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 recombinant virus and its application of porcine circovirus cap protein chimeric B cell epitope of classical swine fever virus. In the invention, the artificially synthesized cap-24B gene is expressed under the pH promoter of a baculovirus transfer carrier to obtain Recombinant baculovirus, CCTCC NO:V201432. The subunit vaccine prepared from the chimeric PCV2 VLP protein expressed by the recombinant baculovirus of the present invention can induce the body to produce specific humoral immunity after immunizing mice, and can produce specific antibody levels against CSFV and PCV2. It provides a good candidate vaccine for clinical prevention and treatment of these two diseases, and also provides feasibility test data for the study of circovirus as a vector for expressing other foreign antigens.

Description

A recombinant virus of porcine circovirus cap protein chimeric classical swine fever virus B cell epitope and applications

technical field

The invention belongs to the technical field of veterinary medicine, and in particular relates to a recombinant virus and application of porcine circovirus cap protein chimeric B cell epitope of classical swine fever virus.

Background technique

Porcine circovirus (PCV) is a member of the Circovirus family (Circoviridae), including PCV1 and PCV2, and is the smallest vertebrate virus found in the world so far. PCV1 is nonpathogenic, while PCV2 is pathogenic. PCV2 can clinically infect pigs aged 5-12 weeks to produce postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy (PDNS), porcine respiratory disease complex (porcine respiratory disease complex, PRDC), proliferative and necrotizing pneumonia (proliferative and necrotizingpneumonia, PNP) and other diseases. At present, for the convenience of research in the world, these diseases are collectively referred to as porcine circovirus disease (postweaning multisystemic wasting syn drome associated disease, PCVAD). The full length of ORF2 gene of PCV is 702bp (nt 1,735-1,034), and the Cap protein encoded by it is composed of 234 amino acids and its size is 27.8kDa. Cap is the main immunogenic protein and can induce specific PCV2 neutralizing antibodies (Pogranichnyy et al., 2000), so the ORF2 gene is the preferred target gene for designing new PCV2 vaccines. Liu et al. (2001) expressed ORF2 gene in Escherichia coli and proved that it has strong immunogenicity; after expressing ORF2 gene in baculovirus, circovirus-like particles (VLP) could be formed (Nawagitgul, 2000). The N segment of the Cap protein is a highly conserved and arginine-rich sequence, and the first 41 amino acid fragments of the N segment are the nuclear localization signal sequence (NLS) of the Cap protein. Studies have shown that the deletion of the N segment nuclear localization sequence does not affect its assembly into a virus-like Particle properties.

Classical swine fever (CSF) is a highly contagious, hemorrhagic and fatal infectious disease caused by Classical swine fever virus (CSFV) of the Flaviviridae family. The World Organization for Animal Health (OIE) lists it as an animal disease that must be reported, and my country lists it as a first-class animal disease. At present, swine fever is prevalent in many pig farms in my country, clinically manifested as premature birth, abortion, stillbirth, mummified fetus and decreased pig production capacity. At the same time, multiple infections and mixed infections often occur between the disease and pseudorabies, circovirus infection-related diseases. E2 protein is the main component of the main protective antigen that induces the body to produce neutralizing antibodies, and can resist virulent challenge protection experiments (Dong et al., 2006; Chen et al., 2011; Llilianne Gangesa et al., 2012) . E2 protein has 4 relatively independent antigen regions: A, B, C and D, and the amino acid sequence between 693-716 of B region (CKEDYRYAISSTNEIGLLGAGGLT) belongs to B cell epitope, which can produce neutralizing antibody and provide complete protection rate (Dong XN, et al., 2006, 2007).

In recent years, baculovirus expression vectors are easy to operate, high in safety, can accommodate large target genes, express foreign proteins with high efficiency, have post-translational modification, and express proteins with immunogenicity, biological activity and natural The advantages of protein similarity to other proteins have occupied a dominant position in expression vectors (Anderson et al., 1995; Wang et al., 2001; Ri beiro et al., 2001). The virus-like particle vaccine developed in recent years contains one or more antigenic proteins of the virus, particles with a structure similar to the virus particle, does not contain the genetic material of the virus, cannot replicate autonomously, has no infectivity, and can be used in a natural conformation and The pattern presents antigens, effectively stimulates humoral and cellular immunity, and induces good immune protection in the body. Based on the ORF2 gene of PCV2, the present invention deletes 39 amino acid sequences in its N-segment nuclear localization, inserts CSFV B cell epitopes or gene sequences coupled with T cell amino acids, and expresses chimeric B cell epitopes with a baculovirus expression system The Cap protein of PCV2, to study the ability of B-cell epitopes of PCV2 to form virus-like particle vaccines. At the same time, the formed chimeric virus-like particles are used to prepare subunit bivalent vaccines, and the immunogenicity effect against swine fever virus and porcine circovirus is evaluated through animal experiments, which provides an ideal for clinical prevention and control of swine fever and porcine circovirus disease candidate vaccine strains.

Contents of the invention

One object of the present invention is to provide a kind of cDNA gene sequence cap-24B of artificially modified and synthesized porcine circovirus cap protein gene and classical swine fever virus B cell epitope gene sequence chimerism, and its sequence is SEQ ID NO.1 respectively As shown, the encoded protein is shown in SEQ ID NO.2.

Another object of the present invention is to provide a transfer vector pFBD-cap-24B, which is a baculovirus transfer vector pFBDPHmHNM1P10eGFP containing 3 copies of the chimeric cap-24B cDNA gene sequence.

Another object of the present invention is to provide a recombinant baculovirus expressing porcine circovirus cap protein and B cell epitope gene, which was submitted to China Center for Type Culture Collection (CCTCC) on October 19, 2014 Preservation, its preservation number is CCTCC NO:V201432, classification name: recombinant baculovirus Recominant Autographa californica multiple Nucleopolyhedrovirus Ac-cap-24B, address: Wuhan University, Wuhan, China.

Another object of the present invention is to provide the application of recombinant baculovirus Ac-cap-24B in the preparation of subunit vaccines, which can induce the body to produce specific anti-swine fever virus and The immune response to porcine circovirus, and can produce specific antibody responses against classical swine fever and porcine circovirus.

For a more detailed technical solution, refer to the description in "Detailed Embodiments".

The beneficial effects of the present invention are:

1. According to the preference of baculovirus codons, the present invention first artificially modified and synthesized the gene sequence Cap of the N-segment nuclear localization sequence (NLS) of porcine circovirus Cap gene to replace the cDNA gene sequence of the B cell epitope of classical swine fever virus -24B.

2. In the present invention, the artificially synthesized target gene Cap-24B is placed under the baculovirus pH promoter to express, thereby increasing the expression level of the exogenous gene. Inserted into the baculovirus transfer vector and placed under the baculovirus pH promoter for expression, because the insertion is in the form of 3 copies, which can increase the expression of foreign genes.

3. After the porcine circovirus Cap gene chimeric classical swine fever virus B cell epitope expressed by the recombinant baculovirus of the present invention, PCV2 sample virus particles can be successfully formed equally, indicating that the porcine circovirus Cap gene N segment nuclear localization sequence ( NLS) was replaced without affecting its ability to form virus-like particles, which provided a feasible basis for the further development of chimeric porcine circovirus.

4. The subunit vaccine prepared by the chimeric VLP expressed by the recombinant baculovirus of the present invention can induce the body to produce specific immune responses against classical swine fever virus and porcine circovirus after immunizing mice.

Description of drawings

Figure 1 Schematic diagram of artificial synthesis of cap-24B gene and construction of baculovirus transfer vector

A: Schematic diagram of cap-24B gene artificial synthesis;

B: Schematic diagram of the construction of pFBD-cap-24B baculovirus transfer vector.

Fig. 2 is an enzyme digestion identification map of the baculovirus transfer vector pFBD-cap-24B.

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

Figure 3 Schematic diagram of PCR identification of recombinant bacmid rBac-cap-24B

The correct identified baculovirus transfer vector pFBD-cap-24B was transformed into DH10Bac, positive colonies were obtained by blue-white screening, and the bacmid was extracted for PCR identification.

M: 2000bp; 1: Amplified by Cap-24B 4F and Cap-R primers; 2: Amplified by Cap-24B 4F and M13R primers; 3: Negative control.

Figure 4 is a schematic diagram of Western blot analysis of recombinant baculovirus Ac-cap-24B protein expression

M: protein marker; 1: SF9 cell control; 2: Ac-cap infected sf9 cells (positive control); 3: Ac-cap-24B infected sf9 cells;

Figure 5 is a schematic diagram of indirect immunofluorescence analysis of recombinant baculovirus Ac-cap-24B protein expression

Anti-Cap: use PCV2 cap monoclonal antibody to detect the expression of the target protein, and observe the red fluorescence; EGFP: the baculovirus carries EGFP marker, and observe the green fluorescence; DAPI: observe the nucleus; Merge: the combined picture of red fluorescence and rate fluorescence

Ac-cap: Ac-cap infected sf9 cells; Ac-cap-24B: Ac-cap-24B infected sf9 cells

Figure 6 is an electron microscope schematic diagram of the formation of virus-like particles in the supernatant of sf9 cells infected with baculovirus Ac-cap-24B

Recombinant baculovirus Ac-cap-24B was inoculated into sf9 cells. After 84 hours, the diseased cells were collected, ultrasonically lysed and ultracentrifuged for electron microscope observation. A large number of non-enveloped viruses with a size of about 17-20nm were seen, and the morphology was the same as that of typical PCV2. Demonstrated assembly into virus-like particles VLP.

Figure 7 is a schematic diagram of indirect ELISA antibodies and neutralizing antibodies against PCV2 at different times after immunization of mice

A: ELISA antibody levels against PCV2 cap produced by mice at different time points after immunization; B: Neutralizing antibodies against PCV2 produced by mice at 14 days and 40 days after immunization;

Figure 8 is a schematic diagram of anti-CSFV antibodies at different times after immunization of mice

The IgG content of B cell epitopes produced by mice against CSFV at different times after immunization.

detailed description

The present invention can be better understood from the following examples. However, those skilled in the art can easily understand that the content described in the embodiments is only for illustrating the present invention, and should not and will not limit the present invention described in the claims. The technical solutions described in the present invention, unless otherwise specified, are conventional solutions in the art, and the described practices, unless otherwise specified, are commercial or disclosed reagent materials.

Example 1:

A recombinant baculovirus Ac-cap-2 expressing porcine circovirus cap protein chimeric classical swine fever virus B cell epitope gene

Construction of 4B

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

1. Obtaining the target gene of porcine circovirus cap chimeric CSFV B cell epitope

Refer to the ORF2 gene sequence of PCV2 WH isolate (Master's thesis of Huazhong Agricultural University: Isolation and Identification of Porcine Circovirus Type 2 (PCV2) and Research and Application of Inactivated Vaccine, Master Student: Yan Weidong; Supervisor: He Qigai ; Graduation Date: June 2009), the 117 nucleotide sequence of the 39 amino acids of the N segment nuclear localization signal was deleted and replaced with the 72 nucleotide sequence sequence of the classical swine fever virus B cell epitope (CKEDYRYAISSTNEIGLLGAGGLT) (as shown in Figure 1) According to the preference of baculovirus codons, a 666bp cap-24B gene sequence is artificially synthesized, its nucleotide sequence is shown in SEQ ID NO.1, and its encoded amino acid sequence is shown in SEQ ID NO.2. This sequence was subcloned into vector pUC to obtain recombinant plasmid pUC-cap-24B (made by Shanghai Bioengineering Company).

2. Construction of baculovirus transfer vector pFBD-cap-24B

The schematic diagram of the construction of the baculovirus transfer vector pFBD-cap-24B is shown in FIG. 1 .

Using the plasmid pUC-57cap-24B as a template, three pairs of different primers (see Table 1) were used to amplify the cap-24B gene fragment, and cloned into the baculovirus transfer vector pFBDPHmHNM1P10eGFP plasmid (referred to as pFBD. Qian Ping, an artificial expression Modified recombinant baculovirus of influenza A H1N1 virus HA-NA-M1 gene, patent publication number CN101624580A, patent number ZL200910063217.6). Furthermore, the cap-24B gene fragment was cloned into the corresponding site of pFFBD by BamHI and EcoRI, SpeI and NotI, SalI and HindIII, respectively, to obtain the transfer plasmid pFBD-cap-24B. The pFBD-cap-24B vector was identified by double enzyme digestion with BamHI and EcoRI. The results showed that there were 1129bp and 750bp fragments after digestion, and the size was consistent with the expected result, indicating that the transfer plasmid pFBD-cap-24B was successfully constructed. The results of enzyme digestion and identification are shown in Figure 2 shown. The transfer vector contains 3 copies of chimeric cap-24B cDNA gene sequence.

Table 1 Primers used for cloning the target gene

Note: The boldface is the restriction site sequence.

3. Construction of recombinant virus baculovirus Ac-cap-24B

(1) Acquisition and identification of recombinant bacmid Ac-cap-24B

Take 1.0 μg of the transfer plasmid pFBD-cap-24B and add it to 100 μL DH10Bac Escherichia coli competent cells (products of GiBCO BRL Company) to mix. After 30 minutes in ice bath, heat shock in 42°C water bath for 45 seconds, then ice bath for 2 minutes, and then add 900 μL SOC Liquid culture medium, cultured with shaking at 37°C for 4 hours, diluted according to 10 ^-1 , 10 ^-2 , 10 ^-3 , 100 μL each was spread on high-salt LB plates containing kanamycin, gentamicin and tetracycline, Use the concentration according to the Bac-to-Bac B aculovirusExpression System operating instructions (Invitrogen Company), culture at 37°C for 24-48h, screen and purify positive colonies through blue and white spots, extract recombinant bacmid, and then carry out PCR-specific primer amplification identification: vBac- The cap-24B bacmid was identified and detected by two pairs of primers, Cap-24B 1-F and Cap-1R; Cap-24B 1-F and M13R, and the estimated amplification sizes were 750bp and 1500bp, respectively.

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

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

M13R: 5'-AGCGGATAACAATTTCACACAGG-3';

Its PCR amplification system is as follows:

According to the reaction conditions of 95°C for 5 minutes; then 94°C for 45 seconds, 56°C for 30 seconds, 72°C for 1 minute, a total of 35 cycles, and finally 72°C for 10 minutes, the reaction conditions were amplified on the ABI PCR instrument. The amplified PCR product was subjected to 1.0% (weight/volume, w/v) agarose gel electrophoresis, and the results showed that two specific purpose bands of 1500bp and 750bp could be amplified, and the size was consistent with the expectation, indicating that the purpose The gene cap-24B fragment has been successfully recombined into the baculovirus genome (as shown in Figure 3).

(2) Recombinant baculovirus Ac-cap-24B obtained

Aseptically pick the positive colonies containing the recombinant bacmid rBac-cap-24B in high-salt LB liquid medium, culture at 37°C for 16 hours, add 0.3mL solution I (50mmol/L glucose, 10mmol/L EDTA, 25mmol /L Tris-Cl (pH8.0)) to resuspend, add 0.3mL solution II (0.2mol/L NaOH, 1% SDS, ready-to-use) and mix slightly, let stand at room temperature for 5min, slowly add 0.3mL solution III (3mol/L potassium acetate, pH5.0) mix well, ice bath for 5-10min, centrifuge at 12000r/min for 10min, add supernatant to 0.5mL isopropanol, mix well and ice bath for 5-10min, room temperature 12000r/min After centrifugation for 20 min, the precipitate was washed with 75% ethanol, dried, dissolved in 100 μL TE, and stored at -80°C.

Utilize the liposome-mediated transfection method (operate according to Invitrogen company Bac-to-Bac BaculovirusExpression System instruction manual), the recombinant bacmid rBac-cap-24B after screening and purifying is passed through Reagent transfected sf9 cells (purchased from Wuhan University China Type Collection Center), cultured at 28°C, placed a fluorescent microscope 72 hours after transfection to observe whether there is green fluorescence, collected the cell supernatant to obtain the recombinant baculovirus Ac-cap -24B, store at -80°C. The recombinant baculovirus Ac-cap-24B has the same morphological and cultural characteristics as the parental strain of Spodoptera californica multigrain embedded nuclear polyhedrosis virus (AcMNPV), but the recombinant baculovirus has a green fluorescent marker gene eGFP can observe virus amplification under a fluorescent microscope. The virus was sent to China Collection Center for Typical Microorganisms for preservation on October 19, 2014. Its classification name is: recombinant baculovirus Recominant Autographa californica multiple Nucleopoly hedrovirus Ac-cap-24B; preservation number: CCTCC NO: V201432. Address: Wuhan University, Wuhan, China.

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

1. Western blot analysis to detect the expression of exogenous genes

Inoculate sf9 cells into 6-well cell culture plates, and when the cells grow into 80-90% monolayer, use 1.0moi of Ac-cap-24B and Ac-cap (positive control, master's thesis: Peng Bo ; PCV2 Cap protein in Pichia Expression in yeast and baculovirus and preparation of subunit vaccine. Supervisor: Qian Ping ) inoculated sf9 cells, collected cells 84 hours after inoculation and lysed cell samples with NP-40 lysate. After SDS-PAGE, the membrane was transferred, and the Cap protein monoclonal antibody prepared by Professor He Qigai in our laboratory was used as the primary antibody (Master's thesis: Chen Donghuan ; Construction and identification of recombinant Salmonella expressing porcine circovirus type 2 Cap protein. Supervisor: He Qi Cap), HRP-labeled goat anti-mouse IgG (Sigma) was used as the secondary antibody for Western-blotting to detect the expression of Cap protein. The results showed that a specific band with a size of about 27KDa appeared in the cell samples infected by the recombinant virus Ac-cap and Ac-cap-24B respectively, and the size was consistent with the expectation, while the sf9 cell control did not have this specific band, confirming that the Cap protein was expressed. The result is shown in Figure 4.

2. Identification of exogenous gene expression by indirect immunofluorescence

The recombinant baculovirus Ac-cap-24B and Ac-cap were inoculated at a dose of 1.0 moi on the sf9 cells that had just grown into a monolayer. After 48 hours, the cells were fixed and the expression of Cap protein was detected by indirect immunofluorescence. The Cap protein monoclonal antibody prepared by Professor He Qigai in our laboratory was used as the primary antibody (Master's thesis: Chen Donghuan ; construction and identification of recombinant Salmonella expressing porcine circovirus type 2 Cap protein. Supervisor: He Qigai ), and the secondary antibody was CY3 - Goat anti-mouse IgG (Sigma) was used for indirect immunofluorescence, and finally observed under a confocal microscope. The results showed that the sf9 cells infected with Ac-cap-24B had a strong red fluorescent signal, which was mainly expressed in the nucleus, while the normal sf9 cells did not have any red fluorescent signal, indicating that the recombinant baculovirus Ac-cap and Ac-cap- 24B can effectively express cap protein without changing the protein localization. The result is shown in Figure 5.

3. Whether the insertion of exogenous genes affects the electron microscope observation of PCV2 cap protein assembled into virus-like particles in vitro

The F4 generation Ac-cap-24B was infected with 1.0 m.o.i dose to the suspension cultured sf9 cells, and the cells were collected 84 hours after infection, added to the lysate and frozen and thawed 3 times. Centrifuge at 8000g for 30min at 4°C, then concentrate with sucrose to 1/10 of the original volume, and the supernatant contains expressed VLP particles. Ultracentrifuge the supernatant at 27,000rpm/min for 3 hours, resuspend the pellet in 2.0ml of PBS, and blow it repeatedly with a 1.0ml syringe, then use a 20-60% sucrose density gradient to perform ultracentrifugation at 27,00rpm/min for the virus suspension After centrifugation for 16 hours, the uppermost protein band was collected for electron microscope observation. The results showed that cap-24B protein could form virus liquid particles (Virus-like particles, VLPs) under the electron microscope (H-7000FA), with a diameter of about 17– 25nm, its shape and size are similar to PCV2 whole virions (Fig. 6). It shows that we delete the NLS sequence in the N segment of PCV2 cap protein and replace it with the B epitope sequence of CSFV, which does not affect the assembly of cap protein to form VLP. Example 2:

The application of recombinant baculovirus Ac-cap-24B of the present invention in the preparation of subunit vaccines, the process is as follows:

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

The recombinant baculovirus Ac-cap-24B obtained in Example 1 was used to inoculate suspension-cultured insect cells High Five ^TM (purchased from Invitrogen) at a dose of 1.0 moi, and the cells and supernatant were collected after 84 hours, and processed by a cell disruptor After centrifugation to remove cell debris, the protein expression of cap-24B calculated by quantitative SDS-PAGE ligand gel is 52 micrograms per milliliter, and then emulsified with ISA-206 oil adjuvant (purchased from SEPPIC company) to prepare sub-vaccine. The cap-24B protein content per milliliter was 40 micrograms, stored at 4°C for use in the following examples.

(1) Physical shape

Appearance This product is milky white homogeneous emulsion.

The dosage form is a two-way dosage form (water-in-oil-in-water). Take a clean straw to suck up a small amount of vaccine and drop it in cold water, both on the water surface and in the water.

Viscosity Use a pipette with an outlet diameter of 1.2mm to draw 1mL of the vaccine at a room temperature of about 25°C, let it flow out vertically, and flow out 0.5mL within 8 seconds, which is qualified.

(2) Sterility test

Carry out according to " Chinese Veterinary Pharmacopoeia " appendix 169-171 pages, the result shows that there is no bacterial growth.

(2), the safety test of the chimeric VLPs subunit vaccine prepared by recombinant baculovirus Ac-cap-24B of the present invention

(1) Newborn piglet safety test

Take 4 batches of chimeric VLPs subunit vaccine prepared in the laboratory, take 3 bottles of each batch immediately, mix well, select healthy 1-day-old newborn piglets, vaccinate 10 pigs in each batch, and inject 2 times the dose of 4mL chimeric vaccine intramuscularly into each batch VLPs subunit vaccine, another 4 pigs were used as blank control. Before the vaccination and 1 week after the vaccination, the body temperature of all piglets was measured twice a day and the growth status, spirit and appetite of the piglets were observed for 14 consecutive days. On the day of vaccination, body temperature, spirit and appetite were normal, and there were no adverse reactions, indicating that the vaccine is safe for newborn piglets.

(2) Safety test of weaned piglets

Take 4 batches of chimeric VLPs subunit vaccine prepared in the laboratory, take 3 bottles from each batch, mix well, select healthy 30-day-old weaned piglets, vaccinate 10 pigs in each batch, and inject 2 times the dose of 4 mL chimeric vaccine intramuscularly into each batch For the VLPs subunit vaccine, another 4 pigs were used as blank controls and observed for 14 days as above. On the day of vaccination, the body temperature, spirit and appetite were normal, and there were no adverse reactions, indicating that the vaccine was safe for weaned piglets.

(3) Safety test on pregnant sows

Four batches of chimeric VLPs subunit vaccine prepared in the laboratory were taken, and 3 bottles were randomly taken from each batch. After mixing evenly, healthy 80-day-old pregnant sows were selected, and 10 pigs were vaccinated in each batch, and each batch was injected intramuscularly with 2 times the dose of 4 mL of chimeric VLPs subunit vaccine. Combined with VLPs subunit vaccine, another 2 pigs were used as blank control. Feed to farrowing under the same conditions, observe the effect of the vaccine on the reproductive performance of pregnant sows and the development of newborn piglets. After vaccination, the body temperature, spirit and appetite are normal, and there are no abortions, stillbirths, mummies, etc., indicating that the vaccine has a positive effect on pregnant sows. is safe.

(3), Immunogenicity experiment of recombinant baculovirus Ac-cap-24B chimeric VLP subunit vaccine in mice

In order to evaluate the immunogenicity of the chimeric VLPs subunit vaccine prepared above against porcine circovirus and classical swine fever virus, 18 6-week-old female Ba/bc mice were purchased and randomly divided into 2 groups, 6 in each group. PBS was negative, Ac-cap-24B group. The dose of immunization was 200 ul for each mouse in the Ac-cap-24B group, so that the amount of cap-24B protein was 40 micrograms per mouse. The route of immunization was intramuscular injection in the hind leg, followed by a booster immunization with the same dose 3 weeks later. Blood was collected from the tail vein at 0, 14, 28, and 40 days after the first immunization to evaluate the immune effect.

(1) The body produces antibody levels against PCV2 cap protein

0.1 μg of prokaryotic expression product of PCV2 cap protein was used to coat the enzyme-linked reaction plate, and the level of anti-cap antibody in mouse serum was detected by indirect ELISA method. The results showed that the mice group immunized with the recombinant baculovirus Ac-cap-24B chimeric VLP subunit vaccine could produce specific ELISA antibodies against PCV2 cap protein 14 days after the first immunization. The antibody level continued to rise after immunization, and the ELISA antibody reached the highest level at 40 days, and the antibodies reached 0.953 and 0.968 respectively. There was no significant difference in the ELISA antibody level against cap between the two groups (P<0.05), and the negative control group did not produce specific Anti-PCV2 antibody (as shown in Figure 7).

(2) The body produces immunogenicity against CSFV

The CSFV B cell epitope polypeptide (CKEDYRYAISSTNEIGLLGAGGLT) (synthesized by Shanghai Shenggong Co., Ltd.) was used to couple the KLH protein carrier, and the enzyme-linked reaction plate was coated with 2 micrograms of protein, and the anti-CSFV B cell expression in mouse serum was detected by indirect ELISA method. Bit IgG content. The results showed that the mice in the Ac-cap-24B immunized group could detect the production of IgG targeting B cell epitopes 14 days after immunization, and reached the highest level at 40 days after immunization; while the negative control group could not detect the production of IgG targeting B cell epitopes. IgG production at the bit (as shown in Figure 8).

The above animal test results show that the chimeric VLPs subunit vaccine prepared by Ac-cap-24B can effectively induce the body to generate immune responses against PCV2 and CSFV.

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

3-month-old pigs with positive porcine circovirus and classical swine fever virus serology were selected and randomly divided into 4 groups: PBS-negative group, Ac-cap-24B chimeric VLP subunit vaccine group, porcine circovirus type 2 inactivated vaccine and pig The pestivirus lapinized attenuated vaccine group and the Ac-cap-24B chimeric VLP subunit vaccine group were immunized with 40 μg protein dose in pig neck muscles, and immunized twice with an interval of 4 weeks. Eight weeks after pig immunization, use 100RID50 of CSFV C strain or 10 ⁷ TCID ₅₀ of porcine circovirus type 2 WH strain (Yan Weidong, master's thesis, isolation, identification and inactivation of porcine circovirus type 2 (PCV2) Research and Application of Vaccine) Infection, 5 pigs were challenged with each virus, and then the clinical symptoms and body temperature changes of the pigs after the challenge were observed.

After the pigs in each immunization group were inoculated with 100 RID50 CSFV C strains, none of the 5 pigs in the CSFV commercial vaccine immunization group had significant body temperature changes, and there were 3 pigs in the Ac-cap-24B chimeric VLP subunit vaccine immunization group The body temperature of the pigs did not change, while the body temperature of the pigs in the negative group and the porcine circovirus type 2 inactivated vaccine immunized group all increased and showed a stereotyped thermal reaction. The above results show that the Ac-cap-24B chimeric VLP subunit vaccine has 60% protection against the challenge of classical swine fever virus (as shown in Table 2).

Table 2: Changes in body temperature after challenge with CSFV strain C

After porcine circovirus type 2 WH strain challenged (the challenge dose was 1×10 ⁷ TCID ₅₀ porcine circovirus type 2 WH strain/head), the PBS negative group and the CSFV rabbitized attenuated vaccine immunized group started to have One pig was depressed and returned to normal on the 8th day, with no increase in body temperature. The Ac-cap-24B chimeric VLP subunit vaccine immunization group and the porcine circovirus type 2 inactivated vaccine immunization group had no abnormalities during the period after the challenge, and their food intake, mental state, and body temperature were all normal. Weighed before the challenge and at the end of the test, and the average relative daily gain results showed that the average relative daily gain of the Ac-cap-24B chimeric VLP subunit vaccine immunized group was equivalent to that of the porcine circovirus type 2 inactivated vaccine immunized group, Both were significantly higher than those in the negative control group and the attenuated CSFV vaccine immunization group. In addition, all the pigs were sacrificed and autopsyed to observe the pathological changes of the pigs. The results showed that the lymph nodes, kidneys and spleens of the Ac-cap-24B chimeric VLP subunit vaccine immunized group and the porcine circovirus type 2 inactivated vaccine immunized group were all normal. The pigs in the negative control group and the vaccinated group immunized with the attenuated CSFV vaccine showed consolidation and hemorrhagic spots, edge bleeding of inguinal lymph nodes, and severe hemorrhage of hilar lymph nodes. It shows that the Ac-cap-24B chimeric VLP subunit vaccine immunization group can completely protect the challenge of porcine circovirus intensity.

Although the content of the present invention is described in conjunction with this embodiment, it should not be regarded as limiting the scope of the present invention, which is defined by the appended claims. In addition, those skilled in the art may make various changes or modifications to the present invention within the scope defined by the appended claims, and these changes or modifications also fall within the scope of the present invention.

SEQUENCE LISTING

<110> Huazhong Agricultural University

<120> A recombinant virus of cap protein of porcine circovirus chimeric with B cell epitope of classical swine fever virus and its application

<130> A recombinant virus of cap protein of porcine circovirus chimeric with B cell epitope of classical swine fever virus and its application

<160> 2

<170> PatentIn version 3.1

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atgtgcaagg aggactacag gtacgccatc tcctccacca acgagatcgg cctgctgggc 60

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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

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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

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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.