are exempted from Epidemic disease mouse group exempt from one after 14 days, can produce the ELISA antibody for PCV2 cap albumen of characteristic.Antibody horizontal after immunity Persistently rise, when 40 days, ELISA antibody reaches highest, antibody is respectively and reaches 0.953,0.968, produces anti-cap's between two groups ELISA antibody horizontals do not have obvious difference (P<0.05), and negative control group does not produce the antibody of the anti-PCV2 of characteristic type (as shown in Figure 7).

(2) body produces the immunogenicity for CSFV

B cell epitope polypeptide (CKEDYRYAISSTNEIGLLGAGGLT) (synthesis of Shanghai Sheng Gong companies) using CSFV is even Connection KLH protein carriers, are coated with enzyme-linked reaction plate with 2 micrograms of protein, are detected by indirect ELISA method and are resisted in mice serum respectively The IgG content of CSFV B cell epi-positions.As a result show that Ac-cap-24B immune group mouse can detect generation in 14 days after immunity Produce for the IgG of B cell epi-position, when 40 days after immunity, reach highest level；And negative control group can't detect it is thin for B The IgG of born of the same parents' epi-position produces (as shown in Figure 8).

Above-mentioned animal test results show that chimeric VLPs subunit vaccines prepared by Ac-cap-24B can effectively induce machine Body is produced for PCV2 and CSFV immune responses.

(4), immune efficacy experiment of the chimeric VLP subunit vaccines of recombinant baculovirus Ac-cap-24B in pig body

Pig circular ring virus and 3 monthly age of CSFV Serology Negative pig is selected, it is random to divide 4 groups：PBS feminine gender groups, Ac-cap- The chimeric VLP subunit vaccine groups of 24B, porcine circovirus 2 type inactivated vaccine and CSFV rabbitization attenuated vaccine group, Ac-cap- With 40 μ g/ albumen dosage immune swine musculi collis, immunity 2 times altogether are spaced 4 weeks the chimeric VLP subunit vaccines groups of 24B.Pig body Use 100RID50 CSFV C strains or 10 within the 8th week after immunity⁷ TCID₅₀Porcine circovirus 2 type WH strain (Yan Weidong, Shuo Shixue Degree thesis whole-length, the separation identification and the research of inactivated vaccine of porcine circovirus 2 type (PCV2) and application) poison is attacked, every kind of virus attacks poison 5 Head pig, and then the clinical symptoms of pig and detection Temperature changing etc. after poison are attacked in observation.

Each immune group pig after the CSFV C strains of 100 RID50 are shot, 5 pigs of CSFV commercially available vaccine immune group Without obvious Temperature changing, the chimeric VLP subunit vaccine immune groups of Ac-cap-24B have 3 pigs not have Temperature changing, and negative The body temperature of group group and porcine circovirus 2 type inactivated vaccine immune group pig raises and sizing thermal response is presented.Result above shows Chimeric attacks of the VLP subunit vaccines to CSFV of Ac-cap-24B has 60% protection (as shown in table 2).

Table 2：The Temperature changing that CSFV C strains are attacked after poison

(toxic agent amount is attacked for 1 × 10 after poison is attacked in porcine circovirus 2 type WH strains⁷ TCID₅₀Porcine circovirus 2 type WH strains/head), The negative groups of PBS and CSFV rabbitization attenuated vaccine immunity group start respectively have 1 pig spirit more depressed, recover normal when the 8th day, Without body temperature rising condition.The chimeric VLP subunit vaccine immune groups of Ac-cap-24B and porcine circovirus 2 type inactivated vaccine immunity Group does not all occur abnormal conditions, feeding, the state of mind and body temperature etc. after poison is attacked in this period all normal.Attack before poison and Off-test time-division another name weight, average relative daily gain result show that the chimeric VLP subunit vaccine immune groups of Ac-cap-24B are put down It is suitable with porcine circovirus 2 type inactivated vaccine immune group with respect to daily gain, obviously higher than negative control group and CSFV Rabbitization attenuated vaccine immunity group.In addition, by after death cut open inspection observes the pathology situation of pig at all pigs, as a result showing Ac-cap-24B The lymph node of chimeric VLP subunit vaccine immune groups and porcine circovirus 2 type inactivated vaccine immune group pig, kidney and spleen compared with Normally；And negative control group and CSFV rabbitization attenuated vaccine immunity Zu Zhu lungs are presented consolidation, blutpunkte, inguinal lymph Knot edge bleeding, hilar lymph node bleeding are extremely serious.Show that the chimeric VLP subunit vaccine immune groups of the Ac-cap-24B can be complete The attack of full guard pig circular ring virus intensity.

Although present disclosure is illustrated with reference to the present embodiment, the limit to the scope of the invention is not construed as System, it is intended that the scope of the present invention be defined by the claims appended hereto.In addition, those skilled in the art is limited in appended claims In the range of various changes or modification are carried out to the present invention, these change or modified forms also fall within the scope of the invention.

SEQUENCE LISTING

<110>Hua Zhong Agriculture University

<120>A kind of recombinant virus of the chimeric CSFV B cell epi-position of pig circular ring virus cap albumen and application

<130>A kind of recombinant virus of the chimeric CSFV B cell epi-position of pig circular ring virus cap albumen and application

<160> 2

<170> PatentIn version 3.1

<210> 1

<211> 666

<212> DNA

<213>Artificial sequence

<400> 1

atgtgcaagg aggactacag gtacgccatc tcctccacca acgagatcgg cctgctgggc 60

gccggcggcc tgacctggcg tagaaagaac ggtatcttca acacaagatt gtctcgtaca 120

atcggttaca ctgtcaagaa gaccaccgtc agaaccccat cttggaacgt tgacatgatg 180

agattcaaca tcaacgactt cctgccacct ggtggtggta gcaacccact caccgtccct 240

ttcgagtact acagaatccg taaggttaag gttgagttct ggccatgctc tcctatcacc 300

caaggagaca gaggtgtcgg tagcactgct gtcatcctgg acgacaactt cgttactaag 360

gccaacgctc tgacctacga cccttacgtc aactacagct ctagacacac cattactcaa 420

ccattcagtt accactcccg ttacttcacc ccaaagcctg tcctggaccg tactattgac 480

tacttccaac caaacaacaa gcgtaaccag ctgtggctga gactgcaaac tacaggaaac 540

gtcgaccacg ttggtctggg aaccgccttc gagaactcca tctacgacca ggactacaac 600

atcagaatca caatgtacgt ccagttcaga gagttcaacc tgaaggaccc tccactgaac 660

ccaaag 666

<210> 2

<211> 222

<212> PRT

<213>Artificial sequence

<400> 2

Met Cys Lys Glu Asp Tyr Arg Tyr Ala Ile Ser Ser Thr Asn Glu Ile

1 5 10 15

Gly Leu Leu Gly Ala Gly Gly Leu Thr Trp Arg Arg Lys Asn Gly Ile

20 25 30

Phe Asn Thr Arg Leu Ser Arg Thr Ile Gly Tyr Thr Val Lys Lys Thr

35 40 45

Thr Val Arg Thr Pro Ser Trp Asn Val Asp Met Met Arg Phe Asn Ile

50 55 60

Asn Asp Phe Leu Pro Pro Gly Gly Gly Ser Asn Pro Leu Thr Val Pro

65 70 75 80

Phe Glu Tyr Tyr Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys

85 90 95

Ser Pro Ile Thr Gln Gly Asp Arg Gly Val Gly Ser Thr Ala Val Ile

100 105 110

Leu Asp Asp Asn Phe Val Thr Lys Ala Asn Ala Leu Thr Tyr Asp Pro

115 120 125

Tyr Val Asn Tyr Ser Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr

130 135 140

His Ser Arg Tyr Phe Thr Pro Lys Pro Val Leu Asp Arg Thr Ile Asp

145 150 155 160

Tyr Phe Gln Pro Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln

165 170 175

Thr Thr Gly Asn Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn

180 185 190

Ser Ile Tyr Asp Gln Asp Tyr Asn Ile Arg Ile Thr Met Tyr Val Gln

195 200 205

Phe Arg Glu Phe Asn Leu Lys Asp Pro Pro Leu Asn Pro Lys

210 215 220

Claims (4)

1. a kind of gene order of manually modified synthesis, its sequence are shown in SEQ ID NO.1.

2. the baculovirus transfer vector of gene order described in claim 1 is included.

It is 3. a kind of to express pig circular ring virus and CSFV mosaic gene Cap-24B gene recombined virus, it is characterised in that：Restructuring Baculoviral Recominant Autographa californica multiple Nucleopolyhedrovirus Ac- Cap-24B, CCTCC NO:V201432.

4. application of the recombinant virus described in claim 3 in subunit vaccine is prepared.