阅读说明：本技术 一种可溶性猪圆环病毒2型Cap蛋白及应用 (Soluble porcine circovirus type 2 Cap protein and application thereof ) 是由 于江 吴家强 张玉玉 陈智 孙文博 韩红 杨杰 于 2020-03-13 设计创作，主要内容包括：本发明涉及生物技术中蛋白表达技术领域,特别涉及一种可溶性猪圆环病毒2型Cap蛋白。该可溶性猪圆环病毒2型Cap蛋白制成的铝胶佐剂疫苗比油佐剂疫苗安全效果好,免疫后猪体吸收快,应激小,比201佐剂免疫保护率高。(The invention relates to the technical field of protein expression in biotechnology, in particular to a soluble porcine circovirus type 2 Cap protein. The alumina gel adjuvant vaccine prepared from the soluble porcine circovirus type 2 Cap protein has better safety effect than an oil adjuvant vaccine, the absorption of a immunized pig body is fast, the stress is small, and the immune protection rate is higher than that of an adjuvant 201.)

1. The porcine circovirus type 2 ORF2 gene has the nucleotide sequence shown as sequence 1 in the sequence table.

2. A soluble porcine circovirus type 2 Cap protein expressed by a sequence 1 in a sequence table.

3. The soluble porcine circovirus type 2 Cap protein of claim 1, wherein the amount of the water soluble porcine circovirus type 2 Cap protein expressed in 1L of bacterial culture is 36.75 mg.

4. Use of the soluble porcine circovirus type 2 Cap protein of claim 2 or 3 in the preparation of an immunological formulation.

5. The use according to claim 4, characterized in that the immunological preparation is a porcine circovirus type 2 Cap protein inactivated vaccine.

6. The use according to claim 5, characterized in that the adjuvant in the inactivated vaccine is an oil adjuvant, a 201 adjuvant or an alumina gel adjuvant.

7. The use according to claim 5, wherein the concentration of soluble porcine circovirus type 2 Cap protein in the inactivated vaccine is 100 ug/ml.

Technical Field

The invention relates to the technical field of protein expression in biotechnology, in particular to a soluble porcine circovirus type 2 Cap protein, and also relates to application of the soluble porcine circovirus type 2 Cap protein.

Background

Porcine circovirus type 2 (PCV 2) is one of the more serious viruses which have been newly discovered in recent years and endanger the swine industry, and is closely related to various disease syndromes of pigs, including Post-weaning multisystemic wasting syndrome (PMWS) of pigs, reproductive failure (SAMS) of sows, and the like. Since PMWS was first reported in Canada in 1991, porcine circovirus infection has now spread around the world in the 2010 International Pig Veterinary Society (IPVS), and is one of the most important infectious diseases recognized globally as harming the swine industry. In recent years, domestic porcine circovirus infection is on the rise, and the domestic porcine circovirus infection is very popular in domestic swinery, thereby causing great economic loss to the domestic pig industry.

PCV2 genome is 1767bp or 1768bp in total length and comprises 11 Open Reading Frames (ORF), wherein ORF1 and ORF2 are the two largest open reading frames and respectively code replication-related proteins (Rep proteins) and capsid proteins (Cap proteins). The Cap protein is a main structural protein, and researches show that the N end of the Cap protein contains a Nuclear Localization Signal (NLS) consisting of 41 amino acid residues and contains a large number of rare codons of Escherichia coli, which seriously influences the expression of foreign proteins. Related studies have demonstrated that animals immunized with Cap protein can produce neutralizing antibodies to the virus. The ORF2 gene has been the focus of developing genetically engineered vaccines. However, the expression condition of Cap protein in prokaryotic expression system is often expressed in the form of inclusion body, which brings inconvenience to the purification of protein, and the reactogenicity and immunogenicity are not as good as those of soluble protein, so the development of soluble porcine circovirus type 2 Cap protein is an important research direction for expanding the immune application.

Disclosure of Invention

In order to solve the problem of inclusion bodies of the porcine circovirus type 2 Cap protein in the prior art, the application provides a soluble porcine circovirus type 2 Cap protein, and a porcine circovirus type 2 ORF2 gene expressing the soluble porcine circovirus type 2 Cap protein. If the protein is soluble, the proportion of the active protein can be enhanced without complicated processes such as protein renaturation and the like, and the method is suitable for large-scale production of biological product enterprises.

The invention also provides application of the soluble porcine circovirus type 2 Cap protein.

The invention is obtained by the following steps:

the porcine circovirus type 2 ORF2 gene has the nucleotide sequence shown as sequence 1 in the sequence table.

A soluble porcine circovirus type 2 Cap protein expressed by a sequence 1 in a sequence table.

Preferably, the expression quantity of the soluble porcine circovirus type 2 Cap protein in 1L of bacterial culture solution is 36.75 mg.

Preferably, the soluble porcine circovirus type 2 Cap protein is applied to the preparation of an immune preparation.

Preferably, the immune preparation is porcine circovirus type 2 Cap protein inactivated vaccine.

Preferably, the adjuvant in the inactivated vaccine is an oil adjuvant, a 201 adjuvant or an alumina gel adjuvant.

Preferably, the concentration of the soluble porcine circovirus type 2 Cap protein in the inactivated vaccine is 100 ug/ml.

The invention has the beneficial effects that:

the optimized porcine circovirus type 2 ORF2 gene is obtained by splicing and modifying the ORF2 gene and optimizing rare codons, the expressed porcine circovirus type 2 Cap protein has solubility, the proportion of active protein is enhanced, complex processes such as protein renaturation and the like are not needed, and the method is suitable for large-scale production of biological product enterprises. The alumina gel adjuvant vaccine prepared from the soluble porcine circovirus type 2 Cap protein has better safety effect than an oil adjuvant vaccine, the absorption of a immunized pig body is fast, the stress is small, and the immune protection rate is higher than that of an adjuvant 201.

Drawings

FIG. 1 is a comparison of nucleotide sequences before and after optimization of a target gene, the top being a pre-optimized sequence and the bottom being an optimized sequence,

FIG. 2 shows the results of restriction enzyme identification of recombinant plasmid, M: DL2000 Marker; 1: BL21(DE3) recombinant plasmid; 2: BL21Star (DE3) recombinant plasmid; 3: a Rosetta recombinant plasmid;

FIG. 3 shows the result of PCR identification of recombinant plasmid, M: DL2000 Marker; 1: BL21(DE3) recombinant plasmid; 2: BL21Star (DE3) recombinant plasmid; 3: a Rosetta recombinant plasmid;

FIG. 4 is a diagram of SDS-PAGE analysis of the expression of Cap protein in BL21(DE3), BL21Star (DE3) and Rosetta in whole cells, M: a protein Marker; 1: pET-30a vector; 2: 0.1mM IPTG, 15 ℃, 16 h, BL21(DE 3); 3: 0.1 miptg, 37 ℃, 16 hours, BL21(DE 3); 4: 0.25mM IPTG, 37 ℃, 16 h, BL21(DE 3); 5: 0.25mM IPTG, 37 ℃, 4 hours, BL21(DE 3); 6: 0.1mM IPTG, 15 ℃, 16 h, BL21Star (DE 3); 7: 0.1mM IPTG, 37 ℃, 16 hours, BL21Star (DE 3); 8: 0.25mM IPTG, 37 ℃, 16 h, BL21Star (DE 3); 9: 0.25mM IPTG, 37 ℃, 4 hours, BL21Star (DE 3); 10: 0.1mM IPTG, 15 ℃, 16 hours, Rosetta; 11: 0.1mM IPTG, 37 ℃, 16 hours, Rosetta; 12: 0.25mM IPTG, 37 ℃, 16 hours, Rosetta; 13: 0.25 miptg, 37 ℃, 4 hours, Rosetta;

FIG. 5 shows the results of SDS-PAGE analysis of Cap protein expression analysis in BL21(DE3), BL21Star (DE3) and Rosetta, A: (iii) supernatant expression; b: inclusion body expression, M: a protein Marker; 1: pET-30a vector; 2: 0.1mM IPTG, 15 ℃, 16 h, BL21(DE 3); 3: 0.1mM IPTG, 37 ℃, 16 h, BL21(DE 3); 4: 0.25mM IPTG, 37 ℃, 16 h, BL21(DE 3); 5: 0.25mM IPTG, 37 ℃, 4 hours, BL21(DE 3); 6: 0.1mM IPTG, 15 ℃, 16 h, BL21Star (DE 3); 7: 0.1mM IPTG, 37 ℃, 16 h, BL21Star (DE 3); 8: 0.25mM IPTG, 37 ℃, 16 h, BL21Star (DE 3); 9: 0.25mM IPTG, 37 ℃, 4 hours, BL21Star (DE 3); 10: 0.1mM IPTG, 15 ℃, 16 hours, Rosetta; 11: 0.1mM IPTG, 37 ℃, 16 hours, Rosetta; 12: 0.25mM IPTG, 37 ℃, 16 hours, Rosetta; 13: 0.25mM IPTG, 37 ℃, 4 hours, Rosetta;

FIG. 6 shows the purification results of Cap protein supernatant analyzed by SDS-PAGE, M: a protein Marker; 1: centrifuging the whole bacteria to obtain supernatant; 2: discharging liquid after the supernatant is incubated with Ni-IDA; 3: an eluted fraction of 50mM imidazole; 4: an eluted fraction of 100mM imidazole; 5: an eluted fraction of 500mM imidazole;

fig. 7 shows the results of recombinant protein identification, a: SDS-PAGE identification; b: identifying by Western-blot; m1: SDS-PAGEMARKER; m2: western-blot Marker; 1: BSA (1.5. mu.g); 2: protein of interest (1.5 μ g);

FIG. 8 is the average relative daily gain results;

fig. 9 shows the results of immunohistochemical assays, a: a toxin counteracting group; b: blank control group; c: an oil adjuvant group; d: 201 adjuvant group; e: an alumina gel adjuvant group;

Detailed Description

The invention is further illustrated by the following specific examples: