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

文档序号：431193 发布日期：2021-12-24 浏览：6次中文

阅读说明：本技术 猪圆环病毒2b型Capsid-Fc融合蛋白及其制备方法、基因及构建方法和应用 (Porcine circovirus type 2b Capsid-Fc fusion protein, and preparation method, gene, construction method and application thereof ) 是由郭美锦罗清平周云飞于 2020-06-24 设计创作，主要内容包括：本发明公开一种猪圆环病毒2b型Capsid-Fc融合蛋白及其制备方法、基因及构建方法和应用,通过对现有PCV 2b 41513病毒毒株的衣壳蛋白改造去除N端41个氨基酸,C端连接多肽linker并融合猪IgG Fc片段,得到新型猪圆环病毒2b型Capsid-Fc融合蛋白基因的N端依次添加Kozak序列和人IgG2H重链信号肽后进行全基因密码子优化,得到编码猪圆环病毒2b型Capsid-Fc融合蛋白的表达基因,依次经重组质粒构建、转染、表达、分离纯化等得猪圆环病毒2b型Capsid-Fc融合蛋白,其作为活性成分所得猪圆环病毒2b型Capsid-Fc融合蛋白亚单位疫苗能够很好刺激小鼠机体产生高浓度的抗体。(The invention discloses a porcine circovirus type 2b Capsid-Fc fusion protein, a preparation method, a gene, a construction method and application thereof, removing 41 amino acids at the N end by modifying the Capsid protein of the existing PCV2b 41513 virus strain, connecting a polypeptide linker at the C end, fusing a porcine IgG Fc fragment to obtain a novel porcine circovirus type 2b Capsid-Fc fusion protein gene, sequentially adding a Kozak sequence and a human IgG2H heavy chain signal peptide at the N end, then carrying out whole-gene codon optimization to obtain an expression gene for encoding the porcine circovirus type 2b Capsid-Fc fusion protein, sequentially carrying out recombinant plasmid construction, transfection, expression, separation and purification and the like to obtain the porcine circovirus type 2b Capsid-Fc fusion protein, the porcine circovirus type 2b Capsid-Fc fusion protein subunit vaccine obtained by using the porcine circovirus type 2b as an active ingredient can well stimulate a mouse body to generate high-concentration antibodies.)

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

2. The porcine circovirus type 2b Capsid-Fc fusion protein according to claim 1, wherein the amino acid sequence SEQ ID No.2 is formed by sequentially joining end to end the amino acid sequence SEQ ID No.4 encoding the Capsid segment of PCV2, the amino acid sequence SEQ ID No.6 encoding the linker segment and the amino acid sequence SEQ ID No.8 encoding the Fc segment.

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

4. The method for constructing the gene expressing the porcine circovirus type 2b Capsid-Fc fusion protein as claimed in claim 3, which is characterized in that Capsid protein of PCV2b 41513 virus strain failed in the immunity of the existing porcine circovirus type2 vaccine is modified, 41 amino acids at the N end of the Capsid protein are removed, a polypeptide linker is connected to the C end, a porcine IgG Fc fragment is fused, a Kozak sequence and a human IgG2H heavy chain signal peptide are sequentially added to the N end, and then the whole gene codon optimization is carried out to obtain the expression gene of the porcine circovirus type 2b Capsid-Fc fusion protein.

5. A recombinant vector for expressing a recombinant porcine circovirus type 2b Capsid-Fc fusion protein is characterized by comprising a base sequence table shown as SEQ ID No. 13.

6. The recombinant vector for expressing the porcine circovirus type 2b Capsid-Fc fusion protein of claim 5, wherein the plasmid used for the preparation of the recombinant vector is a pet plasmid or a pcdna3.1(+) plasmid.

7. The method for preparing the porcine circovirus type 2b Capsid-Fc fusion protein according to claim 1 or 2, which comprises the following steps:

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

(2) constructing pEM-PCV2b Capsid-Fc recombinant plasmid or pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmid containing porcine circovirus type 2b Capsid-Fc fusion protein expression gene;

(3) transfection of the recombinant plasmid into E.coli DH5 α: transfecting the pEM-PCV2b Capsid-Fc recombinant plasmid or the pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmid obtained in the step (2) into Escherichia coli DH5 alpha to obtain Escherichia coli DH5 alpha containing the pEM-PCV2b Capsid-Fc recombinant plasmid or Escherichia coli DH5 alpha containing the pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmid;

(4) respectively amplifying by recombinant escherichia coli DH5 alpha to obtain a transfection-grade pEM-PCV2b Capsid-Fc recombinant plasmid or a transfection-grade pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmid;

(5) and transfecting the transfection-grade pEM-PCV2b Capsid-Fc recombinant plasmid or the transfection-grade pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmid to a mammalian cell HEK293F, sequentially carrying out suspension culture, and then separating and purifying to obtain a target porcine circovirus 2b type Capsid-Fc fusion protein product.

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

Technical Field

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

Background

Porcine Circovirus type2 (PCV2 virus) belongs to the family of circoviridae, the genome of which is single-stranded circular DNA, virions are free of envelope membrane, and icosahedron is formed by assembling 60 protein monomers, and the virus is one of the smallest viruses known at present. PCV2 genome is 1767bp or 1768bp long, and two main open reading frames ORF1 and ORF2 of genome encode Cap protein and Rep protein, respectively. The Pep protein is an essential protein for the replication of PCV2 virus. The Cap protein is a capsid protein forming the PCV2 virus coat, has the capacity of binding host cell receptors, is a main immune antigen, and is often used for preparing PCV2 virus-specific vaccines and target proteins for serological diagnosis. PCV2 viral capsid protein is icosahedral symmetric.

PCV2 virus was first discovered in Canada in 1998 and was identified as an important pathogen responsible for a variety of diseases such as sow dysgenesis, granulomatous enteritis, congenital tremor interstitial pneumonia, postweaning multisystemic wasting syndrome, porcine dermatitis and nephrotic syndrome.

At present, a plurality of PCV2 virus vaccines are developed in the market, and inactivated vaccines, attenuated vaccines and recombinant vaccines are mainly developed by porcine circovirus type 2a virus (hereinafter referred to as PCV 2a virus) strains. There are also some inactivated vaccines developed as strains of porcine circovirus type 2b virus (hereinafter referred to as PCV2b virus).

These PCV2b virus vaccines play an important role in controlling PCV2b virus infection, but the immune effect still needs to be further improved. And because the PCV2b virus variant has more genotypes, the cases of immune failure are frequently seen, and even in a pig farm inoculated with PCV2b virus vaccine, the problems of PCV2b virus infection epidemic outbreak and the like still exist.

In summary, the conventional PCV2b virus vaccine also has the technical problems of low antibody titer, insufficient protection on immune objects and the like. Therefore, the existing PCV2b virus vaccine needs to be updated and upgraded, the antibody titer is improved, better immunogenicity is obtained, the immune coverage rate is expanded, and the protection of immune objects is improved.

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

5. CN201480045584.2 porcine circovirus type2 (PCV2) 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 Capsid-Fc fusion protein subunit vaccine which has the ability to produce high antibody titer and at the same time has the ability to respond to humoral and cellular immunity enhancement of an immune subject, in order to solve the technical problems of low antibody titer produced by the existing PCV2b virus vaccine, insufficient protection of the immune subject, and the like.

The invention also aims to provide a porcine circovirus type 2b Capsid-Fc fusion protein used in the porcine circovirus type 2b Capsid-Fc fusion protein subunit vaccine and a preparation method thereof.

The invention also aims to provide an expression gene of the porcine circovirus type 2b Capsid-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 type2 vaccine, engineering and removing 41 amino acids at the N end of the Capsid protein, connecting a polypeptide linker at the C end, and fusing a porcine IgG Fc fragment to generate a novel porcine circovirus type 2b Capsid-Fc fusion protein, namely PCV2b Capsid-Fc fusion protein;

and (2) carrying out whole-gene codon optimization on the DNA for coding the novel porcine circovirus type 2b Capsid-Fc fusion protein to obtain an expression gene of the porcine circovirus type 2b Capsid-Fc fusion protein, namely the expression gene of the porcine circovirus type 2b Capsid-Fc fusion protein, wherein the gene sequence is shown in SEQ ID No.1, and then inserting the gene into a pEM expression plasmid through whole-gene synthesis and an EcoRI/HindIII enzyme double-enzyme cutting method to obtain the pEM-PCV2b Capsid-Fc recombinant plasmid. In addition, PCV2b Capsid-Fc fusion protein gene is inserted into pCDNA3.1(+) plasmid by primer amplification and NheI/HindIII enzyme double-enzyme cutting method to obtain pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmid; transfecting the recombinant plasmid to escherichia coli DH5 alpha to obtain a recombinant escherichia coli DH5 alpha containing pEM-PCV2b Capsid-Fc recombinant plasmid or pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmid; respectively carrying out amplification through recombinant escherichia coli DH5 alpha to obtain transfection-level recombinant plasmids;

and transfecting the transfection-grade pEM-PCV2b Capsid-Fc recombinant plasmid or the transfection-grade pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmid to a mammalian cell HEK293F, sequentially carrying out suspension culture, and then separating and purifying to obtain a target porcine circovirus 2b type Capsid-Fc fusion protein product.

Note: the Capsid in the PCV2b Capsid-Fc fusion protein is the PCV2b Capsid protein described as N-terminal cut of 41 amino acids.

The PCV2b Capsid-Fc fusion protein can form nanoparticles with the average size of about 41nm when observed by a transmission electron microscope. The results of experiments on BLAB/c mouse immunization show that the composition has good effect of stimulating the body of the mouse to generate humoral immunity and cellular immune response.

The technical scheme of the invention is as follows:

a porcine circovirus type 2b virus Capsid-Fc fusion protein is PCV2b Capsid-Fc fusion protein, the PCV2b Capsid-Fc fusion protein is expressed in a host, preferably the host is mammalian cell HEK293F cell for expression, pEM plasmid or pcDNA3.1(+) plasmid is used as a vector for constructing a recombinant vector for encoding the PCV2b Capsid-Fc fusion protein, in the embodiment of the invention, pEM-PCV2b Capsid-Fc fusion protein recombinant plasmid is used for transfecting mammalian cell HEK293F for expression, and pcDNA3.1(+) plasmid is used as a vector for constructing the recombinant vector for encoding the PCV2b Capsid-Fc fusion protein for transfecting mammalian cell HEK293F for expression, meanwhile, the host is not limited to mammalian cells, and can also adopt eukaryotic Pichia pastoris for expression, preferably adopts Pichia pastoris GSl15 for expression, in this case, the preferred vector is pIC9K, and in the examples of the present invention, only recombinant vectors are transfected into HEK293F cells to express PCV2b Capsid-Fc fusion proteins in HEK293F cells, but the application of expression using eukaryotic pichia pastoris as host bacteria is not limited.

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

(1) synthesis of expression gene sequence of PCV2b Capsid-Fc fusion protein

Expression gene synthesis of novel PCV2b Capsid-Fc fusion protein

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

based on that the gene of PCV2 capsid protein is coded by a Porcinecroviridype 2-BstrainnDSU41513 strain, namely the sequence ID is GenBank accession number rALD62452.1, 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 which is shown in SEQ ID No. 3;

the Fc region gene of the pig IgG heavy chain is used, the sequence ID is the Fc segment sequence of Gen Bank accession number rAAD38418.1, see SEQ ID No. 7;

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

carrying out codon optimization on the three-part segment sequence according to the codon preference of a mammalian cell to obtain a novel PCV2 bPasid-Fc fusion protein gene; the sequence is shown in SEQ ID No. 1;

② synthesis of PCV2b Capsid-Fc gene

Enzyme cutting sites EcoRI-HindIII are contained at the front end and the rear end of the novel PCV2b Capsid-Fc fusion protein obtained in the step I, a Kozak sequence and a human IgG2H heavy chain signal peptide sequence are inserted, the sequences are 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 Capsid-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 human IgG2H heavy chain signal peptide sequence is shown in SEQ ID No. 12;

(2) construction of pEM-PCV2b Capsid-Fc recombinant plasmid containing PCV2b Capsid-Fc Gene

Designing enzyme cutting sites EcoRI and HindIII at the two ends of the N end and the C end of the gene of the PCV2b Capsid-Fc fusion protein, synthesizing the sequences in an artificial gene synthesis mode, and inserting the gene of the PCV2b Capsid-Fc fusion protein into an expression vector pEM plasmid by a double-enzyme cutting method to obtain a pEM-PCV2b Capsid-Fc recombinant plasmid;

(3) construction of pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmid containing the Gene of PCV2b Capsid-Fc

Designing enzyme cutting 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 for synthesizing PCV2b Capsid-Fc, such as SEQ ID No.1, through the primers PcfF and PcfR to obtain an expression fragment containing the enzyme cutting sites NheI and HindIII, carrying out NheI/HindIII double enzyme cutting on the PCR amplification fragment, and then connecting the PCR amplification fragment to a pCDNA3.1 plasmid (+) expression vector subjected to the same NheI/HindIII double enzyme cutting treatment to obtain a pCDNA3.1(+) -2 b Capsid-Fc fusion protein recombinant plasmid;

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

sucking 80-100ng of the pEM-PCV2b Capsid-Fc recombinant plasmid obtained in the step (2) or 80-100ng of the pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmid obtained in the step (3) into a prepared DH5 alpha competent cell, then adding the mixture into an LB liquid culture medium, placing the mixture into a shaking table, controlling the temperature to be 37 ℃ and the rotating speed to be 200rpm, shaking and culturing for about 30min, and then separating out a DH5 alpha competent cell to obtain a recombinant Escherichia coli DH5 alpha containing the pEM-PCV2b Capsid-Fc recombinant plasmid or the pCDNA3.1(+) -PCV 2bCapsid-Fc recombinant plasmid;

(5) extraction of transfection-grade plasmids

And (3) selecting a single clone in a fresh culture plate from the Escherichia coli DH5 alpha containing the pEM-PCV2b Capsid-Fc recombinant plasmid or the pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmid obtained in the step (4) to culture in a 2-5mLLB culture medium at the temperature of 37 ℃ and the rotation speed of 200rpm for 8 hours to obtain a seed solution. Then inoculating the seed liquid into 200mLLB culture medium according to the proportion of 1/500 by volume ratio, continuously controlling the temperature at 37 ℃ and culturing for 16h at the rotating speed of 200rpm to obtain bacterial liquid containing the recombinant plasmid, then centrifuging the bacterial liquid at the rotating speed of 4000r/min for 10min to remove supernatant, and adopting E.Z.N.A. of omega Biotek company to obtain the precipitate.Extracting transfection-grade plasmids by using an Endo-Free Plasmid DNA Maxi Kit Plasmid large-extraction Kit D6926-01 according to the method of the Kit to finally obtain transfection-grade pEM-PCV2b Capsid-Fc recombinant plasmids or transfection-grade pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmids;

(6) mammalian cell expression of PCV2b Capsid-Fc fusion protein

Recovery and amplification culture of HEK293 cell

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 cryopreserved tube filled with HEK293F cells from the liquid nitrogen tank, immediately putting the cryopreserved cells into a water bath kettle at 37 ℃, slightly shaking to rapidly melt the cryopreserved cells within 1min, taking out the cryopreserved cells, sterilizing the outer wall of the cryopreserved tube filled with HEK293F cells by using 75% ethanol, putting the cryopreserved cells into a biological safety cabinet, transferring the cryopreserved cells into a 15mL centrifugal tube filled with 10mL LUNIon-293 serum-free culture medium, centrifuging the cells at the controlled rotation speed of 800rpm for 5min, removing the supernatant after centrifugation, taking out fresh cells, and taking out the cellsResuspending HEK293F cells in a centrifugal tube by Union-293 serum-free medium, transferring to a culture flask, adding Union-293 serum-free medium, shaking to disperse the cells uniformly, counting the cells, detecting the activity, and controlling the density of the viable cells at 6-8 × 10⁵Measuring cell viability per mL, recovering when cell viability is lower than 80%, and placing cell culture solution in CO when cell viability is higher than 80%₂CO at a concentration of 5% by volume₂Culturing in an incubator at 37 deg.C and 110rpm for 2-3 days, removing 10mL of cell culture medium, and adding 10mL of fresh culture medium until cell activity is greater than 90% to obtain culture solution containing revived HEK293 cells;

b: placing the culture solution containing the resuscitated HEK293 cells obtained in the step A in CO₂CO at a concentration of 5% by volume₂Culturing in an incubator at 37 deg.C and 110rpm for 2-3 days until cell concentration reaches 2-3 × 10⁶And (3) completing the first cell amplification culture of the HEK293 cells in the culture solution per mL of HEK293 cells:

30mL of fresh medium was added to the above-mentioned HEK293 cell culture solution obtained after 15mL of the first cell expansion culture, and then CO was added thereto₂CO at a concentration of 5% by volume₂Continuously culturing in an incubator at 37 deg.C and 110rpm, detecting HEK293 cell concentration and cell activity every day, and determining cell concentration to 2-3 × 10⁶Performing secondary cell amplification culture on the HEK293 cells by using the HEK293 cells/mL culture solution, wherein the culture volume reaches about 45 mL;

90mL of fresh medium was added to the above-mentioned HEK293 cell culture solution obtained after 45mL of the first cell expansion culture, followed by CO addition₂CO at a concentration of 5% by volume₂Continuously culturing in an incubator at 37 deg.C and 110rpm, detecting HEK293 cell concentration and cell activity every day, and determining cell concentration to 2-3 × 10⁶Completing the third cell amplification culture of HEK293 cells by using HEK293 cells/mL culture solution to obtain HEK-293F cell culture solution subjected to the third cell amplification culture, wherein the culture volume isTo about 135 mL;

preparing Union-293 serum-free culture solution containing transfection-grade pEM-PCV2b Capsid-Fc recombinant plasmid:

adding the transfection-grade pEM-PCV2b Capsid-Fc recombinant plasmid obtained in the step (5) into a Union-293 serum-free culture medium to obtain a Union-293 serum-free culture solution containing the transfection-grade pEM-PCV2b Capsid-Fc recombinant plasmid with 20 mu g/ml;

preparation of Union-293 serum-free culture solution containing polyethyleneimine

Adding a transfection reagent polyethyleneimine into a Union-293 serum-free culture medium to obtain 80 microgram/ml Union-293 serum-free culture solution containing polyethyleneimine;

mixing the Union-293 serum-free culture solution containing polyethyleneimine and the Union-293 serum-free culture solution containing transfection-grade pEM-PCV2b Capsid-Fc recombinant plasmid according to the volume ratio of 1:1, and standing at room temperature for 20-30min to obtain a Union-293 serum-free culture solution of PEI/DNA;

fifthly, transfection of recombinant plasmid containing transfection-grade pEM-PCV2b Capsid-Fc to HEK293 cell

Adding the Union-293 serum-free culture solution of the PEI/DNA obtained in the step (r) into the HEK-293F cell culture solution obtained in the step (r) after the three times of amplification culture, and then adding CO₂CO at a concentration of 5% by volume₂Culturing in an incubator at 37 deg.C and 110rpm for 4-6 days, centrifuging the obtained cell culture solution at 1000rpm for 10min, and collecting precipitate which is HEK293F cell containing PCV2b Capsid-Fc fusion protein;

the dosage 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 as follows: the volume ratio of HEK-293F cell culture solution after three times of amplification culture is 1: 10, calculating the proportion;

(7) isolation of PCV2b Capsid-Fc fusion protein

Resuspending HEK293F cells containing PCV2b Capsid-Fc fusion protein obtained in the step (6) by using an ultrasonic lysis buffer solution, then using an ultrasonic disruptor of a type Q125 of Qsonica LLC company, controlling the output of amplitude intensity to be 34%, working for 3 seconds, stopping for 7 seconds, carrying out ultrasonic lysis on the cells for 30min of total working time, then controlling the temperature to be 4 ℃ and centrifuging at the rotating speed of 12,000rpm for 20min, and collecting supernatant;

the ultrasonic lysis buffer solution contains 50mM Tris, 300mM NaCl, 20mM imidazole with pH8.0, 10g TritonX-100, 1mM phenylmethylsulfonyl fluoride (PMSF) and the balance of water in terms of per liter;

the dosage of HEK293F cells containing PCV2b Capsid-Fc fusion protein and ultrasonic lysis buffer is calculated according to the proportion of the wet weight of HEK293F cells containing PCV2b Capsid-Fc fusion protein to the ultrasonic lysis buffer of 1g:10 mL;

the wet weight of the HEK293F cells containing the PCV2b Capsid-Fc fusion protein is the actual mass of the precipitate obtained by the centrifugation in the fifth step in the step (5);

(8) purification of PCV2b Capsid-Fc fusion protein

And (3) loading the supernatant collected after centrifugation in the step (7) onto a Protein A sepharose chromatography column (Detai Biotechnology (Nanjing) Co., Ltd.), sequentially carrying out equilibrium chromatography column, loading, impurity washing, elution, collection, sample neutralization and filtration through a 0.22 micron filter according to the use requirement of the Protein A sepharose chromatography column to obtain a filtrate containing PCV2b Capsid-Fc fusion Protein.

The resulting filtrate containing PCV2b Capsid-Fc fusion protein was stored at-70 ℃ for subsequent experiments.

The application of the PCV2b Capsid-Fc fusion protein in preparing a PCV2b Capsid-Fc fusion protein subunit vaccine for resisting porcine circovirus type 2b infection, wherein the PCV2b Capsid-Fc fusion protein subunit vaccine consists of an active ingredient PCV2b Capsid-Fc fusion protein and an adjuvant according to mass-to-volume ratio, wherein the PCV2b Capsid-Fc fusion protein: adjuvant 3.3 mg: 100 mL; wherein the adjuvant is complete Freund's adjuvant or incomplete Freund's adjuvant;

the active ingredient is PCV2b Capsid-Fc fusion protein which is self-assembled into virus-like nanoparticles.

The preparation method of the PCV2b Capsid-Fc fusion protein subunit vaccine comprises the following specific steps:

according to PCV2b Capsid-Fc fusion protein subunit vaccine PCV2b Capsid-Fc fusion protein: adjuvant 3.3 mg: the preparation method comprises the following steps of preparing 100mL, namely diluting the filtrate obtained after filtering the filtrate by using a 0.22 micron filter by using a 1xPBS solution with the pH value of 7.4 for one time, and then mixing the diluted filtrate with a Freund complete adjuvant preheated to 37 ℃ or a Freund incomplete adjuvant preheated to 37 ℃ in a shaking way to realize the emulsification of PCV2b Capsid-Fc fusion protein until a stable emulsification system is formed, thus obtaining the PCV2 Capsid-Fc fusion protein subunit vaccine.

In the PCV2b Capsid-Fc fusion protein subunit vaccine, the active ingredient PCV2b Capsid-Fc fusion protein is self-assembled into virus-like nanoparticles with the particle size of about 41nm by the detection of a scanning electron microscope.

The porcine circovirus type 2b Capsid-Fc fusion protein can also be applied to preparation of antibody drug 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 Capsid-Fc fusion protein for coding PCV2b Capsid-Fc fusion protein, and a porcine circovirus type 2b Capsid-Fc fusion protein subunit vaccine obtained by using the protein as an active ingredient can well stimulate a mouse body to generate high-concentration antibodies so as to improve the immunity of cells and body fluid. Particularly, the subunit vaccine taking PCV2b Capsid-Fc fusion protein as an active substance shows that the antibody level is slightly increased on the 12 th day after primary immunization through a BLAB/c mouse immunization experiment result, a specific antibody aiming at PCV2 Capsid can be generated at a high level after secondary boosting immunization, the antibody concentration exceeds 500ng/mL, the antibody concentration is improved by more than 1 time compared with that of a BLAB/c mouse group immunized by commercial subunit vaccine, and meanwhile, the subunit vaccine taking PCV2b Capsid-Fc fusion protein as an active substance can well stimulate a mouse body to generate humoral immunity and cellular immune response.

Drawings

FIG. 1: the PCR identification result of the pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmid obtained in example 2 is shown schematically; wherein M, DNA marker 1kb DNA; PCV2b Capsid-Fc PCR product;

FIG. 2: schematic representation of the nucleic acid fragment of pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmid obtained in example 2 after NheI/HindIII enzyme double cleavage of PCV2b Capsid-Fc, wherein M.DNA Marker; 1. extracting recombinant plasmids; NheI/HindIII enzyme double digestion recombinant plasmid pCDNA3.1(+) -PCV2b Capsid-Fc;

FIG. 3: schematic representation of the nucleic acid fragment obtained by double restriction of PCV2b Capsid-Fc of the pEM-PCV2b Capsid-Fc recombinant plasmid obtained in example 2 with NcoI/ApaI, wherein M.DNA Marker; 1. extracting recombinant plasmids; 2. double-enzyme digestion of pEM-PCV2b Capsid-Fc recombinant plasmid by NcoI/ApaI enzyme;

FIG. 4: a schematic structural diagram of the PCV2b Capsid-Fc fusion protein obtained in example 4;

FIG. 5: 3D predictive structural drawing of PCV2b Capsule-Fc fusion protein obtained in example 4;

FIG. 6: SDS-PAGE and Western Blot analysis of the PCV2b Capsid-Fc fusion protein obtained in example 4;

FIG. 7: the transmission electron microscope of the inactivated virus selected in the application example 1 is observed under the multiplying power of 4K-300K (200 nm);

FIG. 8: the transmission electron microscope observation image of the PCV2b Capsid-Fc fusion protein selected in the application example 1 under the magnification of 4K-300K (200 nm);

FIG. 9: transmission electron microscopy of the commercial subunit vaccine selected in application example 1 at 4K-300K magnification (200 nm);

FIG. 10: the transmission electron microscope of the inactivated virus selected in the application example 1 is observed under the multiplying power of 4K-300K (200 nm);

FIG. 11: a transmission electron microscope observation image of the PCV2b Capsid-Fc fusion protein selected in the application example 1 under the magnification of 4K-300K (100 nm);

FIG. 12: transmission electron microscopy images of the commercial subunit vaccine selected in application example 1 at 4K-300K magnification (100 nm);

FIG. 13: the transmission electron microscope of the inactivated virus selected in the application example 1 is observed under the multiplying power of 4K-300K (50 nm);

FIG. 14: an observation image of a transmission electron microscope applying the PCV2b Capsid-Fc fusion protein selected in the embodiment 1 under the multiplying power of 4K-300K (50 nm);

FIG. 15: transmission electron microscopy of the commercial subunit vaccine selected in application example 1 at 4K-300K magnification (50 nm);

FIG. 16: lymphocyte proliferation assay analysis in animal cell experiments on immunized mouse spleen cells, where EFSV is PCV2b Capsid-Fc fusion protein subunit vaccine test group of the present invention, CSV is commercial subunit vaccine group, ConA is 5 μ g/ml ConA as positive control, data are expressed as SI mean ± SD, × P <0.05, × P < 0.01;

FIG. 17: a schematic diagram of the change of the specific antibody titer of a PCV2b Capsid-Fc fusion protein subunit vaccine immunized mouse in the antibody titer determination;

FIG. 18: PCV2b Capsid-Fc fusion protein subunit vaccine and commercial subunit vaccine immunization mouse specific antibody titer schematic diagram in antibody titer determination.

FIG. 19: and (3) a schematic diagram of the difference of the secretion concentration of the cytokine IFN-gamma of the mice immunized by the PCV2b Capsid-Fc fusion protein subunit vaccine and the commercial subunit vaccine in the determination of the secretion concentration of the cytokine IFN-gamma.

FIG. 20: schematic diagram of difference of secretion concentration of the PCV2b Capsid-Fc fusion protein subunit vaccine and the commercial subunit vaccine in the measurement of the secretion concentration of the cytokine IL-10 in the immunized mouse cytokine IL-10.

Detailed Description

The invention will be further elucidated by means of specific embodiments, without being limited thereto, in conjunction with the accompanying drawings.

Used in the examples of the present invention:

starting strain name: porcine circovirus type2-B strain NDSU41513, the capsid protein amino acid sequence from protein _ id ═ ALD 62452.1;

pEM plasmid, available from Detai Biotechnology (Nanjing) Ltd

Plasmid pcDNA3.1(+), Detai Biotechnology (Nanjing) Ltd

Escherichia coli DH5 α, Detai Biotech (Nanjing) Ltd;

HEK293F (human embryonic kidney cell 293F) from university of eastern china laboratory;

the primer sequences are as follows:

the LB liquid medium used for culturing the Escherichia coli DH5 alpha cells comprises, by mass, 1.0% of sodium chloride, 1.0% of peptone, 0.5% of yeast extract, and the balance of water.

The culture medium used for HEK293F cell culture is commercially available serum-free Union-293 medium, and the serum-free Union-293 medium adopted in the embodiment of the invention is produced by UnionTech Biotech (Shanghai) Co., Ltd.

Cell transfection was carried out using 40-kDa linear Polyethylenimine (PEI) transfection reagent supplied by Shanghai diligent Bio-technology Co., Ltd, according to the instructions.

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

and (3) separating and identifying cell lysate and an enriched PCV2b Capsid-Fc fusion protein component protein band on 12.5% polyacrylamide gel by adopting a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method. The polyacrylamide gel was stained with coomassie brilliant blue G250 to reveal protein bands. On the protein gel separated on SDS-PAGE gel, the separated protein was transferred to PVDF membrane by wet Western blot transfer mode. Proteins that had been transferred to PVDF membrane were blocked overnight at 4 ℃ in QuickBlockTM Western blocking buffer, 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 the secondary antibody. After washing 3 times with TBST, the imaged protein bands were detected using the ECL chemiluminescence system BeyoECL Plus (hypersensitivity ECL chemiluminescence kit) (P0018S), the Gel Image System (GIS) software package of the pica yunnan biotechnology limited and Tanon, the full-automatic digital gel image analysis system of shanghai sky science limited.

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

the method for measuring the number of cells in the examples of the present invention is a method for measuring the number of cells, which is the most common method for measuring the number of cells by trypan blue staining, and an instrument for measuring the number of cells is a CountStar automatic counting cell analyzer.

BCA protein quantification kit method (Biyuntian Biotech Co., Ltd.) was carried out 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. Graphical and data statistical analysis was performed using GraphPadPrism 8. P value<0.05 was considered statistically significant. *0.05<P<0.01，**0.01<P<0.001

Example 1

An expression gene of porcine circovirus type 2b Capsid-Fc fusion protein, the base sequence of which is shown in SEQ ID No.1, and the code of which consists of three parts, namely PCV2 Capsid segment sequence, linker segment sequence and Fc segment sequence; wherein the PCV2 coat segment sequence is SEQ ID No. 3; the linker section sequence is SEQ ID No. 5; the Fc segment sequence is a coding sequence of a Pig IgG heavy chain Fc segment is SEQ ID No.7, and the construction process comprises the following specific steps:

(1) gene sequence codon optimization for expressing novel PCV2b Capsid-Fc fusion protein

Firstly, selecting a PCV2 capsid protein gene coded by a portane circovirus type2-B strain NDSU41513 strain, namely, taking Gen Bank accession number ALD62452.1 as a sequence ID as a basis, removing 41 amino acids at the N end of the capsid protein, and then adopting Destai biotechnology (Nanjing) Limited company codon optimization software to perform codon optimization according to the codon preference of mammalian cells to obtain a PCV2B capsid segment sequence; 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 the mammalian cells to obtain a linker segment sequence; taking a porcine IgG heavy chain Fc region gene, wherein the sequence ID is an Fc segment sequence of GenBank access number AAD 38418.1; adopting codon optimization software of Detai biotechnology (Nanjing) Limited company to perform codon optimization according to codon preference of mammalian cells, and performing whole-gene synthesis to obtain a novel PCV2b Capsid-Fc fusion protein gene; the sequence is shown in SEQ ID No. 1.

(2) Adding N-terminal and C-terminal expression sequence elements of gene for expressing novel PCV2b Capsid-Fc fusion protein

Designing and adding EcoRI-HindIII containing enzyme cutting sites at the front end and the rear end of the novel PCV2b Capsid-Fc fusion protein obtained in the step (1), and inserting a Kozak sequence and a human IgG2H heavy chain signal peptide sequence which are sequentially a Kozak sequence and a human IgG2H heavy chain signal peptide sequence. The gene of the novel expression PCV2b Capsid-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 is carried out by adopting codon optimization software of the Deltay Biotechnology (Nanjing) Co., Ltd according to the codon preference of a mammalian cell.

(3) Sequence gene synthesis of gene N-terminal and C-terminal expression sequence elements added with novel PCV2b Capsid-Fc fusion protein

And carrying out whole-gene synthesis on the sequence to obtain a gene sequence containing expression recombinant porcine circovirus type 2b Capsid-Fc fusion protein, wherein the sequence is shown in SEQ ID No. 13. The Kozak sequence is shown in SEQ ID No. 11; the human IgG2H heavy chain signal peptide sequence is shown in SEQ ID No. 12; the obtained gene expressing the porcine circovirus type 2b Capsid-Fc fusion protein was sequenced using an 3730XL sequencer from Applied Biosystems, Inc., and the result was consistent with that of SEQ ID No. 13.

Example 2

A recombinant plasmid containing a gene of PCV2b Capsid-Fc is constructed by the following specific steps:

(1) construction of pEM-PCV2b Capsid-Fc recombinant plasmid containing PCV2b Capsid-Fc Gene

(2) construction of pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmid containing the Gene of PCV2b Capsid-Fc

Designing enzyme cutting 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 for synthesizing and expressing PCV2b Capsid-Fc, such as SEQ ID No.1, through the primers PcfF and PcfR to obtain an amino acid expression fragment containing the enzyme cutting sites NheI and HindIII, carrying out NheI/HindIII double enzyme cutting on the PCR amplification fragment, and connecting the PCR amplification fragment to a pCDNA3.1(+) plasmid expression vector subjected to the same NheI/HindIII double enzyme cutting treatment to obtain a pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmid;

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

The schematic diagram of the nucleic acid fragment obtained by double digestion with the NheI/HindIII enzyme of PCV2b Capsid-Fc of the pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmid is shown in FIG. 2, wherein M.DNA Marker; 1. extracting recombinant plasmids; the PCV2b Capsid-Fc of the pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmid was double digested with NheI/HindIII enzyme, and it can be seen from FIG. 2 that the expression plasmid pCDNA3.1(+) -PCV2b Capsid-Fc was successfully constructed.

The schematic diagram of the nucleic acid fragment obtained by double digestion with the NcoI/ApaI enzyme of PCV2b Capsid-Fc of the pEM-PCV2b Capsid-Fc recombinant plasmid obtained above is shown in FIG. 3, wherein M.DNA Marker; 1. extracting recombinant plasmids; 2. the pEM-PCV2b Capsid-Fc recombinant plasmid was successfully constructed by double digestion with NcoI/ApaI enzymes, as shown in FIG. 3.

Example 3

The preparation of the transfection-grade pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmid or the transfection-grade pEM-PCV2b Capsid-Fc recombinant plasmid comprises the following steps:

(1) the pEM-PCV2b Capsid-Fc recombinant plasmid or the pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmid is transfected into Escherichia coli DH5 alpha; sucking 80-100ng of the pEM-PCV2b Capsid-Fc recombinant plasmid or pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmid obtained in the example 2, adding the pEM-PCV2b Capsid-Fc recombinant plasmid or the pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmid into the prepared DH5 alpha competent cell, adding the mixture into an LB liquid culture medium, placing the mixture into a shaker, controlling the temperature to be 37 ℃ and the rotating speed to be 200rpm, performing shake culture for about 30min, and separating the DH5 alpha competent cell to obtain recombinant Escherichia coli DH5 alpha containing the pEM-PCV2b Capsid-Fc recombinant plasmid or the pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmid;

(2) extraction of transfection-grade plasmids

And (2) selecting a single clone of the escherichia coli DH5 alpha containing the transfection-grade pEM-PCV2b Capsid-Fc recombinant plasmid or pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmid obtained in the step (1) from a fresh culture plate into 2-5mL of LB culture medium, culturing for 8h at the rotating speed of 200rpm under the condition of controlling the temperature to be 37 ℃ to obtain a seed solution, then inoculating the seed solution into 200mL of LB culture medium according to the volume ratio of 1/500, culturing for 16h at the temperature to be further controlled to be 37 ℃ and the rotating speed of 200rpm to obtain a bacterial solution containing the recombinant plasmid, then centrifuging the bacterial solution at the rotating speed of 4000rpm for 10min, removing the supernatant, and harvesting the obtained precipitate by adopting E.Z.N.A of the Omega Biotek company.Extracting transfection-grade plasmids according to the method of the Kit by using an Endo-Free Plasmid DNA Maxi Kit Plasmid large extraction Kit D6926-01 to finally obtain transfection-grade pEM-PCV2b Capsid-Fc recombinant plasmids or transfection-grade pCDNA3.1(+) -PCV2b Capsid-Fc recombinant plasmids.

Practice ofExample 4

The PCV2b Capsid-Fc fusion protein is prepared by transiently transfecting the transfection-grade pEM-PCV2b Capsid-Fc recombinant plasmid obtained in example 3 to HEK293F suspension cells by using 40kDa linear Polyethyleneimine (PEI) as a transfection reagent, and then sequentially culturing, separating and purifying to finally obtain the PCV2b Capsid-Fc fusion protein, wherein the preparation process comprises the following steps:

(1) reviving and passaging HEK293 cell

Opening a water bath kettle at 37 ℃ in advance, and preheating Union-293 serum-free fresh culture medium at 37 ℃ in advance; taking out a cryopreserved HEK293F cell cryopreservation tube from a liquid nitrogen tank, immediately putting the cryopreserved cells into a 37 ℃ water bath kettle, slightly shaking to quickly melt the cryopreserved cells within about 1min, taking out, sterilizing the outer wall of the HEK293F cell cryopreservation tube with 75% ethanol, putting the cryopreserved cells into a biosafety cabinet, transferring the cells into a 15mL centrifuge tube filled with a culture medium in a 10mLUNion-293 serum-free culture medium, centrifuging for 5min at the controlled rotation speed of 800rpm, removing the supernatant from the centrifuge tube after centrifugation, taking a small amount of HEK293F cells in a fresh culture medium centrifuge tube resuspension, transferring the cells into a culture bottle, adding a Union-293 serum-free culture medium, slightly shaking to uniformly disperse the cells, taking out cell counting and activity detection, and controlling the viable cell density to be 6-8 × 10⁵Measuring cell viability (the cell viability is the ratio of the living cells to the total cells), recovering when the cell viability is lower than 80%, and placing the cell culture solution in CO when the cell viability is higher than 80%₂CO at a concentration of 5% by volume₂Culturing in incubator at 37 deg.C and 110rpm for 2-3 days until the density reaches 2-3 × 10⁶When HEK293 cells are cultured in each mL of culture solution, 10mL of cell culture solution is removed, 10mL of fresh culture medium is added until the cell viability is more than 90%, and culture solution containing revived HEK293 cells is obtained; ② placing the culture solution containing the recovered HEK293 cells in CO₂CO at a concentration of 5% by volume₂Culturing in an incubator at 37 deg.C and 110rpm for 2-3 days until cell concentration reaches 2-3 × 10⁶HEK29 was prepared from HEK293 cells/mLHEK 293 cells3 cells were cultured for the first cell expansion: 30mL of fresh medium was added to the above-mentioned HEK293 cell culture solution obtained after 15mL of the first cell expansion culture, and then CO was added thereto₂CO at a concentration of 5% by volume₂Continuously culturing in an incubator at 37 deg.C and 110rpm, detecting HEK293 cell concentration and cell activity every day, and determining cell concentration to 2 × 10⁶Performing secondary cell amplification culture on the HEK293 cells by using the HEK293 cells/mL culture solution, wherein the culture volume reaches about 45 mL; 90mL of fresh medium was added to the above-mentioned HEK293 cell culture solution obtained after the second cell expansion culture of 45mL, and then CO was added₂CO at a concentration of 5% by volume₂Continuously culturing in an incubator at 37 deg.C and 110rpm, detecting HEK293 cell concentration and cell activity every day, and determining cell concentration to 2-3 × 10⁶Completing the third cell amplification culture of the HEK293 cells by using the HEK293 cells/mL culture solution, wherein the culture volume reaches about 135 mL;

(2) preparation of Union-293 serum-free culture Medium containing pEM-PCV2b Capsule-Fc or pCDNA3.1(+) -PCV2b Capsule-Fc transfection-grade plasmid:

adding the transfection-grade pEM-PCV2b Capsid-Fc recombinant plasmid obtained in the example 3 into a Union-293 serum-free culture medium to obtain a Union-293 serum-free culture solution containing the transfection-grade pEM-PCV2b Capsid-Fc recombinant plasmid with 20 mu g/ml;

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

adding a transfection reagent polyethyleneimine into a Union-293 serum-free culture medium to obtain 80 microgram/ml Union-293 serum-free culture solution containing polyethyleneimine;

(4) preparation of Union-293 serum-free culture solution of PEI/DNA:

mixing the obtained 20 mu g/ml Union-293 serum-free culture solution containing the transfection-grade pEM-PCV2b Capsid-Fc recombinant plasmid with 80 mu g/ml polyethyleneimine-containing Union-293 serum-free culture solution according to the volume ratio of 1:1, and standing at room temperature for 20-30min to obtain a PEI/DNA Union-293 serum-free culture solution;

(5) plasmid-transfected HEK293 cells:

adding 13.5mL of Union-293 serum-free culture solution of PEI/DNA into 135mL of HEK-293F culture solution obtained by the third amplification culture of the step (1), and then adding CO₂CO at a concentration of 5% by volume₂Culturing in an incubator at 37 deg.C and 110rpm for 4-6 days, centrifuging the obtained cell culture solution at 1000rpm for 10min, and collecting precipitate which is HEK293F cell containing PCV2b Capsid-Fc fusion protein;

(6) isolation of PCV2b Capsule-Fc fusion protein:

resuspending HEK293F cells containing PCV2b Capsid-Fc fusion protein obtained in the step (5) by using an ultrasonic lysis buffer solution, then using an ultrasonic disruptor of a type Q125 of a Qsonica LLC company, controlling the output of amplitude intensity to be 34%, working for 3 seconds, stopping for 7 seconds, carrying out ultrasonic lysis on the cells for 30min of total working time, then controlling the temperature to be 4 ℃ and centrifuging at the rotating speed of 12,000rpm for 20min, and collecting supernatant;

the wet weight of the HEK293F cells containing the PCV2b Capsid-Fc fusion protein is the actual mass of the precipitate obtained after the centrifugation in the step (5);

(7) purification of PCV2 Capsid-Fc fusion protein:

and (3) loading the supernatant collected after centrifugation in the step (6) onto a Protein A sepharose chromatography column (Detai Biotechnology (Nanjing) Co., Ltd.), sequentially carrying out equilibrium chromatography column, loading, impurity washing, elution, collection, sample neutralization and filtration through a 0.22 micron filter according to the use requirement of the Protein A sepharose chromatography column to obtain a filtrate containing PCV2b Capsid-Fc fusion Protein.

The amino acid sequence of the PCV2b Capsid-Fc fusion protein in the filtrate containing the PCV2b Capsid-Fc fusion protein obtained in the above way was determined, and the expected amino acid sequence thereof was consistent with the sequence shown in SEQ ID No. 2.

The schematic structural diagram of the PCV2b Capsid-Fc fusion protein obtained in the above is shown in FIG. 4, and can be seen from FIG. 4; the PCV2b Capsid-Fc fusion protein can form a monomer PCV2b Capsid-Fc fusion protein into PCV2b Capsid-Fc fusion protein dimer through the disulfide bond of the Fc fragment;

the 3D structure of the PCV2b Capsid-Fc fusion protein obtained by the method is predicted by using an I-TRASSER Server through a ribbon model, and the predicted structural diagram is shown in FIG. 5 and can be seen in FIG. 5; the Capsid protein portion and the Fc portion of the PCV2b Capsid-Fc fusion protein do not structurally interfere with each other, and each has enough space to be thought of as a self-functioning part.

The SDS-PAGE and Western Blot analysis chart of the PCV2b Capsid-Fc fusion protein obtained in the above is shown in FIG. 6, wherein A is an electrophoretogram of a cell lysate expressed by HEK293F cells and purified PCV2b Capsid-Fc fusion protein on 12.5% polyacrylamide gel under a denaturing condition, wherein M is marker, lane 1, a supernatant lane 2 of the cell lysate after ultrasonic treatment is a flow-through solution, and lanes 3-8 are eluents; lane a supernatant of the cell lysate after sonication, lane B flow through, lanes c-e eluent, SDS-PAGE analysis of the gel with Cookland staining, and Western Blot analysis, detecting a specific band of about 59kDa protein. As can be seen in FIG. 6, there is the expected band at the position of about 59 kDa. Thus, it is shown that the cell-expressed lysate is purified to finally obtain the target protein product, PCV2b Capsid-Fc fusion protein.

Application examples

The PCV2b Capsid-Fc fusion protein is applied to preparing PCV2b Capsid-Fc fusion protein subunit vaccine for resisting porcine circovirus type 2b infection, and the PCV2 Capsid-Fc fusion protein subunit vaccine consists of an active ingredient PCV2b Capsid-Fc fusion protein and an adjuvant according to the mass-to-volume ratio, wherein the PCV2b Capsid-Fc fusion protein to the adjuvant is 3.3mg to 100 mL;

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

the active ingredient is PCV2b Capsid-Fc fusion protein which is self-assembled into virus-like nanoparticles.

The preparation method of the PCV2b Capsid-Fc fusion protein subunit vaccine comprises the following specific steps:

the filtrate containing PCV2b Capsid-Fc fusion protein obtained in step (7) of example 4 was dialyzed, replaced with 1xPBS having a concentration of 10% by volume of glycerol and pH7.4, and then filtered through a 0.22 μm filter, and the filtrate obtained after filtration through the 0.22 μm filter was measured by using the BCA protein concentration measurement kit, whereby the PCV2b Capsid-Fc fusion protein concentration was 66 μ g/mL.

PCV2b is obtained by taking 1 volume of filtrate obtained after the filtration by a 0.22 micron filter, diluting the filtrate by 1xPBS solution with pH7.4 for one time, dividing the filtrate into two equal parts, and respectively mixing the two equal parts with 1 volume of Freund complete adjuvant preheated to 37 ℃ and 1 volume of Freund incomplete adjuvant preheated to 37 ℃ in an equal-volume oscillating way to realize the emulsification of PCV2b Capsid-Fc fusion protein until a stable emulsification system is formed, namely PCV2b Capsid-Fc fusion protein subunit vaccine is obtained.

According to the PCV2b Capsid-Fc fusion protein subunit vaccine, the active ingredient PCV2b Capsid-Fc fusion protein is self-assembled into virus-like nanoparticles through the detection of a scanning electron microscope, and the particle size of the nanoparticles is about 41 nm.

Using a commercial subunit vaccine (hereinafter referred to as CSV, which is an anti-PCV 2 subunit vaccine produced by Qingdao Yibang bioengineering Co., Ltd.) and an inactivated virus (produced by animal health products GmbH, Boringer Yiger, Germany) as a control, scanning the obtained PCV2b Capsule-Fc fusion protein subunit vaccine by transmission electron microscopy respectively to obtain scanning transmission electron microscopy images as shown in FIGS. 7, 8, 9, 10, 11 and 12, 13. 14, 15, the forms of the inactivated virus, the PCV2b Capsid-Fc fusion protein subunit vaccine and the commercial subunit vaccine are different from each other in comparison of the graphs in FIGS. 7, 8 and 9, and the forms of the inactivated virus and the PCV2b Capsid-Fc fusion protein subunit vaccine are regular particles and irregular spikes of the commercial subunit vaccine in comparison of the graphs in FIGS. 10, 11 and 12; from the comparison of FIGS. 13, 14, and 15, it can be seen that the inactivated virus size is around 15-20 nm, and the PCV2b Capsid-Fc fusion protein subunit vaccine nanoparticle size is about 41 nm; therefore, the PCV2b Capsid-Fc fusion protein can spontaneously form nanoparticles with regular morphology, the average size is about 41n m, and a structural basis is provided for better stimulating the body to generate immune response.

Animal cell experiments

The obtained PCV2b Capsid-Fc protein subunit vaccine is respectively subjected to animal cell immunization experiments by taking a commercial subunit vaccine (namely an anti-PCV 2 subunit vaccine produced by Qingdao Yibang bioengineering GmbH and Germany) and an inactivated virus (produced by Boringer Yiger Han (Shanghai) animal health products GmbH and the like) as controls;

female BLAB/c mice (6-8 weeks old) were used as subjects, were housed in a specific pathogen free animal (SPF) grade animal laboratory at the bioengineering institute of university of eastern physics, 7 days prior to the official experiment, and then animals were randomly divided into three experimental groups (n-3 mice/group) and maintained in a temperature and light controlled environment and optionally food and water conditions;

mice were immunized intraperitoneally with an amount of 6.6ug vaccine/mouse, a subunit vaccine of PCV2b Capsid-Fc fusion protein of the invention and a commercially available subunit vaccine against PCV 2. The control group received sterile PBS solution only. The PBS solution is prepared as follows, according to the content of each substance contained in each liter; NaCl, 8.0 g; KH (Perkin Elmer)₂PO₄，0.2g；Na₂HPO₄.12H₂O, 2.9 g; KCl, 0.2 g; then the volume is determined to be 1000mL, and the pH value is adjusted to 7.4;

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

Lymphocyte proliferation assay and cytokine assay

Splenocytes were isolated from immunized mice 70 days after immunization of the mice. Spleen cells (5X 10)⁵Cells/well) were inoculated into a 96-well round plate, and 200. mu.l of RPMI 1640 containing 10% FBS was added thereto for culture. These splenocytes were stimulated with concanavalin ConA (5. mu.g/mL) or vaccine. After incubation at 37 ℃ for 72 hours, then in an equal volume of 100uLReagents were added to each well and incubated at 37 ℃ for 15 minutes. Used inThe proliferation of lymphocytes was measured using an automated cell analyzer in 96-well, flat, clear-bottom, opaque-wall microplates according to the manufacturer's protocol using a luminescence cell viability assay kit (Promega, usa) and the results are expressed as Stimulation Index (SI) and calculated according to the following formula: SI ═ (number of cells in the immune group-number of cells in the blank)/(number of cells in the negative control group-number of cells in the blank).

Specifically, fig. 16 shows the proliferation of lymphocytes, in which EFSV is the PCV2b Capsid-Fc fusion protein subunit vaccine of the present invention, CSV is the commercial subunit vaccine, ConA is the positive Control, and Control is the RPMI 1640 Control group, and it can be seen from fig. 16 that both the PCV2b Capsid-Fc fusion protein subunit vaccine and the commercial subunit vaccine of the present invention have strong lymphocyte stimulation and cause proliferation reactions. The SI of two groups of mice immunized by the PCV2b Capsid-Fc fusion protein subunit vaccine and the commercial subunit vaccine is 1.728 +/-0.259 and 1.430 +/-0.212 respectively, which are higher than that of an RPMI 1640 control group (P <0.05), while the SI value of the PCV2b Capsid-Fc fusion protein subunit vaccine immunization group is also higher than that of the commercial subunit vaccine immunization group. The SI value of the ConA (positive control) control was 2.967 ± 0.713, indicating that ConA effectively exerted a function. These results indicate that the Capsid-Fc fusion protein subunit vaccine of the present invention induces a significant cellular immune response in mice.

Determination of antibody Titers

To further investigate the extent of immune response of the PCV2b Capsid-Fc fusion protein subunit vaccine of the present invention, i.e. PCV2b Capsid-Fc fusion protein subunit vaccine and commercial subunit vaccine, the level of specific antibodies of PCV2 Capsid protein was measured on serum obtained from immunized mice by indirect ELISA analysis, and the specific results are shown in fig. 17, fig. 18;

as can be seen from fig. 17, the antibody level was slightly increased at day 12 after the primary immunization, and after the secondary booster immunization, specific antibodies against PCV2 Capsid were produced at a high level, the antibody concentration was over 500ng/ml, and the antibody concentration was kept at a high level throughout the experiment, thereby indicating that the PCV2b Capsid-Fc fusion protein subunit vaccine of the present invention was able to stimulate mice to produce high concentrations of PCV2 Capsid-specific antibody, and 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 Capsid protein in the groups immunized with the PCV2 Capsid-Fc fusion protein subunit vaccine of the present invention increased rapidly in antibody concentration 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 of 540ng/mL or more.

In FIG. 18, the control group is PBS solution immunized mice, the EFSV immunized mice group is PCV2bFc fusion protein subunit vaccine immunized mice group of the present invention, CSV represents commercial subunit vaccine immunized mice group, wherein P <0.05 and P < 0.01, it can be seen from FIG. 18 that both the PCV2b Capsid-Fc fusion protein subunit vaccine of the present invention and the commercial subunit vaccine can induce the production of anti-mouse PCV2 antibody, while the PCV2b Capsid-Fc fusion protein subunit vaccine immunized mice group of the present invention induces the production of antibody at a concentration significantly higher than that of the commercial subunit vaccine immunized mice group, and in the mice group immunized with the commercial subunit vaccine, the level of specific antibody of PCV2 Capsid protein is at 200-250ng/mL, so that the PCV2b Capsid-Fc fusion protein subunit vaccine of the present invention has an antibody concentration level increased by more than 1-fold compared to the commercial subunit vaccine immunized mice group, statistical analysis shows that the PCV2b Capsid-Fc fusion protein subunit vaccine has significant difference (P <0.05), thereby showing that the PCV2b Capsid-Fc fusion protein subunit vaccine can well stimulate a mouse body to generate high-concentration antibodies so as to improve the immunity of cells and body fluid.

To further characterize the antigen-specific cellular immune properties of mice immunized with the PCV2b Capsid-Fc fusion protein subunit vaccine (EFSV) and the Commercial Subunit Vaccine (CSV) of the present invention, the inventors evaluated the use of the PCV2b Capsid-Fc fusion protein subunit vaccine and the commercial subunit vaccine of the present invention to stimulate cytokines secreted by splenic T cells of immunized mice. The assay was carried out using a commercial mouse IFN-. gamma.and IL-10ELISA kit (Jiangsu enzyme Immunity Co., Ltd.). Compared with a PBS control group, the secretion concentration of the cytokine IFN-gamma is 230.76 +/-16.97 and 237.28 +/-20.11 pg/ml, when the PCV2b Capsid-Fc fusion protein subunit vaccine and the immunized mouse T cell stimulated by the commercial subunit vaccine are used, the IFN-gamma cytokine secreted by the T cell reaches 292.17 +/-13.80 and 293.80 +/-30.50 pg/ml respectively, and the data result shows that the statistic analysis shows that the data has significant difference (P < 0.01, P <0.05, and the result is shown in a figure 19;

furthermore, when spleen T cells of mice immunized with EFSV were stimulated with the culture medium, compared with spleen T cells of mice immunized with EFSV stimulated with PCV2b Capsid-Fc fusion protein (PCFFP), the average cytokine IL-10 secretion concentrations of both were 17.17. + -. 0.85ng/mL and 19.11. + -. 0.26ng/mL, respectively, and the statistical analysis of the two sets of data showed significant differences (P <0.05), 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 results show that the PCV2b Capsid-Fc fusion protein subunit vaccine can better induce the humoral and cellular immune response of mice.

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

The porcine circovirus type 2b Capsid-Fc fusion protein can form viroid-like nanoparticles, has good structural attributes, and is easy to stimulate the immune system of an organism to generate stress immune response. The data result shows that the porcine circovirus type 2b Capsid-Fc fusion protein can not only induce the humoral immunity and cellular immune response of mice, but also be superior to the commercial subunit vaccine in some indexes compared with the commercial subunit vaccine in the experimental vaccine taking the porcine circovirus type 2b Capsid-Fc fusion protein as the active component. Therefore, the porcine circovirus type 2b Capsid-Fc fusion protein is a candidate vaccine with good potential for preventing and treating porcine circovirus infection.

The foregoing is merely an example of the embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Shanghai eagle technology industries Ltd

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

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1605

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

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

<210> 2

<211> 535

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

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