CN113831414B

CN113831414B - Porcine circovirus 2b type Capid-Fc fusion protein, preparation method, gene and construction method and application thereof

Info

Publication number: CN113831414B
Application number: CN202010592316.XA
Authority: CN
Inventors: 郭美锦; 罗清平; 周云飞
Original assignee: Shanghai Hawk In Science And Technology Industrial Co ltd
Current assignee: Shanghai Hawk In Science And Technology Industrial Co ltd
Priority date: 2020-06-24
Filing date: 2020-06-24
Publication date: 2023-08-01
Anticipated expiration: 2040-06-24
Also published as: CN113831414A

Abstract

The invention discloses a porcine circovirus type 2b Capid-Fc fusion protein, a preparation method, a gene, a construction method and an application thereof, wherein the N-terminal 41 amino acids are removed by modifying the Capsid protein of the existing PCV 2b 41513 virus strain, the C-terminal is connected with a polypeptide linker and fused with a porcine IgG Fc fragment to obtain a novel porcine circovirus type 2b Capid-Fc fusion protein gene, a Kozak sequence and a human IgG2H heavy chain signal peptide are sequentially added to the N-terminal, and then whole-gene codon optimization is carried out to obtain an expression gene for encoding the porcine circovirus type 2b Capid-Fc fusion protein, and recombinant plasmid construction, transfection, expression, separation and purification are sequentially carried out to obtain the porcine circovirus type 2b Capid-Fc fusion protein, and the porcine circovirus type 2b Capid-Fc fusion protein subunit vaccine obtained by taking the porcine circovirus type 2b Capid-Fc fusion protein as an active ingredient can well stimulate a mouse organism to produce high-concentration antibodies.

Description

Porcine circovirus 2b type Capid-Fc fusion protein, preparation method, gene and construction method and application thereof

Technical Field

The invention relates to the field of bioengineering and immunology, in particular to a porcine circovirus 2b type Capsd-Fc fusion protein, a preparation method, a gene and a construction method and application thereof.

Background

Porcine circovirus type 2 (Porcine Circovirus type, PCV2 virus for short) belongs to the family of circoviridae, the genome of the porcine circoviridae is single-stranded circular DNA, the virus particles have no envelope, and an icosahedron is formed by assembling 60 protein monomers, so that the porcine circoviridae is one of the smallest viruses known at present. PCV2 genome is 1767bp or 1768bp long, and the two main open reading frames ORF1 and ORF2 of the genome encode Cap protein and Rep protein, respectively. The Pep protein is an essential protein for PCV2 virus replication. Cap proteins are capsid proteins that form the outer shell of PCV2 viruses, have the ability to bind host cell receptors, and are the primary immune antigens, often used to prepare PCV2 virus-specific vaccines and serodiagnostic target proteins. PCV2 viral capsid proteins are icosahedral symmetric.

PCV2 virus was first discovered in Canada in 1998 and was identified as an important pathogen responsible for a number of diseases such as sow reproductive impairment, granulomatous enteritis, congenital tremor interstitial pneumonia, postweaning multisystemic failure syndrome, swine dermatitis and nephrotic syndrome.

Various PCV2 virus vaccines, such as inactivated vaccines, attenuated vaccines and recombinant vaccines, developed mainly by porcine circovirus type 2a virus (hereinafter abbreviated as PCV 2a virus) strain are developed in the market. There are also some inactivated vaccines developed with strains of porcine circovirus type 2b (hereinafter referred to as PCV 2b virus).

These PCV 2b virus vaccines play an important role in controlling PCV 2b virus infection, but the immune effect still remains to be further improved. And because of the large genotype of PCV 2b virus variety, cases of immune failure appear many times at present, even in pig farms vaccinated with PCV 2b virus vaccine, there are still problems such as epidemic outbreaks of PCV 2b virus infection.

In summary, the present PCV 2b virus vaccine also has the technical problems of low antibody titer, insufficient protection of immune subjects, and the like. Therefore, it is needed to update and upgrade the existing PCV 2b virus vaccine, improve the antibody titer, obtain better immunogenicity and expand the immune coverage, thereby improving the protection of immune subjects.

Reference to the literature

1、Allan,G.,Meehan,B.,Todd,D.,Kennedy,S.,McNeilly,F.,Ellis,J.,Clark,E.G.,Harding,J.,Espuna,E.,Botner,A.,Charreyre,C.,1998.Novel porcine circoviruses from pigs with wasting disease syndromes.Vet.Rec.142,467–468；

2、de Boisseson C,Beven V,Bigarre L,Thiery R,Rose N,Eveno E,et al.Molecular characterization of Porcine circovirus type 2isolates from post-weaning multisystemic wasting syndrome-affected and non-affected pigs.J Gen Virol2004；85(February(Pt 2)):293–304；

3、Constans M,Ssemadaali M,Kolyvushko O,et al.Antigenic Determinants of Possible Vaccine Escape by Porcine Circovirus Subtype 2b Viruses[J].Bioinformatics and Biology Insights,2015,9s2:BBI.S30226；

4. Construction of CN201510413407.1 porcine circovirus double subtype ORF2 co-expression vector and vaccine preparation;

5. CN201480045584.2 porcine circovirus type 2 (PCV 2) subunit vaccine;

6. CN201180024467.4 live attenuated chimeric porcine circovirus vaccine.

Disclosure of Invention

One of the purposes of the present invention is to provide a porcine circovirus type 2b Capid-Fc fusion protein subunit vaccine, which has the capability of generating high antibody titer and the capability of enhancing the response of humoral and cellular immunity of an immune subject, in order to solve the technical problems of low antibody titer, insufficient protection of the immune subject, etc. of the existing PCV 2b virus vaccine.

The second purpose of the invention is to provide a porcine circovirus type 2b Capid-Fc fusion protein used in the porcine circovirus type 2b Capid-Fc fusion protein subunit vaccine and a preparation method thereof.

The invention also provides an expression gene of the porcine circovirus type 2b Capid-Fc fusion protein and a construction method thereof.

The technical principle of the invention is as follows:

adopting Capsid protein of PCV2b 41513 virus strain which fails to immunize the existing porcine circovirus type 2 vaccine, engineering to remove 41 amino acids at N end of the Capsid protein, connecting polypeptide linker at C end, and fusing pig IgG Fc fragment to generate a novel porcine circovirus type 2b Capsd-Fc fusion protein, namely PCV2b Capsd-Fc fusion protein;

the DNA of the novel porcine circovirus type 2b Capid-Fc fusion protein is subjected to total gene codon optimization to obtain the expression gene of the porcine circovirus type 2b Capid-Fc fusion protein, namely the expression gene of the porcine circovirus type 2b Capid-Fc fusion protein, the gene sequence of the gene is specifically shown as SEQ ID No.1, and then the gene is inserted into a pEM expression plasmid through total gene synthesis and EcoRI/HindIII enzyme double enzyme digestion to obtain the pEM-PCV 2b Capid-Fc recombinant plasmid. In addition, PCV2b Capid-Fc fusion protein genes are inserted into pCDNA3.1 (+) plasmid by a primer amplification method through NheI/HindIII enzyme double-enzyme digestion method to obtain pCDNA3.1 (+) -PCV 2b Capid-Fc recombinant plasmid; the recombinant plasmid is transfected into escherichia coli DH5 alpha to obtain recombinant escherichia coli DH5 alpha containing pEM-PCV 2b Capid-Fc recombinant plasmid or pCDNA3.1 (+) -PCV 2b Capid-Fc recombinant plasmid; amplifying by recombinant escherichia coli DH5 alpha to obtain transfection-grade recombinant plasmids;

The transfection-grade pEM-PCV2b Capid-Fc recombinant plasmid or the transfection-grade pCDNA3.1 (+) -PCV2b Capid-Fc recombinant plasmid is transfected into a mammalian HEK293F, and then suspension culture is carried out sequentially, and separation and purification are carried out to obtain the target porcine circovirus 2b type Capid-Fc fusion protein product.

Note that: capid in the PCV2b Capid-Fc fusion protein is a PCV2b Capsid protein described as N-terminally cleaved by 41 amino acids.

The PCV2b Capid-Fc fusion protein was found to be capable of forming nanoparticles of about 41nm average size by transmission electron microscopy. The results of the BLAB/c mouse immunization experiment show that the mouse vaccine has the function of well stimulating the mouse organism to generate humoral immunity and cellular immunity response.

The technical scheme of the invention is as follows:

a porcine circovirus type 2b Capid-Fc fusion protein is PCV2b Capid-Fc fusion protein, the PCV2b Capid-Fc fusion protein is expressed in a host, preferably in a manner that the host is a mammalian cell HEK293F, a recombinant vector encoding the PCV2b Capid-Fc fusion protein is constructed by taking a pEM plasmid or a pcDNA3.1 (+) plasmid as a vector, the recombinant vector encoding the PCV2b Capid-Fc fusion protein is transfected into the mammalian cell HEK293F for expression, the pcDNA3.1 (+) plasmid is taken as the vector for constructing the recombinant vector encoding the PCV2b Capid-Fc fusion protein for transfection into the mammalian cell HEK293F, and the host is not limited to the mammalian cell, but eukaryotic yeast is taken as a host for expression, preferably in a manner that pichia pastoris GSl15 strain is taken as a preferred vector, and the recombinant vector is also transfected into the mammalian cell HEK293F in the embodiment of the invention, and the recombinant vector is not limited to the PCV2b Capid-Fc fusion protein is expressed in the manner that the host is expressed in the eukaryotic yeast cell.

The preparation process of the porcine circovirus type 2b Capid-Fc fusion protein, namely PCV2b Capid-Fc fusion protein, specifically comprises the following steps:

(1) Expression Gene sequence Synthesis of PCV2b Capid-Fc fusion protein

(1) Expression Gene Synthesis of novel PCV2b Capid-Fc fusion protein

The expression gene sequence of PCV2b Capid-Fc fusion protein comprises the following three segment sequences:

based on the gene of Porcinecircovirus type2-BstrainNDSU41513 strain for encoding PCV2 capsid protein, namely the sequence ID is GenBank accession ionniumberALD 62452.1, after 41 amino acids at the N end of the capsid protein are removed, codon optimization is carried out according to the codon preference of a mammalian cell to obtain a PCV2 capsid segment sequence, wherein the sequence is shown in SEQ ID No.3;

the Fc region gene of the heavy chain of the pig IgG has the sequence ID of Gen Bank accession number AAD38418.1, and the sequence of the Fc region gene is shown in SEQ ID No.7;

taking a DNA sequence shown as SEQ ID No.5 as a linker connecting section sequence;

carrying out codon optimization on the three-section sequence according to the codon preference of the mammalian cells to obtain a novel PCV2bCAPSid-Fc fusion protein gene; the sequence of the polypeptide is shown in SEQ ID No.1;

(2) synthesis of PCV2b Capid-Fc Gene

The front end and the rear end of the novel PCV2b Capid-Fc fusion protein obtained in the step (1) contain enzyme digestion sites EcoRI-HindIII, a Kozak sequence and a human IgG2H heavy chain signal peptide sequence are inserted, the sequence is a Kozak sequence and a human IgG2H heavy chain signal peptide sequence in sequence, and thus the expression gene sequence of the porcine circovirus type 2b Capid-Fc fusion protein is obtained, and the sequence is shown in SEQ ID No.1;

The Kozak sequence is shown in SEQ ID No.11; the sequence of the heavy chain signal peptide of the human IgG2H is shown in SEQ ID No.12;

(2) Construction of pEM-PCV 2b Capid-Fc recombinant plasmid containing Gene of PCV 2b Capid-Fc

Designing cleavage sites EcoRI and HindIII at the N end and the C end of the gene of the PCV 2b Capid-Fc fusion protein, synthesizing the sequences by an artificial gene synthesis mode, and inserting the gene of the PCV 2b Capid-Fc fusion protein into an expression vector pEM plasmid by a double enzyme cleavage method to obtain a pEM-PCV 2b Capid-Fc recombinant plasmid;

(3) Construction of pCDNA3.1 (+) -PCV 2b Capid-Fc recombinant plasmid containing the Gene of PCV 2b Capid-Fc

Designing cleavage sites NheI and HindIII in a primer PcfF sequence with a sequence shown as SEQ ID No.9 and a primer PcfR sequence with a sequence shown as SEQ ID No.10, carrying out PCR amplification on an expression gene sequence which synthesizes PCV 2b Capid-Fc by the primers PcfF and PcfR as shown as SEQ ID No.1 to obtain an expression fragment containing cleavage sites NheI and HindIII, carrying out double cleavage on the PCR amplified fragment, and then connecting the PCR amplified fragment to a pCDNA3.1 (+) plasmid expression vector subjected to the double cleavage treatment of the same NheI/HindIII to obtain a recombinant plasmid of the pCDNA3.1 (+) -2 b Capid-Fc fusion protein;

(4) The pEM-PCV2b Capid-Fc recombinant plasmid or the pCDNA3.1 (+) -PCV2b Capid-Fc recombinant plasmid is transfected into escherichia coli DH5 alpha;

sucking the pEM-PCV2b Capid-Fc recombinant plasmid obtained in the step (2) or 80-100ng of the pCDNA3.1 (+) -PCV2b Capid-Fc recombinant plasmid obtained in the step (3), adding the plasmid into the prepared DH5 alpha competent cells, adding the cells into an LB liquid medium, putting the liquid medium into a shaking table to control the temperature to 37 ℃ and shaking and culturing the liquid medium at the speed of 200rpm for about 30min, and separating the DH5 alpha competent cells to obtain recombinant escherichia coli DH5 alpha containing the pEM-PCV2b Capid-Fc recombinant plasmid or the pCDNA3.1 (+) -PCV2 bCapsild-Fc recombinant plasmid;

(5) Extraction of transfection-grade plasmid

E.coli DH5 alpha containing pEM-PCV2b Capid-Fc recombinant plasmid or pCDNA3.1 (+) -PCV2b Capid-Fc recombinant plasmid obtained in the step (4) is selected and monoclonal in a 2-5mLLB culture medium in a fresh culture plate, and the temperature is controlled to be 37 ℃ and the rotating speed is controlled to be 200rpm, so as to obtain seed liquid. Then inoculating the seed solution into 200mLLB culture medium according to the volume ratio of 1/500, continuously controlling the temperature to 37 ℃ and the rotating speed to 200rpm for 16 hours to obtain bacterial solution containing the recombinant plasmid, centrifuging for 10 minutes to remove the supernatant, and adopting E.Z.N.A. of omega Biotek company to harvest the obtained precipitate. Extracting transfection-grade plasmid according to the method described by the kit from Endo-Free Plasmid DNA Maxi Kit plasmid large sampling kit and D6926-01 to obtain transfection-grade pEM-PCV2b Capid-Fc recombinant plasmid or transfection-grade pCDNA3.1 (+) -PCV2b Capid-Fc recombinant plasmid;

(6) Expression of PCV2b Capsd-Fc fusion protein by mammalian cells

(1) HEK293 cell resuscitating and amplifying culture

A: the water bath kettle is opened at 37 ℃ in advance, and the Union-293 serum-free fresh culture medium is preheated at 37 ℃ in advance; taking out the frozen HEK293F cell-containing frozen tube from a liquid nitrogen tank, immediately putting the frozen cell into a water bath kettle at 37 ℃ and slightly shaking to enable the frozen cell to melt rapidly within 1min, sterilizing the outer wall of the HEK293F cell-containing frozen tube with 75% ethanol after taking out, putting the frozen cell-containing frozen tube into a biosafety cabinet, transferring the biosafety cabinet into a 15mL centrifuge tube filled with 10mLUNIon-293 serum-free medium, controlling the rotating speed to be 800rpm for centrifugation for 5min, removing the supernatant after centrifugation, taking the HEK293F cell in the fresh Union-293 serum-free medium resuspension centrifuge tube, transferring the HEK293F cell into a culture bottle, adding the Union-293 serum-free medium for shaking to enable the cell to disperse uniformly, taking cell count and activity detection, and controlling the activityCell density is 6-8×10 ⁵ Cell viability was measured for individual cells/mL, resuscitated when cell viability was below 80%, and cell culture medium was placed in CO when cell viability was above 80% ₂ CO with volume percentage concentration of 5% ₂ Culturing in an incubator at 37 deg.C and 110rpm for 2-3 days, removing 10mL of cell culture solution, and adding 10mL of fresh culture medium until the cell activity is greater than 90%, to obtain culture solution containing recovered HEK293 cells;

b: placing the culture solution containing the recovered HEK293 cells obtained in the step A in CO ₂ CO with volume percentage concentration of 5% ₂ Culturing in incubator at 37deg.C and rotational speed of 110rpm for 2-3 days until cell concentration reaches 2-3×10 ⁶ Performing first cell expansion culture on HEK293 cells by using a culture solution of HEK293 cells/mL:

30mL of fresh medium was added to the 15mL of HEK293 cell broth obtained after the first cell expansion culture, and then CO was added ₂ CO with volume percentage concentration of 5% ₂ Continuously culturing in incubator at 37deg.C and rotation speed of 110rpm, detecting HEK293 cell concentration and cell activity every day, and detecting when cell concentration reaches 2-3×10 ⁶ Performing secondary cell expansion culture on HEK293 cells by using the HEK293 cell/mL culture solution, wherein the culture volume reaches about 45mL;

90mL of fresh medium was added to the 45mL of HEK293 cell broth obtained after the first cell expansion culture, and then CO was added ₂ CO with volume percentage concentration of 5% ₂ Continuously culturing in incubator at 37deg.C and rotation speed of 110rpm, detecting HEK293 cell concentration and cell activity every day, and detecting when cell concentration reaches 2-3×10 ⁶ Performing third cell expansion culture on HEK293 cells by using the HEK293 cell/mL culture solution to obtain HEK-293F cell culture solution subjected to third expansion culture, wherein the culture volume reaches about 135mL;

(2) preparation of Union-293 serum-free Medium containing transfection-grade pEM-PCV 2b Capid-Fc recombinant plasmid:

adding the transfection-grade pEM-PCV 2b Capid-Fc recombinant plasmid obtained in the step (5) into a Union-293 serum-free medium to obtain a Union-293 serum-free medium containing the transfection-grade pEM-PCV 2b Capid-Fc recombinant plasmid, wherein the concentration of the Union-293 serum-free medium is 20 mug/ml;

(3) preparation of Union-293 serum-free culture solution containing polyethyleneimine

The transfection reagent polyethyleneimine is added into Union-293 serum-free medium to obtain 80 mug/ml Union-293 serum-free medium containing polyethyleneimine;

(4) mixing the Union-293 serum-free culture solution containing the polyethyleneimine obtained in the step (3) and the Union-293 serum-free culture solution containing the transfection-grade pEM-PCV 2b Capid-Fc recombinant plasmid obtained in the step (2) according to the volume ratio of 1:1, and standing for 20-30min at room temperature to obtain the Union-293 serum-free culture solution of PEI/DNA;

(5) Transfection of transfection-grade pEM-PCV 2b Capid-Fc recombinant plasmid into HEK293 cells

Adding the Union-293 serum-free culture solution of PEI/DNA obtained in the step (4) into the HEK-293F cell culture solution obtained in the step (1) after three amplification culture, and then adding CO ₂ CO with volume percentage concentration of 5% ₂ Culturing in an incubator at 37 ℃ and 110rpm for 4-6 days, centrifuging the obtained cell culture solution at 1000rpm for 10min, and obtaining precipitate, namely HEK293F cells containing PCV 2b Capid-Fc fusion protein;

the usage amount of the Union-293 serum-free culture solution of PEI/DNA and the HEK-293F cell culture solution after three times of amplification culture is the same as that of the Union-293 serum-free culture solution of PEI/DNA: the volume ratio of HEK-293F cell culture solution after three times of amplification culture is 1:10, calculating the proportion;

(7) Isolation of PCV 2b Capsd-Fc fusion proteins

Resuspending HEK293F cells containing PCV 2b Capid-Fc fusion protein obtained in the step (6) with an ultrasonic lysis buffer, controlling the amplitude intensity output to 34% by using an ultrasonic breaker of model Q125 of Qsonic LLC company, working for 3 seconds, stopping for 7 seconds, performing ultrasonic lysis for 30 minutes, controlling the temperature to 4 ℃, centrifuging at a rotating speed of 12,000rpm for 20 minutes, and collecting supernatant;

The ultrasonic lysis buffer comprises 50mM Tris, 300mM NaCl, 20mM imidazole with pH of 8.0, 10g Triton X-100, 1mM phenylmethylsulfonyl fluoride (PMSF) and the balance of water according to each liter;

the amounts of HEK293F cells containing PCV 2b Capid-Fc fusion protein and the amount of the ultrasonic lysis buffer were calculated as the ratio of wet weight of HEK293F cells containing PCV 2b Capid-Fc fusion protein to 1g:10mL of ultrasonic lysis buffer;

the wet weight of HEK293F cells containing PCV 2b Capid-Fc fusion protein is the actual mass of the precipitate obtained by centrifugation in the step (5);

(8) Purification of PCV 2b Capsd-Fc fusion proteins

Loading the supernatant collected after centrifugation in the step (7) onto a Protein A agarose gel chromatographic column (Detai biotechnology (Nanjing) limited company), carrying out equilibrium chromatographic column, loading, washing impurities, eluting, collecting and collecting samples in sequence according to the use requirement of the Protein A agarose gel chromatographic column, neutralizing the collected samples, and filtering by a 0.22-micrometer filter to obtain filtrate containing PCV 2b Capsd-Fc fusion Protein.

The resulting filtrate containing PCV 2b Capid-Fc fusion protein was stored at-70℃for use in subsequent experiments.

The application of the PCV 2b Capid-Fc fusion protein in preparing the PCV 2b Capid-Fc fusion protein subunit vaccine for resisting porcine circovirus type 2b infection, wherein the PCV 2b Capid-Fc fusion protein subunit vaccine consists of an active ingredient PCV 2b Capid-Fc fusion protein and an adjuvant, and the mass volume ratio is calculated, wherein the PCV 2b Capid-Fc fusion protein: adjuvant was 3.3mg:100mL; wherein the adjuvant is complete Freund's adjuvant or incomplete Freund's adjuvant;

The active ingredient is PCV 2b Capid-Fc fusion protein which is self-assembled into virus-like nano particles.

The preparation method of the PCV 2b Capid-Fc fusion protein subunit vaccine comprises the following specific steps:

PCV 2b Capid-Fc fusion protein in PCV 2b Capid-Fc fusion protein subunit vaccine: adjuvant was 3.3mg:100mL of the solution is prepared, namely, the filtrate obtained after filtration by a 0.22 micron filter is diluted by one time by 1xPBS solution with pH of 7.4 and is mixed with Freund complete adjuvant preheated to 37 ℃ or Freund incomplete adjuvant preheated to 37 ℃ in an oscillating way, so that the PCV 2b Capsd-Fc fusion protein is emulsified until a stable emulsifying system is formed, and the PCV2 Capsd-Fc fusion protein subunit vaccine is obtained.

In the PCV 2b Capid-Fc fusion protein subunit vaccine, the active ingredient PCV 2b Capid-Fc fusion protein is self-assembled into virus-like nano particles with the particle size of about 41nm by the measurement of a scanning electron microscope.

The porcine circovirus type 2b Capid-Fc fusion protein can also be applied to the preparation of antibody pharmaceutical reagents and/or kits for resisting porcine circovirus type 2b infection.

The invention has the beneficial effects that:

The invention discloses an expression gene of porcine circovirus type 2b Capid-Fc fusion protein for encoding PCV2b Capid-Fc fusion protein, which is used as an active ingredient to obtain porcine circovirus type 2b Capid-Fc fusion protein subunit vaccine which can well stimulate a mouse organism to generate high-concentration antibodies so as to improve the immunity of cells and body fluids. Particularly, the subunit vaccine taking PCV2b Capid-Fc fusion protein as an active substance shows that the antibody level is slightly increased on the 12 th day after primary immunization through BLAB/c mouse immunization experiment results, and the specific antibody aiming at PCV2 capsids can be produced at a high level after secondary boost immunization, the antibody concentration exceeds 500ng/mL, the antibody concentration is increased by more than 1 time compared with that of a BLAB/c mouse group immunized with commercial subunit vaccine, and meanwhile, the subunit vaccine taking PCV2b Capid-Fc fusion protein as an active substance can well stimulate the mouse organism to produce humoral immunity and cellular immune response.

Drawings

Fig. 1: PCR identification results of the pCDNA3.1 (+) -PCV 2b Capid-Fc recombinant plasmid obtained in example 2 are schematically shown; wherein M, DNA marker 1kb DNA; PCV2b Capid-Fc PCR product;

fig. 2: schematic representation of the nucleic acid fragment of pcdna3.1 (+) -PCV 2b hepsd-Fc recombinant plasmid obtained in example 2 after double cleavage with NheI/HindIII enzyme of PCV2b hepsd-Fc, wherein m.dna Marker;1. extracting recombinant plasmid; nheI/HindIII enzyme double-digested recombinant plasmid pCDNA3.1 (+) -PCV 2b Capid-Fc;

Fig. 3: schematic representation of the NcoI/ApaI double digested nucleic acid fragment of PCV 2b Capid-Fc of the pEM-PCV 2b Capid-Fc recombinant plasmid obtained in example 2, wherein M.DNA Marker;1. extracting recombinant plasmid; 2. double-enzyme digestion of pEM-PCV 2b Capid-Fc recombinant plasmid with NcoI/ApaI;

fig. 4: schematic structural representation of PCV 2b Capid-Fc fusion protein obtained in example 4;

fig. 5: 3D prediction structure of PCV 2b Capid-Fc fusion protein obtained in example 4;

fig. 6: SDS-PAGE and Western Blot analysis of PCV 2b Capid-Fc fusion proteins obtained in example 4;

fig. 7: a transmission electron microscope observation chart of the inactivated virus selected in the example 1 under the magnification of 4K-300K (200 nm) is applied;

fig. 8: a transmission electron microscope observation chart of the PCV 2b Capid-Fc fusion protein selected in the example 1 under the magnification of 4K-300K (200 nm) is applied;

fig. 9: a transmission electron microscope observation chart at a magnification of 4K-300K (200 nm) of the commercial subunit vaccine selected in example 1 was applied;

fig. 10: a transmission electron microscope observation chart of the inactivated virus selected in the example 1 under the magnification of 4K-300K (200 nm) is applied;

fig. 11: a transmission electron microscope observation chart of the PCV 2b Capid-Fc fusion protein selected in the example 1 under the magnification of 4K-300K (100 nm) is applied;

Fig. 12: a transmission electron microscope observation chart at a magnification of 4K-300K (100 nm) of the commercial subunit vaccine selected in the example 1 is applied;

fig. 13: a transmission electron microscope observation chart of the inactivated virus selected in example 1 under a magnification of 4K-300K (50 nm) is applied;

fig. 14: a transmission electron microscope observation chart of the PCV 2b Capid-Fc fusion protein selected in the example 1 under the magnification of 4K-300K (50 nm) is applied;

fig. 15: a transmission electron microscope observation chart at a magnification of 4K-300K (50 nm) of the commercial subunit vaccine selected in example 1 was applied;

fig. 16: lymphocyte proliferation assay analysis of spleen cells of immunized mice in animal cell experiments, wherein EFSV is PCV 2b Capsd-Fc fusion protein subunit vaccine test group of the invention, CSV is commercial subunit vaccine group, conA is ConA of 5 μg/ml as positive control, and data are expressed as SI average value + -SD, P < 0.05, P < 0.01;

fig. 17: schematic of changes in titers of PCV 2b hepsd-Fc fusion protein subunit vaccine immunized mice specific antibodies in antibody titer assays;

fig. 18: schematic of PCV 2b hepsd-Fc fusion protein subunit vaccine and commercial subunit vaccine immunization mice specific antibody titers in antibody titer assays.

Fig. 19: the difference of cytokine IFN-gamma secretion concentration of immunized mice of PCV 2b Capsd-Fc fusion protein subunit vaccine and commercial subunit vaccine in cytokine IFN-gamma secretion concentration measurement is shown schematically.

Fig. 20: cytokine IL-10 secretion concentration in PCV 2b Capsd-Fc fusion protein subunit vaccine and commercial subunit vaccine immunization mice were tested for differential cytokine IL-10 secretion concentration.

Detailed Description

The invention will now be further illustrated by means of specific examples in conjunction with the accompanying drawings without limiting the invention.

The embodiments of the present invention use:

starting strain name: porcine circovirus type 2-B strain NDSU 41513, capsid protein amino acid sequence from protein_id= "ALD62452.1";

pEM plasmid from Detai Biotechnology (Nanjing) Co., ltd

Plasmid pcDNA3.1 (+), detai Biotech (Nanjing) Co., ltd

Coli DH 5. Alpha., deltay Biotechnology (Nanjing) Co., ltd;

HEK 293F (human embryonic kidney cells 293F) from the university of eastern university of china laboratory;

the primer sequences were as follows:

the composition and content of LB liquid medium used for culturing E.coli DH5 alpha cells are 1.0% sodium chloride, 1.0% peptone, 0.5% yeast extract and the balance of water according to mass percentage.

The culture medium used for HEK 293F cell culture is a commercially available serum-free Union-293 culture medium, and the serum-free Union-293 culture medium is produced by the company Shanghai Corp.

Cell transfection was performed according to the instructions using the 40-kDa linear Polyethylenimine (PEI) transfection reagent available from Shanghai Kangbio technology Co.

The SDS-PAGE and Western Blot analysis methods used in the examples of the present invention were as follows:

the cell lysate and the protein bands of the enriched PCV 2b Capid-Fc fusion protein component were separated and identified on a 12.5% polyacrylamide gel by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Polyacrylamide gel was stained with coomassie brilliant blue G250 to reveal protein bands. The separated proteins were transferred to PVDF membrane by wet Western blot transfer mode on protein gels separated on SDS-PAGE gels. Proteins were transferred to PVDF membrane in QuickBlockTM Western blocking buffer overnight at 4℃and then incubated with anti-porcine circovirus mab (1:1000 dilution; abcam, USA) in TBST for 2h at room temperature. HRP-labeled goat anti-rabbit antibody (SAB, china) was used as secondary antibody. After washing 3 times with TBST, the imaged protein bands were detected using an ECL chemiluminescent system BeyoECL Plus (hypersensitive ECL chemiluminescent kit) (P0018S), a Gel Imaging System (GIS) software package from Biyun Biotechnology Co., ltd and Tanon, and a full-automatic digital gel image analysis system from Shanghai Technical Co., ltd.

The names, specifications and manufacturer information of the main reagents used in the invention are as follows:

measurement of cell number in the examples of the present invention the method of measuring cell number using trypan blue staining is the most common method of cell number measurement, and the apparatus used for measurement is the CountStar automatic counting cell analyzer.

BCA protein assay kit method (bi yun biotechnology limited) was performed according to the product instructions.

Data statistics

Data are expressed as mean ± standard errorStudent's t-test (GraphPad Software inc., san Diego, CA, USA) was used. Graphic and data statistical analysis was performed using GraphPadPrism 8. P value<0.05 is considered statistically significant. *0.05<P<0.01，**0.01<P<0.001

Example 1

The gene for expressing the porcine circovirus 2b type Capsd-Fc fusion protein has a base sequence shown in SEQ ID No.1, and the coding of the gene consists of three parts, namely a PCV2 Capsid segment sequence, a linker segment sequence and an Fc segment sequence; wherein the PCV2 capsid segment sequence is SEQ ID No.3; the sequence of the linker section is SEQ ID No.5; the Fc segment sequence is the Pic IgG heavy chain Fc fragment, the coding sequence is SEQ ID No.7, and the construction process comprises the following specific steps:

(1) Gene sequence codon optimization for expression of novel PCV 2b Capid-Fc fusion proteins

Firstly, selecting a Porcine circovirus type-B strain NDSU 41513 strain to code PCV2 capsid protein gene, namely based on a sequence ID of Gen Bank accession number ALD62452.1, removing 41 amino acids at the N end of the capsid protein, and adopting code optimization software of Detai biotechnology (Nanjing) limited company to obtain a PCV 2B capsid segment sequence by carrying out code optimization according to the codon preference of a mammalian cell; taking a linker as a connecting peptide (the amino acid sequence of the connecting peptide is SEQ ID No. 4), and carrying out codon optimization according to the codon preference of mammalian cells to obtain a linker segment sequence; an Fc region sequence with a sequence ID of Gen Bank accession number AAD38418.1 is obtained by using a pig IgG heavy chain Fc region gene; the three-section sequence adopts the codon optimization software of Detai biotechnology (Nanjing) limited company, carries out codon optimization according to the codon preference of mammalian cells, and carries out total gene synthesis to obtain a novel PCV 2b Capsd-Fc fusion protein gene; the sequence is shown in SEQ ID No.1.

(2) Adding gene N-terminal and C-terminal expression sequence elements for expressing novel PCV 2b Capid-Fc fusion protein

And (2) designing and adding EcoRI-HindIII containing enzyme cutting sites at the front end and the rear end of the novel PCV 2b Capid-Fc fusion protein obtained in the step (1), and inserting a Kozak sequence and a human IgG2H heavy chain signal peptide sequence, wherein the sequence is the Kozak sequence and the human IgG2H heavy chain signal peptide sequence. The novel gene for expressing PCV 2b Capsd-Fc fusion protein added with the Kozak sequence and the human IgG2H heavy chain signal peptide sequence element is subjected to codon optimization, and codon optimization software of De Tai Biotechnology (Nanjing) is adopted for codon optimization according to the codon preference of mammalian cells.

(3) Sequence gene synthesis by adding novel gene N-terminal and C-terminal expression sequence elements for expressing PCV 2b Capid-Fc fusion protein

The sequence is subjected to total gene synthesis to obtain a gene sequence containing the recombinant porcine circovirus 2 b-type Capid-Fc fusion protein, and the sequence is shown in SEQ ID No.13. The Kozak sequence is shown in SEQ ID No.11; the sequence of the heavy chain signal peptide of the human IgG2H is shown in SEQ ID No.12; the gene expressing the porcine circovirus type 2b Capid-Fc fusion protein obtained above was sequenced by using a 3730XL sequencer from Applied Biosystems company, and the result was consistent with that of SEQ ID No.13.

Example 2

A recombinant plasmid containing PCV 2b Capid-Fc gene is constructed by the following steps:

(1) Construction of pEM-PCV 2b Capid-Fc recombinant plasmid containing Gene of PCV 2b Capid-Fc

(2) Construction of pCDNA3.1 (+) -PCV 2b Capid-Fc recombinant plasmid containing the Gene of PCV 2b Capid-Fc

Designing cleavage sites NheI and HindIII in a primer PcfF sequence with a sequence shown as SEQ ID No.9 and a primer PcfR sequence with a sequence shown as SEQ ID No.10, carrying out PCR amplification on a gene sequence expressing PCV2b Capid-Fc by the primers PcfF and PcfR to obtain an amino acid expression fragment containing cleavage sites NheI and HindIII, carrying out double cleavage on the PCR amplified fragment, and then connecting the PCR amplified fragment to a pCDNA3.1 (+) plasmid expression vector subjected to double cleavage treatment by the same NheI/HindIII to obtain a pCDNA3.1 (+) -PCV 2b Capid-Fc recombinant plasmid;

the PCR identification result of the pCDNA3.1 (+) -PCV 2b Capsd-Fc recombinant plasmid obtained above is schematically shown in FIG. 1; wherein M, DNA marker 1kb DNA; the PCV2b Capid-Fc PCR product, as can be seen from FIG. 1, contains the PCV2b Capid-Fc fusion protein gene.

The nucleic acid fragment obtained after double digestion of NheI/HindIII enzyme of PCV2b Capid-Fc of the pCDNA3.1 (+) -PCV 2b Capid-Fc recombinant plasmid obtained above is shown in FIG. 2, wherein M.DNA Marker;1. extracting recombinant plasmid; the successful construction of the expression plasmid pCDNA3.1 (+) -PCV 2b Capid-Fc can be seen from FIG. 2 by double cleavage of the PCDNA3.1 (+) -PCV 2b Capid-Fc with NheI/HindIII enzyme.

The schematic diagram of the nucleic acid fragment obtained after double digestion of NcoI/ApaI enzyme of PCV 2b Capid-Fc of the pEM-PCV 2b Capid-Fc recombinant plasmid obtained above is shown in FIG. 3, wherein M.DNA Marker;1. extracting recombinant plasmid; 2. the successful construction of pEM-PCV 2b Capid-Fc recombinant plasmid was seen from FIG. 3 by double cleavage with NcoI/ApaI.

Example 3

The preparation of transfection-grade pCDNA3.1 (+) -PCV 2b Capid-Fc recombinant plasmid or transfection-grade pEM-PCV 2b Capid-Fc recombinant plasmid comprises the following steps:

(1) The pEM-PCV 2b Capid-Fc recombinant plasmid or the pCDNA3.1 (+) -PCV 2b Capid-Fc recombinant plasmid is transfected into escherichia coli DH5 alpha; sucking 80-100ng of pEM-PCV 2b Capid-Fc recombinant plasmid or pCDNA3.1 (+) -PCV 2b Capid-Fc recombinant plasmid obtained in the example 2, adding the plasmid into prepared DH5 alpha competent cells, adding the cells into LB liquid medium, placing the culture medium into a shaking table to control the temperature to 37 ℃ and the rotating speed to 200rpm for shake culture for about 30min, and separating DH5 alpha competent cells to obtain recombinant escherichia coli DH5 alpha containing pEM-PCV 2b Capid-Fc recombinant plasmid or pCDNA3.1 (+) -PCV 2b Capid-Fc recombinant plasmid;

(2) Extraction of transfection-grade plasmid

E.coli DH5 alpha containing the transfection-grade pEM-PCV 2b Capid-Fc recombinant plasmid or pCDNA3.1 (+) -PCV 2b Capid-Fc recombinant plasmid obtained in the step (1) is selected in a fresh culture plate, and is subjected to single clone in 2-5mL LB culture medium, the temperature is controlled to be 37 ℃, the rotation speed is controlled to be 200rpm for 8 hours to obtain seed solution, then the seed solution is inoculated in 200mL LB culture medium according to the proportion of 1/500, the temperature is continuously controlled to be 37 ℃ and the rotation speed is controlled to be 200rpm for 16 hours to obtain bacterial solution containing the recombinant plasmid, the bacterial solution is centrifuged for 10 minutes at the rotation speed of 4000rpm, the supernatant is removed, and the obtained precipitate is obtained by adopting E.Z.N.A of Omega Biotek company. And (3) extracting transfection-grade plasmids by using an Endo-Free Plasmid DNA Maxi Kit plasmid large-sampling kit and D6926-01 according to a method described by the kit to finally obtain transfection-grade pEM-PCV 2b Capid-Fc recombinant plasmids or transfection-grade pCDNA3.1 (+) -PCV 2b Capid-Fc recombinant plasmids.

Example 4

The PCV 2b Capid-Fc fusion protein is prepared by using 40kDa linear Polyethylenimine (PEI) as a transfection reagent, transiently transfecting the transfection-grade pEM-PCV 2b Capid-Fc recombinant plasmid obtained in example 3 into HEK293F suspension cells, and then sequentially culturing, separating and purifying to finally obtain the PCV 2b Capid-Fc fusion protein, wherein the preparation process specifically comprises the following steps:

(1) HEK293 cell resuscitation and passaging

(1) The water bath kettle is opened at 37 ℃ in advance, and the Union-293 serum-free fresh culture medium is preheated at 37 ℃ in advance; taking out frozen HEK293F cell-containing frozen tube from liquid nitrogen tank, immediately putting frozen cell into 37 deg.C water bath, slightly shaking to make it melt quickly within about 1min, sterilizing outer wall of HEK293F cell-containing frozen tube with 75% ethanol, placing into biosafety cabinet, transferring into 15mL centrifuge tube containing 10m lunion-293 serum-free culture medium, centrifuging at 800rpm for 5min, removing supernatant, taking a small amount of fresh culture medium to resuspend HEK293F cell in centrifuge tube, transferring into culture bottle, adding Union-293 serum-free culture medium, slightly shaking to make cell disperse uniformly, taking cell count and activity detection, and controlling living cell density at 6-8X10 ⁵ Cell viability (the cell viability is the ratio of living cells to total cells) is determined per cell/mL, resuscitated when the cell viability is less than 80%, and when the cell viability is greater than 80%, the cell culture solution is placed in CO ₂ CO with volume percentage concentration of 5% ₂ Culturing in incubator at 37deg.C and rotational speed of 110rpm for 2-3 days until density reaches 2-3×10 ⁶ Removing 10mL of cell culture solution when HEK293 cells/mL of culture solution are added, and adding 10mL of fresh culture medium until the cell activity is more than 90%, thus obtaining culture solution containing recovered HEK293 cells; (2) placing the culture solution containing the recovered HEK293 cells obtained in the step (1) in CO ₂ CO with volume percentage concentration of 5% ₂ Culturing in incubator at 37deg.C and rotational speed of 110rpm for 2-3 days until cell concentration reaches 2-3×10 ⁶ HEK293 cells/mLHEK 293 cell culture solution, and performing first cell expansion culture on the HEK293 cells: 30mL of fresh medium was added to the 15mL of HEK293 cell broth obtained after the first cell expansion culture, and then CO was added ₂ The volume percentage concentration is5% CO ₂ Continuously culturing in incubator at 37deg.C and rotation speed of 110rpm, detecting HEK293 cell concentration and cell activity every day, and detecting when cell concentration reaches 2×10 ⁶ Performing secondary cell expansion culture on HEK293 cells by using the HEK293 cell/mL culture solution, wherein the culture volume reaches about 45mL; 90mL of fresh medium was added to the 45mL of HEK293 cell broth obtained after the second cell expansion culture, and then CO was added ₂ CO with volume percentage concentration of 5% ₂ Continuously culturing in incubator at 37deg.C and rotation speed of 110rpm, detecting HEK293 cell concentration and cell activity every day, and detecting when cell concentration reaches 2-3×10 ⁶ Performing a third cell expansion culture on HEK293 cells by using the HEK293 cell/mL culture solution, wherein the culture volume reaches about 135mL;

(2) Preparation of Union-293 serum-free Medium containing pEM-PCV 2b Capid-Fc or pCDNA3.1 (+) -PCV 2b Capid-Fc transfection-grade plasmids:

the transfection-grade pEM-PCV 2b Capid-Fc recombinant plasmid obtained in example 3 was added to Union-293 serum-free medium to obtain 20. Mu.g/ml of Union-293 serum-free medium containing the transfection-grade pEM-PCV 2b Capid-Fc recombinant plasmid;

(3) Preparation of polyethylene imine containing Union-293 serum-free culture broth:

(4) Preparation of Union-293 serum-free Medium of PEI/DNA:

mixing the obtained Union-293 serum-free culture solution containing the transfection-grade pEM-PCV 2b Capid-Fc recombinant plasmid with the Union-293 serum-free culture solution containing polyethyleneimine with the volume ratio of 80 mug/ml at 1:1, and standing at room temperature for 20-30min to obtain the Union-293 serum-free culture solution of PEI/DNA;

(5) Plasmid transfection HEK293 cells:

adding 13.5mL of Union-293 serum-free culture solution of PEI/DNA into 135mL of the culture solution obtained in the step (1) and performing three-time amplification cultureCulturing HEK-293F in culture medium, and adding CO ₂ CO with volume percentage concentration of 5% ₂ Culturing in an incubator at 37 ℃ and 110rpm for 4-6 days, centrifuging the obtained cell culture solution at 1000rpm for 10min, and obtaining precipitate, namely HEK293F cells containing PCV2b Capid-Fc fusion protein;

(6) Isolation of PCV2b Capid-Fc fusion protein:

resuspension of HEK293F cells containing PCV2b Capid-Fc fusion protein obtained in the step (5) with an ultrasonic lysis buffer, then controlling the amplitude intensity output to 34% by using an ultrasonic breaker of model Q125 of Qsonic LLC company, working for 3 seconds, stopping for 7 seconds, performing ultrasonic lysis with the total working time of 30 minutes, controlling the temperature to 4 ℃ and the rotating speed to 12,000rpm for 20 minutes, and collecting supernatant;

the wet weight of HEK293F cells containing PCV 2b Capid-Fc fusion protein is the actual mass of the precipitate obtained after centrifugation in the step (5);

(7) Purification of PCV2 Capid-Fc fusion protein:

loading the supernatant collected after centrifugation in the step (6) onto a Protein A agarose gel chromatographic column (Detai biotechnology (Nanjing) limited company), carrying out equilibrium chromatographic column, loading, washing impurities, eluting, collecting and collecting samples in sequence according to the use requirement of the Protein A agarose gel chromatographic column, neutralizing the collected samples, and filtering by a 0.22-micrometer filter to obtain filtrate containing PCV 2b Capsd-Fc fusion Protein.

The amino acid sequence of PCV 2b Capid-Fc fusion protein in the filtrate containing PCV 2b Capid-Fc fusion protein obtained above was determined, which was identical to the expected amino acid sequence of SEQ ID No. 2.

The structural diagram of the PCV 2b Capid-Fc fusion protein obtained in the above way is shown in FIG. 4, and can be seen from FIG. 4; the PCV 2b Capid-Fc fusion protein can form a PCV 2b Capid-Fc fusion protein dimer from the monomeric PCV 2b Capid-Fc fusion protein through disulfide bonds of the Fc fragment;

predicting the 3D structure of the PCV 2b Capid-Fc fusion protein by using an I-TRASSER Server in a ribbon model, wherein the prediction structure diagram is shown in FIG. 5, and can be seen from FIG. 5; the Capsid protein portion and the Fc portion of PCV 2b Capsd-Fc fusion proteins are structurally non-interfering with each other and each has sufficient spatial design to function independently.

SDS-PAGE and Western Blot analysis of the PCV 2b Capid-Fc fusion protein obtained above are shown in FIG. 6, wherein A is an electrophoresis pattern of a cell lysate after HEK293F cell expression and purified PCV 2b Capid-Fc fusion protein on 12.5% polyacrylamide gel under denaturing conditions, wherein M is marker, lane 1, supernatant lane 2 of the cell lysate after sonication is flow-through, lanes 3-8 are eluents; the supernatant of the cell lysate after sonication in lane a, lane B, the flow-through, lanes c-e, the eluate, SDS-PAGE analysis of the gel by Coke's blue staining, and B, western Blot, detected a specific band of the approximately 59kDa protein. As can be seen from FIG. 6, there is a predicted band at the approximately 59kDa position. Thus, it was demonstrated that the cell-expressed lysate was purified to finally obtain the target protein product, PCV 2b Capsd-Fc fusion protein.

Application examples

The application of the PCV 2b Capid-Fc fusion protein in preparing the PCV 2b Capid-Fc fusion protein subunit vaccine for resisting porcine circovirus type 2b infection comprises an active ingredient PCV 2b Capid-Fc fusion protein and an adjuvant, wherein the mass volume ratio of the PCV 2b Capid-Fc fusion protein to the adjuvant is 3.3 mg/100 mL;

wherein the adjuvant is complete Freund's adjuvant or incomplete Freund's adjuvant;

the filtrate containing PCV 2b Capid-Fc fusion protein obtained in the step (7) of example 4 was replaced by dialysis to 1xPBS having a pH of 7.4 and a glycerol content of 10% by volume, and then filtered with a 0.22 μm filter, and the filtrate obtained after filtration with the 0.22 μm filter was measured using BCA protein concentration measuring kit, and the PCV 2b Capid-Fc fusion protein concentration was 66. Mu.g/mL.

And taking 1 volume of the filtrate obtained after filtering by using a 0.22 micron filter, diluting the filtrate by one time by using a 1xPBS solution with the pH of 7.4, dividing the filtrate into two equal parts, and respectively mixing the equal volumes of Freund complete adjuvant preheated to 37 ℃ and Freund incomplete adjuvant preheated to 37 ℃ by 1 volume in an oscillating manner to realize emulsification of the PCV 2b Capid-Fc fusion protein until a stable emulsifying system is formed, thus obtaining the PCV 2b Capid-Fc fusion protein subunit vaccine.

The PCV 2b Capid-Fc fusion protein subunit vaccine is determined by a scanning electron microscope, and the active ingredient PCV 2b Capid-Fc fusion protein is self-assembled into virus-like nano particles, wherein the particle size of the virus-like nano particles is about 41nm.

Taking commercial subunit vaccine (CSV, namely anti-PCV 2 subunit vaccine produced by Qingdao biological engineering Co., ltd.) and inactivated virus (produced by Shanghai animal health products Co., ltd.) as controls, respectively scanning the obtained PCV 2b Capid-Fc fusion protein subunit vaccine by using a transmission electron microscope, wherein the obtained scanning transmission electron microscope images are shown as figures 7, 8, 9, 10, 11, 12, 13, 14 and 15, the morphology of the inactivated virus is different from that of the PCV 2b Capid-Fc fusion protein subunit vaccine and that of the commercial subunit vaccine are different from each other, and the morphology of the inactivated virus and the PCV 2b Capid-Fc fusion protein subunit vaccine are formed into regular particles and the morphology of the commercial subunit vaccine is irregular from the comparison of figures 10, 11 and 12; from a comparison of FIGS. 13, 14, 15, it can be seen that the inactivated virus size is around 15-20 nm, and the PCV 2b Capsd-Fc fusion protein subunit vaccine nanoparticle size is about 41nm; thus, PCV 2b Capid-Fc fusion protein can spontaneously form nano particles with regular morphology and average size of about 41n m, and provides a structural basis for better stimulating an organism to generate an immune response.

Animal cell experiment

Animal cell immunity experiments are respectively carried out on the PCV 2b Capid-Fc protein subunit vaccine obtained by taking commercial subunit vaccine (namely, anti-PCV 2 subunit vaccine produced by Qingdao biological engineering Co., ltd.) and inactivated virus (produced by Shanghai animal health products Co., ltd.) as controls;

female BLAB/c mice (6-8 weeks old) were used as subjects, and were kept in laboratory for 7 days prior to formal experiments in specific pathogen-free animals (SPF) class animal of university of North China university, and then animals were randomly divided into three experimental groups (n=3 mice/group) and kept in a controlled temperature and light environment and food and water conditions were freely available;

mice were immunized intraperitoneally with 6.6ug vaccine per mouse, PCV 2b hepsd-Fc fusion protein subunit vaccine of the invention and commercially available anti-PCV 2 subunit vaccine. The control group received only sterile PBS solution. The PBS solution is prepared according to the content of each substance contained in each liter; naCl,8.0g; KH (KH) ₂ PO ₄ ，0.2g；Na ₂ HPO ₄ .12H ₂ O,2.9g; KCl,0.2g; then the volume is fixed to 1000mL, and the pH value is adjusted to 7.4;

serum was collected from each group of mice at days 12, 24, 36, 59, 64 and 70 after the primary immunization, and the anti-PCV 2 capsid protein antibody concentration in the serum was determined separately.

Lymphocyte proliferation assay and cytokine assay

Spleen cells were isolated from immunized mice 70 days after immunization from mice. Spleen cells (5X 10) ⁵ Cells/well) were inoculated into 96-well plates, and 200. Mu.l of RPMI 1640 containing 10% FBS was added for culturing. ConA using concanavalin5 μg/mL) or vaccine to stimulate these spleen cells. After incubation at 37℃for 72 hours, the cells were then incubated at an equal volume of 100uLReagents were added to each well and incubated for 15 minutes at 37 ℃. Used +.>Luminescent cell viability assay kit (Promega, usa) the proliferation of lymphocytes was measured in 96-well, flat, transparent bottom, opaque wall microwell plates using an automated cell analyzer according to manufacturer's protocol, the results expressed as Stimulation Index (SI) calculated according to the following formula: si= (number of cells of immune group-number of cells of control)/(number of cells of negative control group-number of cells of control).

The proliferation of lymphocytes is shown in fig. 16, in which EFSV is the PCV 2b hepsd-Fc fusion protein subunit vaccine of the present invention, CSV is a commercial subunit vaccine, conA is a positive Control, and Control is RPMI 1640 Control, and as can be seen from fig. 16, both the PCV 2b hepsd-Fc fusion protein subunit vaccine of the present invention and the commercial subunit vaccine have strong lymphocyte stimulating effect and induce proliferation reaction. The SI of two groups of mice immunized with the PCV 2b Capid-Fc fusion protein subunit vaccine and the commercial subunit vaccine of the present invention was 1.728.+ -. 0.259,1.430.+ -. 0.212, respectively, which were higher than the RPMI 1640 control group (P < 0.05), while the SI of the immunized group of PCV 2b Capid-Fc fusion protein subunit vaccine of the present invention was also higher than the immunized group of the commercial subunit vaccine. SI value of ConA (positive control) control was 2.967± 0.713, indicating that ConA effectively works. These results indicate that the inventive hepsd-Fc fusion protein subunit vaccine induced a significant cellular immune response in mice.

Determination of antibody titres

In order to further study the degree of immune response of the PCV2b Capid-Fc fusion protein subunit vaccine, namely PCV2b Capid-Fc fusion protein subunit vaccine and commercial subunit vaccine of the present invention, specific antibody levels of PCV2 Capid Capsid proteins were measured on the obtained autoimmune mouse serum by indirect ELISA analysis, and specific results are shown in FIG. 17 and FIG. 18;

as can be seen from fig. 17, on day 12 after the primary immunization, the antibody level was weakly increased, the specific antibody against PCV2 hepsd was produced at a high level after the secondary boost, the antibody concentration exceeded 500ng/ml, and the antibody concentration was maintained at a high level throughout the experimental period, thus indicating that the PCV2b hepsd-Fc fusion protein subunit vaccine of the present invention was able to excite mice to produce a high concentration of PCV2 hepsd-specific antibody, the antibody concentration was maintained at a high level;

further, as can be seen from fig. 17, the average level of specific antibodies against PCV2 hepsd Capsid protein in the group immunized with PCV2 hepsd-Fc fusion protein subunit vaccine of the present invention increased rapidly from 222.38ng/mL to 461.23ng/mL at 12 and 24 days. After 36 days, the antibody concentration was maintained at a concentration level above 540 ng/mL.

In fig. 18, the control group is a PBS solution immunized mouse, the EFSV immunized mouse group is a PCV2bFc fusion protein subunit vaccine immunized mouse group of the present invention, CSV represents a commercial subunit vaccine immunized mouse group, wherein P <0.05, P < 0.01, it can be seen from fig. 18 that both the PCV2b hepsd-Fc fusion protein subunit vaccine of the present invention and the commercial subunit vaccine can induce the production of anti-mouse PCV2 antibodies, while the concentration of the antibody induced by the PCV2b hepsd-Fc fusion protein subunit vaccine immunized mouse group of the present invention is significantly higher than that of the commercial subunit vaccine immunized mouse group, and in the commercial subunit vaccine immunized mouse group, the level of the specific antibody of PCV2 hepsd Capsid protein is 200-250ng/mL, so that the level of the PCV2b hepsd-Fc fusion protein subunit vaccine of the present invention is improved by more than 1-fold compared with that of the commercial subunit vaccine immunized mouse group, and the result shows that the PCV2b hepsd-Fc fusion protein of the present invention has significant difference (P0.05), thereby the body cell immunity of the present invention can be significantly improved.

To further characterize the antigen-specific cellular immune properties of mice immunized with the PCV2b hepsd-Fc fusion protein subunit vaccine (EFSV) and Commercial Subunit Vaccine (CSV) of the present invention, the present inventors evaluated cytokines secreted by spleen T cells of immunized mice using the PCV2b hepsd-Fc fusion protein subunit vaccine and commercial subunit vaccine of the present invention. The assay was performed using commercial mouse IFN-gamma and IL-10ELISA kits (Jiangsu enzyme-free Utility Co., ltd.). Compared with the PBS control group, the secretion concentration of the cytokine IFN-gamma is 230.76 +/-16.97 and 237.28 +/-20.11 pg/ml, the secretion of the IFN-gamma cytokine generated by T cells of immunized mice stimulated by the PCV2b Capid-Fc fusion protein subunit vaccine and the commercial subunit vaccine reaches 292.17 +/-13.80 and 293.80 +/-30.50 pg/ml respectively, and the statistical analysis of the results shows that the results of the data show significant differences (P < 0.01 and P <0.05, and the results are shown in FIG. 19;

Furthermore, in the case of stimulating spleen T cells of mice immunized with EFSV with the medium, compared with the case of stimulating spleen T cells of mice immunized with EFSV with PCV 2b Capid-Fc fusion protein (PCFFP), the secretion concentrations of the two average cytokines IL-10 were 17.17.+ -. 0.85ng/mL and 19.11.+ -. 0.26ng/mL, respectively, and the analysis of the two groups of data showed significant differences (P < 0.05) statistically, however, no significant difference in IL-10 secretion was detected in mice immunized with CSV and stimulated with CSV, and the results are shown in FIG. 20; the above results indicate that the PCV 2b Capid-Fc fusion protein subunit vaccine of the present invention can better induce humoral and cellular immune responses in mice.

In summary, the porcine circovirus type 2b Capid-Fc fusion protein encoded by the expression gene of the porcine circovirus type 2b Capid-Fc fusion protein provided by the invention can be used as an active ingredient to obtain the subunit vaccine of the porcine circovirus type 2b Capid-Fc fusion protein, which can better induce body fluid and cells of mice to generate immune response.

The porcine circovirus type 2b Capid-Fc fusion protein can form viroid-like nano particles, has good structural properties, and is easy to excite an immune system of an organism to generate stress immune response. The data result shows that the porcine circovirus type 2b Capid-Fc fusion protein not only can induce humoral immunity and cellular immune response of mice, but also is better than commercial subunit vaccines in some indexes compared with the experimental vaccines which take the porcine circovirus type 2b Capid-Fc fusion protein as an active ingredient. Therefore, the porcine circovirus type 2b Capid-Fc fusion protein is a candidate vaccine with good potential for preventing and treating porcine circovirus infection.

The foregoing is merely illustrative of embodiments of this invention and it will be appreciated by those skilled in the art that changes and modifications may be made without departing from the principles of the invention, which is also intended to be within the scope of the invention.

Sequence listing

<110> Shanghai eagle technology Co.Ltd

<120> porcine circovirus 2b type Capid-Fc fusion protein, and preparation method, gene, construction method and application thereof

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<170> SIPOSequenceListing 1.0

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aacggcatct tcaacaccag gctgtccagg accttcggct acaccatcaa gcggaccacc 60

gtgcggacac cttcttgggc cgtggacatg atgcgcttca acatcaacgc cttcctgccc 120

ccaggcggag gctctaatcc tagaagcgtg cctttcgagt actaccggat ccggaaggtg 180

aaggtggagt tttggccttg cagccccatc acccagggag atagaggagt gggaagtagc 240

gccgtgatcc tggacgacaa cttcgtgacc aaggccacag ccctgaccta cgatccttac 300

gtgaactaca gcagccggca caccatcacc cagcccttca gctaccacag ccgctacttc 360

acccctaaac ccgtgctgga cagcaccatc gactacttcc agcccaacaa caagcggaac 420

cagctctggc tgagactgca gacagcaggc aacgtggatc acgtgggact gggaacagcc 480

ttcgagaaca gcatctacga ccagggctac aacatccgcg tgaccatgta cgtgcagttc 540

cgggagttca acctgaagga cccccccctg aatccaggaa gcggaggagg aagcggagga 600

ggaggaagcg gaggaggaag cgcccctaaa acagctccta gcgtgtatcc tctggcccct 660

tgtggcagag atacaagcgg ccctaacgtg gctctgggct gtctggcctc tagctacttc 720

ccagagcccg tgaccatgac ttggaacagc ggagccctga caagcggagt gcacaccttt 780

cctagcgtgc tgcagcctag cggactgtat agcctgagca gcatggtgac agtgccagcc 840

tctagcctga gcagcaagag ctacacttgc aacgtgaacc accccgccac cacaacaaag 900

gtggacaagc gcgtgggcac caagacaaag cctccttgcc ccatttgtcc aggttgcgag 960

gtggccggcc ctagcgtgtt tatcttccct cctaagccca aggacaccct gatgatcagc 1020

cagaccccag aagtgacttg cgtggtggtg gacgtgtcta aggagcacgc cgaggtgcag 1080

ttcagttggt acgtggacgg cgtggaagtg cacacagccg agacaagacc caaggaggag 1140

cagttcaaca gcacctaccg cgtggtgtcc gtgctgccta tccagcacca ggattggctg 1200

aagggcaagg agttcaagtg caaggtcaac aacgtggacc tgccagcccc tatcaccaga 1260

acaatcagca aggccatcgg acagagcagg gagcctcagg tgtacacact gcctcctcca 1320

gcagaggagc tgagcaggag caaagtgacc gtgacctgcc tggtcatcgg cttttacccc 1380

ccagacatcc acgtcgagtg gaagagtaac gggcagccag agccagaggg caactacaga 1440

accacacctc ctcagcagga cgtggacgga acattcttcc tgtacagcaa gctggccgtg 1500

gacaaagctc gctgggatca cggggagacc ttcgaatgcg cagtgatgca cgaggccctg 1560

cataaccact acacccagaa gagcatcagc aagacccagg gcaag 1605

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<213> Artificial sequence (Artificial Sequence)

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

1 5 10 15

Lys Arg Thr Thr Val Arg Thr Pro Ser Trp Ala Val Asp Met Met Arg

20 25 30

Phe Asn Ile Asn Ala Phe Leu Pro Pro Gly Gly Gly Ser Asn Pro Arg

35 40 45

Ser Val Pro Phe Glu Tyr Tyr Arg Ile Arg Lys Val Lys Val Glu Phe

50 55 60

Trp Pro Cys Ser Pro Ile Thr Gln Gly Asp Arg Gly Val Gly Ser Ser

65 70 75 80

Ala Val Ile Leu Asp Asp Asn Phe Val Thr Lys Ala Thr Ala Leu Thr

85 90 95

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

100 105 110

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

115 120 125

Thr Ile Asp Tyr Phe Gln Pro Asn Asn Lys Arg Asn Gln Leu Trp Leu

130 135 140

Arg Leu Gln Thr Ala Gly Asn Val Asp His Val Gly Leu Gly Thr Ala

145 150 155 160

Phe Glu Asn Ser Ile Tyr Asp Gln Gly Tyr Asn Ile Arg Val Thr Met

165 170 175

Tyr Val Gln Phe Arg Glu Phe Asn Leu Lys Asp Pro Pro Leu Asn Pro

180 185 190

Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala

195 200 205

Pro Lys Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Cys Gly Arg Asp

210 215 220

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

225 230 235 240

Pro Glu Pro Val Thr Met Thr Trp Asn Ser Gly Ala Leu Thr Ser Gly

245 250 255

Val His Thr Phe Pro Ser Val Leu Gln Pro Ser Gly Leu Tyr Ser Leu

260 265 270

Ser Ser Met Val Thr Val Pro Ala Ser Ser Leu Ser Ser Lys Ser Tyr

275 280 285

Thr Cys Asn Val Asn His Pro Ala Thr Thr Thr Lys Val Asp Lys Arg

290 295 300

Val Gly Thr Lys Thr Lys Pro Pro Cys Pro Ile Cys Pro Gly Cys Glu

305 310 315 320

Val Ala Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr

325 330 335

Leu Met Ile Ser Gln Thr Pro Glu Val Thr Cys Val Val Val Asp Val

340 345 350

Ser Lys Glu His Ala Glu Val Gln Phe Ser Trp Tyr Val Asp Gly Val

355 360 365

Glu Val His Thr Ala Glu Thr Arg Pro Lys Glu Glu Gln Phe Asn Ser

370 375 380

Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gln His Gln Asp Trp Leu

385 390 395 400

Lys Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Val Asp Leu Pro Ala

405 410 415

Pro Ile Thr Arg Thr Ile Ser Lys Ala Ile Gly Gln Ser Arg Glu Pro

420 425 430

Gln Val Tyr Thr Leu Pro Pro Pro Ala Glu Glu Leu Ser Arg Ser Lys

435 440 445

Val Thr Val Thr Cys Leu Val Ile Gly Phe Tyr Pro Pro Asp Ile His

450 455 460

Val Glu Trp Lys Ser Asn Gly Gln Pro Glu Pro Glu Gly Asn Tyr Arg

465 470 475 480

Thr Thr Pro Pro Gln Gln Asp Val Asp Gly Thr Phe Phe Leu Tyr Ser

485 490 495

Lys Leu Ala Val Asp Lys Ala Arg Trp Asp His Gly Glu Thr Phe Glu

500 505 510

Cys Ala Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

515 520 525

Ile Ser Lys Thr Gln Gly Lys

530 535

<210> 3

<211> 576

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

aacggcatct tcaacaccag gctgtccagg accttcggct acaccatcaa gcggaccacc 60

gtgcggacac cttcttgggc cgtggacatg atgcgcttca acatcaacgc cttcctgccc 120

ccaggcggag gctctaatcc tagaagcgtg cctttcgagt actaccggat ccggaaggtg 180

aaggtggagt tttggccttg cagccccatc acccagggag atagaggagt gggaagtagc 240

gccgtgatcc tggacgacaa cttcgtgacc aaggccacag ccctgaccta cgatccttac 300

gtgaactaca gcagccggca caccatcacc cagcccttca gctaccacag ccgctacttc 360

acccctaaac ccgtgctgga cagcaccatc gactacttcc agcccaacaa caagcggaac 420

cagctctggc tgagactgca gacagcaggc aacgtggatc acgtgggact gggaacagcc 480

ttcgagaaca gcatctacga ccagggctac aacatccgcg tgaccatgta cgtgcagttc 540

cgggagttca acctgaagga cccccccctg aatcca 576

<210> 4

<211> 192

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

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

1 5 10 15

Lys Arg Thr Thr Val Arg Thr Pro Ser Trp Ala Val Asp Met Met Arg

20 25 30

Phe Asn Ile Asn Ala Phe Leu Pro Pro Gly Gly Gly Ser Asn Pro Arg

35 40 45

Ser Val Pro Phe Glu Tyr Tyr Arg Ile Arg Lys Val Lys Val Glu Phe

50 55 60

Trp Pro Cys Ser Pro Ile Thr Gln Gly Asp Arg Gly Val Gly Ser Ser

65 70 75 80

Ala Val Ile Leu Asp Asp Asn Phe Val Thr Lys Ala Thr Ala Leu Thr

85 90 95

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

100 105 110

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

115 120 125

Thr Ile Asp Tyr Phe Gln Pro Asn Asn Lys Arg Asn Gln Leu Trp Leu

130 135 140

Arg Leu Gln Thr Ala Gly Asn Val Asp His Val Gly Leu Gly Thr Ala

145 150 155 160

Phe Glu Asn Ser Ile Tyr Asp Gln Gly Tyr Asn Ile Arg Val Thr Met

165 170 175

Tyr Val Gln Phe Arg Glu Phe Asn Leu Lys Asp Pro Pro Leu Asn Pro

180 185 190

<210> 5

<211> 45

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

ggaagcggag gaggaagcgg aggaggagga agcggaggag gaagc 45

<210> 6

<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser

1 5 10 15

<210> 7

<211> 984

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

gcccctaaaa cagctcctag cgtgtatcct ctggcccctt gtggcagaga tacaagcggc 60

cctaacgtgg ctctgggctg tctggcctct agctacttcc cagagcccgt gaccatgact 120

tggaacagcg gagccctgac aagcggagtg cacacctttc ctagcgtgct gcagcctagc 180

ggactgtata gcctgagcag catggtgaca gtgccagcct ctagcctgag cagcaagagc 240

tacacttgca acgtgaacca ccccgccacc acaacaaagg tggacaagcg cgtgggcacc 300

aagacaaagc ctccttgccc catttgtcca ggttgcgagg tggccggccc tagcgtgttt 360

atcttccctc ctaagcccaa ggacaccctg atgatcagcc agaccccaga agtgacttgc 420

gtggtggtgg acgtgtctaa ggagcacgcc gaggtgcagt tcagttggta cgtggacggc 480

gtggaagtgc acacagccga gacaagaccc aaggaggagc agttcaacag cacctaccgc 540

gtggtgtccg tgctgcctat ccagcaccag gattggctga agggcaagga gttcaagtgc 600

aaggtcaaca acgtggacct gccagcccct atcaccagaa caatcagcaa ggccatcgga 660

cagagcaggg agcctcaggt gtacacactg cctcctccag cagaggagct gagcaggagc 720

aaagtgaccg tgacctgcct ggtcatcggc ttttaccccc cagacatcca cgtcgagtgg 780

aagagtaacg ggcagccaga gccagagggc aactacagaa ccacacctcc tcagcaggac 840

gtggacggaa cattcttcct gtacagcaag ctggccgtgg acaaagctcg ctgggatcac 900

ggggagacct tcgaatgcgc agtgatgcac gaggccctgc ataaccacta cacccagaag 960

agcatcagca agacccaggg caag 984

<210> 8

<211> 328

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 8

Ala Pro Lys Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Cys Gly Arg

1 5 10 15

Asp Thr Ser Gly Pro Asn Val Ala Leu Gly Cys Leu Ala Ser Ser Tyr

20 25 30

Phe Pro Glu Pro Val Thr Met Thr Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ser Val Leu Gln Pro Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Met Val Thr Val Pro Ala Ser Ser Leu Ser Ser Lys Ser

65 70 75 80

Tyr Thr Cys Asn Val Asn His Pro Ala Thr Thr Thr Lys Val Asp Lys

85 90 95

Arg Val Gly Thr Lys Thr Lys Pro Pro Cys Pro Ile Cys Pro Gly Cys

100 105 110

Glu Val Ala Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Gln Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser Lys Glu His Ala Glu Val Gln Phe Ser Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Thr Ala Glu Thr Arg Pro Lys Glu Glu Gln Phe Asn

165 170 175

Ser Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gln His Gln Asp Trp

180 185 190

Leu Lys Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Val Asp Leu Pro

195 200 205

Ala Pro Ile Thr Arg Thr Ile Ser Lys Ala Ile Gly Gln Ser Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Pro Ala Glu Glu Leu Ser Arg Ser

225 230 235 240

Lys Val Thr Val Thr Cys Leu Val Ile Gly Phe Tyr Pro Pro Asp Ile

245 250 255

His Val Glu Trp Lys Ser Asn Gly Gln Pro Glu Pro Glu Gly Asn Tyr

260 265 270

Arg Thr Thr Pro Pro Gln Gln Asp Val Asp Gly Thr Phe Phe Leu Tyr

275 280 285

Ser Lys Leu Ala Val Asp Lys Ala Arg Trp Asp His Gly Glu Thr Phe

290 295 300

Glu Cys Ala Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

305 310 315 320

Ser Ile Ser Lys Thr Gln Gly Lys

325

<210> 9

<211> 54

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

ctcactatag ggagacccaa gctggctagc ccgccgccac catgggctgg agct 54

<210> 10

<211> 54

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

actagtggat ccgagctcgg taccaagctt ttatcacttg ccctgggtct tgct 54

<210> 11

<211> 13

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

gccgccacca tgg 13

<210> 12

<211> 57

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

atgggctgga gctgcatcat cctgttcctc gtggccacag ccacaggagt gcactct 57

<210> 13

<211> 1691

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

gctagcccgc cgccaccatg ggctggagct gcatcatcct gttcctcgtg gccacagcca 60

caggagtgca ctctaacggc atcttcaaca ccaggctgtc caggaccttc ggctacacca 120

tcaagcggac caccgtgcgg acaccttctt gggccgtgga catgatgcgc ttcaacatca 180

acgccttcct gcccccaggc ggaggctcta atcctagaag cgtgcctttc gagtactacc 240

ggatccggaa ggtgaaggtg gagttttggc cttgcagccc catcacccag ggagatagag 300

gagtgggaag tagcgccgtg atcctggacg acaacttcgt gaccaaggcc acagccctga 360

cctacgatcc ttacgtgaac tacagcagcc ggcacaccat cacccagccc ttcagctacc 420

acagccgcta cttcacccct aaacccgtgc tggacagcac catcgactac ttccagccca 480

acaacaagcg gaaccagctc tggctgagac tgcagacagc aggcaacgtg gatcacgtgg 540

gactgggaac agccttcgag aacagcatct acgaccaggg ctacaacatc cgcgtgacca 600

tgtacgtgca gttccgggag ttcaacctga aggacccccc cctgaatcca ggaagcggag 660

gaggaagcgg aggaggagga agcggaggag gaagcgcccc taaaacagct cctagcgtgt 720

atcctctggc cccttgtggc agagatacaa gcggccctaa cgtggctctg ggctgtctgg 780

cctctagcta cttcccagag cccgtgacca tgacttggaa cagcggagcc ctgacaagcg 840

gagtgcacac ctttcctagc gtgctgcagc ctagcggact gtatagcctg agcagcatgg 900

tgacagtgcc agcctctagc ctgagcagca agagctacac ttgcaacgtg aaccaccccg 960

ccaccacaac aaaggtggac aagcgcgtgg gcaccaagac aaagcctcct tgccccattt 1020

gtccaggttg cgaggtggcc ggccctagcg tgtttatctt ccctcctaag cccaaggaca 1080

ccctgatgat cagccagacc ccagaagtga cttgcgtggt ggtggacgtg tctaaggagc 1140

acgccgaggt gcagttcagt tggtacgtgg acggcgtgga agtgcacaca gccgagacaa 1200

gacccaagga ggagcagttc aacagcacct accgcgtggt gtccgtgctg cctatccagc 1260

accaggattg gctgaagggc aaggagttca agtgcaaggt caacaacgtg gacctgccag 1320

cccctatcac cagaacaatc agcaaggcca tcggacagag cagggagcct caggtgtaca 1380

cactgcctcc tccagcagag gagctgagca ggagcaaagt gaccgtgacc tgcctggtca 1440

tcggctttta ccccccagac atccacgtcg agtggaagag taacgggcag ccagagccag 1500

agggcaacta cagaaccaca cctcctcagc aggacgtgga cggaacattc ttcctgtaca 1560

gcaagctggc cgtggacaaa gctcgctggg atcacgggga gaccttcgaa tgcgcagtga 1620

tgcacgaggc cctgcataac cactacaccc agaagagcat cagcaagacc cagggcaagt 1680

gataaaagct t 1691

Claims

1. A porcine circovirus 2b type Capid-Fc fusion protein is characterized in that the amino acid sequence is shown as SEQ ID No.2.

2. A gene for encoding and expressing porcine circovirus type 2b Capid-Fc fusion protein is characterized in that a base sequence table is shown as SEQ ID No.1.

3. The recombinant vector for expressing recombinant porcine circovirus type 2b Capid-Fc fusion protein is characterized by comprising a gene sequence shown in SEQ ID No.13 for encoding the recombinant porcine circovirus type 2b Capid-Fc fusion protein.

4. The method for constructing a gene encoding recombinant porcine circovirus type 2b Capid-Fc fusion protein according to claim 3, wherein the Capsid protein of the PCV 2b 41513 virus strain of GenBank accession number ALD 62452.1 with failed immunization of the existing porcine circovirus type 2 vaccine is modified, 41 amino acids at the N end of the Capsid protein are removed, a polypeptide linker is connected at the C end, a porcine IgG Fc fragment is fused, a Kozak sequence and a human IgG 2H heavy chain signal peptide are sequentially added at the N end, and then whole-gene codon optimization is performed to obtain the expression gene of the porcine circovirus type 2b Capid-Fc fusion protein.

5. A recombinant vector for expressing a porcine circovirus type 2b hepsd-Fc fusion protein according to claim 3, wherein the plasmid used for the preparation of the recombinant vector is pcdna3.1 (+) plasmid.

6. The method for preparing the porcine circovirus type 2b Capid-Fc fusion protein according to claim 1, comprising the following steps:

(1) Constructing an expression gene of the porcine circovirus type 2b Capsd-Fc fusion protein according to the method of claim 4;

(2) Construction of pCDNA3.1 (+) -PCV 2b Capid-Fc recombinant plasmid containing porcine circovirus 2 b-Capid-Fc fusion protein expression gene;

(3) The recombinant plasmid was transfected into E.coli DH 5. Alpha: transfecting the pCDNA3.1 (+) -PCV 2b Capid-Fc recombinant plasmid obtained in the step (2) into escherichia coli DH5 alpha to obtain escherichia coli DH5 alpha containing the pCDNA3.1 (+) -PCV 2b Capid-Fc recombinant plasmid;

(4) Amplifying by recombinant escherichia coli DH5 alpha to obtain transfection-grade pCDNA3.1 (+) -PCV 2b Capsd-Fc recombinant plasmid;

(5) The transfection-grade pCDNA3.1 (+) -PCV 2b Capid-Fc recombinant plasmid is transfected into a mammalian HEK293F, and then is subjected to suspension culture in sequence, and is separated and purified to obtain a target porcine circovirus type 2b Capid-Fc fusion protein product.

7. The porcine circovirus type 2b hepsd-Fc fusion protein of claim 1 for use in the preparation of a porcine circovirus type 2b hepsd-Fc fusion protein subunit vaccine against porcine circovirus type 2b infection.