阅读说明：本技术 一种二价亚单位疫苗及其制备方法和应用 (Bivalent subunit vaccine and preparation method and application thereof ) 是由 徐高原 周明光 张华伟 曾小燕 朱娴静 孙芳 郝根喜 邵伟 于 2020-06-08 设计创作，主要内容包括：本发明涉及一种二价亚单位疫苗及其制备方法和应用,所述二价亚单位疫苗包括猪圆环病毒2型Cap蛋白和猪圆环病毒3型Cap蛋白；所述猪圆环病毒2型Cap蛋白和猪圆环病毒3型Cap蛋白均为利用昆虫-杆状病毒表达载体系统表达得到。本发明通过昆虫-杆状病毒表达载体系统进行猪圆环病毒2型和3型Cap蛋白的表达,表达效率更高,同时,组合而成的猪圆环病毒2型和3型二价亚单位疫苗可以同时进行猪圆环病毒2型和3型的免疫,且相较于将两者单独使用,显著提升了猪圆环病毒2型Cap蛋白的免疫效果。本发明提供的猪圆环病毒2型和3型二价亚单位疫苗产量高,纯度高,作为制备疫苗的抗原免疫性更强,安全性更高。(The invention relates to a bivalent subunit vaccine and a preparation method and application thereof, wherein the bivalent subunit vaccine comprises porcine circovirus type 2 Cap protein and porcine circovirus type 3Cap protein; the porcine circovirus type 2 Cap protein and the porcine circovirus type 3Cap protein are obtained by expression of an insect-baculovirus expression vector system. According to the invention, the expression of the porcine circovirus type 2 and type 3Cap proteins is carried out through the insect-baculovirus expression vector system, the expression efficiency is higher, meanwhile, the combined porcine circovirus type 2 and type 3 bivalent subunit vaccines can simultaneously carry out the immunization of the porcine circovirus type 2 and type 3, and compared with the single use of the two, the immunization effect of the porcine circovirus type 2 Cap proteins is obviously improved. The bivalent subunit vaccine of porcine circovirus type 2 and type 3 provided by the invention has high yield and high purity, and has stronger antigen immunity and higher safety when being used for preparing the vaccine.)

1. A bivalent subunit vaccine, characterized by: the bivalent subunit vaccine comprises porcine circovirus type 2 Cap protein and porcine circovirus type 3Cap protein;

the porcine circovirus type 2 Cap protein and the porcine circovirus type 3Cap protein are obtained by expression of an insect-baculovirus expression vector system.

2. The bivalent subunit vaccine according to claim 1, wherein the molar ratio of the porcine circovirus type 2 Cap protein to the porcine circovirus type 3Cap protein in the bivalent subunit vaccine is 1: 0.9-1.1.

3. Bivalent subunit vaccine according to claim 1 or 2, characterized in that the expression of the porcine circovirus type 2 Cap protein is performed by Ac-PCV2 Cap recombinant baculovirus; performing expression of the porcine circovirus type 3Cap protein by an Ac-PCV3Cap recombinant baculovirus;

the Ac-PCV2 Cap recombinant baculovirus comprises a nucleotide sequence shown as SEQ ID NO. 1;

the Ac-PCV3Cap recombinant baculovirus comprises a nucleotide sequence shown as SEQ ID NO. 2.

4. The bivalent subunit vaccine according to claim 3, characterized in that the construction of the Ac-PCV2 Cap recombinant baculovirus and the Ac-PCV3Cap recombinant baculovirus is performed by a pFastBac vector.

5. The bivalent subunit vaccine according to claim 3, wherein the porcine circovirus type 2 Cap protein content in the bivalent subunit vaccine is 30-50 μ g/ml; the content of the porcine circovirus type 3Cap protein is 30-50 mug/ml.

6. The bivalent subunit vaccine according to any one of claims 1 to 5, further comprising an adjuvant selected from one or more of the group consisting of aluminum salt series adjuvants, Montanide IMS series adjuvants, Montanide GEL series adjuvants, propolis, immunostimulating complex, cytokine-type adjuvants, nucleic acid and its derivatives-type adjuvants, and lecithin-type adjuvants.

7. The bivalent subunit vaccine according to claim 6, wherein the adjuvant is 201 adjuvant, and the mass ratio of the sum of the porcine circovirus type 2 Cap protein and the porcine circovirus type 3Cap protein to the 201 adjuvant is 2-5: 1.

8. A method of producing a bivalent subunit vaccine as claimed in any one of claims 1 to 7, comprising:

the Cap proteins of the porcine circovirus type 2 and the porcine circovirus type 3 are obtained by expression of an insect-baculovirus expression system, and are prepared into a bivalent subunit vaccine by being matched with an adjuvant.

9. Use of a bivalent subunit vaccine according to any one of claims 1 to 7 for the immunization of a pig against porcine circovirus type 2, and/or for the immunization of a pig against porcine circovirus type 3.

10. The use according to claim 9, wherein the bivalent subunit vaccine is administered in an amount of 30 to 50 μ g of each of the porcine circovirus type 2 Cap protein and the porcine circovirus type 3Cap protein per head.

Technical Field

The invention relates to the technical field of veterinary biology, in particular to a bivalent subunit vaccine and a preparation method and application thereof.

Background

Porcine Circovirus (PCV) is a single-stranded circular DNA virus, is one of the smallest animal DNA viruses, can cause piglet multi-system failure syndrome, Porcine respiratory mixed diseases, Porcine reproductive disorders, Porcine dermatitis nephrotic syndrome and the like, and causes serious harm to the pig industry. Two types were identified early, including circovirus type 1 (PCV1) and circovirus type 2 (PCV 2). In 2015, the clinical symptoms and histological changes of the complex of dermatitis and nephropathy of the sow group in one pig farm in the United states are consistent with those of the diseases related to the circovirus, but PCV2, PRRSV, IAV and the like are all negative. The American scholars RachelPalinski et al found circovirus with different genotypes after metagenome sequencing, and therefore named PCV 3.

The porcine circovirus virus particles are in a regular icosahedral symmetrical structure, have no envelope, have the diameter of 17-20nm, and have single-stranded DNA as a genome. The genome length of the porcine circovirus type 2 is between 1766bp and 1769bp, and the full length of the porcine circovirus type 3 is 1999-2001 bp. For PCV2 and PCV3, three main open reading frames ORF1, ORF2, ORF3 are currently under investigation. ORF2 is located on the complementary strand of the genome to form the nucleocapsid protein (Cap) of the virus, the Cap protein is also the main immunogenic protein of PCV, the C end of the Cap protein contains more corner structures, the hydrophilicity index and the antigenic index are higher, the segment has dominant antigenic epitope, and the N end contains a large number of basic amino acid sequences which are closely related to the intranuclear localization of the Cap protein. Therefore, the Cap protein plays an important role in disease diagnosis and vaccine research, and the research on PCV2 and PCV3 focuses on the Cap protein. In the prior art, the homology between PCV2 and PCV3Cap protein aa is only 30%, the possibility of cross immune protection between the two is low, and no vaccine aiming at PCV2 and PCV3 strains simultaneously exists in the market at present.

Disclosure of Invention

In order to solve at least one problem in the prior art, the invention provides a bivalent subunit vaccine and a preparation method and application thereof. The Cap proteins of the porcine circovirus type 2 and the porcine circovirus type 3 are expressed by an insect-baculovirus expression vector system, so that the porcine circovirus type 2 and the porcine circovirus type 3 can be immunized simultaneously, and a better immunization effect is obtained.

In a first aspect, the invention provides a bivalent subunit vaccine comprising porcine circovirus type 2 Cap protein and porcine circovirus type 3Cap protein;

the porcine circovirus type 2 Cap protein and the porcine circovirus type 3Cap protein are obtained by expression of an insect-baculovirus expression vector system.

Further, the molar ratio of the porcine circovirus type 2 Cap protein to the porcine circovirus type 3Cap protein is 1: 0.9-1.1.

The invention discovers that the immunogenicity of the porcine circovirus type 2 antigen component obtained by using an insect-baculovirus expression vector system for expression is poor, and the immunogenicity is obviously improved when the porcine circovirus type 2 antigen component is combined with the porcine circovirus type 3 antigen component for use.

Further, expression of the porcine circovirus type 2 Cap protein is performed by Ac-PCV2 Cap recombinant baculovirus; performing expression of the porcine circovirus type 3Cap protein by an Ac-PCV3Cap recombinant baculovirus;

the Ac-PCV2 Cap recombinant baculovirus comprises a nucleotide sequence shown as SEQ ID NO. 1;

the Ac-PCV3Cap recombinant baculovirus comprises a nucleotide sequence shown as SEQ ID NO. 2.

The nucleotide sequences for expressing the porcine circovirus type 2 Cap protein and the porcine circovirus type 3Cap protein are optimized, and the optimized sequences are shown as SEQ ID NO.1 and SEQ ID NO. 2; after optimization, the expression levels of the porcine circovirus type 2 Cap protein and the porcine circovirus type 3Cap protein are obviously improved.

Further, the construction of the Ac-PCV2 Cap recombinant baculovirus and the Ac-PCV3Cap recombinant baculovirus was performed by pFastBac vector.

Further, in the bivalent subunit vaccine, the content of the porcine circovirus type 2 Cap protein is 30-50 mug/ml; the content of the porcine circovirus type 3Cap protein is 30-50 mug/ml.

Preferably, the content of Cap protein of the porcine circovirus type 2 is 40 mug/ml; the content of the Cap protein of the porcine circovirus type 3 is 40 mug/ml.

Further, the bivalent subunit vaccine also comprises an adjuvant, wherein the adjuvant is selected from one or more of aluminum salt series adjuvants, Montanide IMS series adjuvants, Montanide GEL series adjuvants, propolis, immunostimulation compound, cytokine type adjuvants, nucleic acid and derivative type adjuvants thereof, and lecithin type adjuvants.

Preferably, the adjuvant is 201 adjuvant, and the mass ratio of the sum of the porcine circovirus type 2 Cap protein and the porcine circovirus type 3Cap protein to the 201 adjuvant is 2-5: 1.

In a second aspect, the present invention provides a method for preparing the bivalent subunit vaccine, comprising:

the Cap proteins of the porcine circovirus type 2 and the porcine circovirus type 3 are obtained by expression of an insect-baculovirus expression system, and are prepared into a bivalent subunit vaccine by being matched with an adjuvant.

The invention further provides the use of the bivalent subunit vaccine in the immunisation of pigs against porcine circovirus type 2 and in the immunisation of pigs against porcine circovirus type 3.

Further, the dosage of the bivalent subunit vaccine is 30-50 mu g/head of each of the porcine circovirus type 2 Cap protein and the porcine circovirus type 3Cap protein when the bivalent subunit vaccine is used.

As a preferred embodiment, the method of preparing the bivalent subunit vaccine comprises:

(1) synthesizing gene sequences of ORF2 of PCV2 and PCV 3;

(2) constructing a pFastdeal-PCV 2ORF2 and pFastdeal-PCV 3 ORF2 recombinant transfer vector;

(3) transferring into escherichia coli competent DH10Bac, culturing, and extracting recombinant bacmid rBac-PCV 2ORF2 and rBac-PCV3 ORF2 as recombinant shuttle vectors;

(4) the extracted recombinant shuttle vector was transfected into sf9 insect cells using lipofection.

(5) Expression and purification of proteins PCV2 Cap and PCV3Cap

(6) The bivalent subunit vaccine is prepared by mixing the PCV2 Cap, the PCV3Cap and an adjuvant according to the proportion of 3: 1.

Further, the gene sequence of step (1) was introduced with AcMNPV GP64 signal peptide sequence at its amino terminus and 6 × His tag peptide sequence at its carboxy terminus.

Furthermore, the content of protein PCV2 Cap in the bivalent subunit vaccine finally prepared in the step (6) is 30-50 mu g/ml, the content of protein PCV3Cap is 30-50 mu g/ml, and the adjuvant is preferably 201 adjuvant.

The invention has the following beneficial effects:

1. the insect-baculovirus eukaryotic expression system used in the invention is efficient and safe, and has no harm to human and livestock, the expressed nucleotide sequences of PCV2 Cap and PCV3Cap proteins are optimized, the target protein can be obtained by expression of the insect-baculovirus eukaryotic expression system, and the obtained protein is completely consistent with the natural state in structure and function and has strong immunogenicity.

2. The PCV2 type and PCV3 type bivalent subunit vaccines have the advantages of simple preparation method, high protein yield, high purity and stronger antigen immunity, can effectively activate the immune response of organisms, and play an ideal immune protection role on PCV2 and PCV 3.

3. Compared with a monovalent vaccine, the PCV2 type and PCV3 type bivalent subunit vaccines provided by the invention can reduce the human co-cost, reduce the vaccination times and reduce the stress response of pigs caused by vaccine immunization.

Drawings

FIG. 1 shows the results of the restriction enzyme identification of KpnI and XhoI of ORF2 of the artificially synthesized genes PCV2 and PCV3 provided in example 1 of the present invention; wherein, 1 in A is DNA marker Dl5000, 2 is pFastdial-PCV 2ORF2, B is DNA marker rDL5000, 2 is pFastdial-PCV 3 ORF 2;

FIG. 2 shows the SDS-PAGE and WesternBlot identification results of the proteins PCV2 Cap and PCV3Cap of interest provided in example 1 of the present invention; wherein A is a target protein PCV2 Cap, and B is a target protein PCV3 Cap;

FIG. 3 shows the SDS-PAGE and Western Blot identification results of the non-optimized and non-optimized PCV2 Cap and PCV3Cap provided in example 1 of the present invention;

FIG. 4 is a graph showing the results of ELISA detection of specific antibodies using coated plates of PCV2 Cap protein and PCV3Cap protein, provided in example 2 of the present invention; wherein A is a target protein PCV2 Cap, and B is a target protein PCV3 Cap;

FIG. 5 is a graph showing the result of detecting specific antibodies of PCV2 Cap protein and PCV3Cap protein by ELISA provided in example 2 of the present invention, wherein A is the target protein PCV2 Cap, and B is the target protein PCV3 Cap;

FIG. 6 is a graph showing the results of antigen-specific antibody detection and neutralizing antibody detection provided in example 2 of the present invention; wherein A is a target protein PCV2 Cap, and B is a target protein PCV3 Cap.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.