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Expression preparation and application of porcine circovirus type 2 recombinant Cap protein

A porcine circovirus and recombinant protein technology, applied in the biological field, can solve problems such as uneven levels of subunit vaccines, unsatisfactory immune effects, and difficult inactivation effects of inactivated vaccines, achieving good biological activity and immunogenicity, The effect of high VLP assembly rate and reduction of production cost

Active Publication Date: 2020-04-10
CHINA ANIMAL HUSBANDRY IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, porcine circovirus vaccines mainly include inactivated vaccines, attenuated live vaccines and subunit vaccines. Most of them are inactivated vaccines in China. However, because PCV2 virus is not easy to reproduce on cells, the titer of the produced virus is generally low, and the immune effect is not ideal. In addition, inactivated vaccines also have problems such as difficult detection of inactivation effect and short immunization period, which increases production costs compared with other virus inactivated vaccines; the safety of subunit vaccines is recognized by the industry, but the level of subunit vaccines produced in China varies Incomplete, and imported subunit vaccines, although the immune effect is good, but the price is relatively high

Method used

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  • Expression preparation and application of porcine circovirus type 2 recombinant Cap protein
  • Expression preparation and application of porcine circovirus type 2 recombinant Cap protein
  • Expression preparation and application of porcine circovirus type 2 recombinant Cap protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1, the preparation of porcine circovirus type 2 recombinant Cap protein

[0034] 1. Experimental materials

[0035] The primers were synthesized from Shanghai Jierui Bioengineering Co., Ltd., and the competent E. coli BL21 (DE3) was preserved in the laboratory;

[0036] Main reagents DNA plasmid small extraction kit; peptone and yeast extract were purchased from Oxoid; IPTG, L-arginine hydrochloride, Tris-HCL were purchased from Amresco; BSA protein standard was purchased from Thermo Fisher Scientific;

[0037] 2. Construction of porcine circovirus type 2 recombinant Cap protein expression plasmid

[0038]The recombinant Cap gene sequence comes from the prevailing strain of PCV2 isolated in our laboratory. The recombinant Cap gene sequence is analyzed and compared, and the structure of the Cap protein sequence is predicted after removing 41 basic amino acids at the N-terminus, and compared with the resolved PCV2Cap protein structure. (PDB: 3R0R) for structu...

Embodiment 2

[0062] The preparation of embodiment 2 porcine circovirus type 2 recombinant Cap protein subunit vaccines

[0063] 1. Protein preparation

[0064] Pick pET-CapR and transform Escherichia coli BL21 (DE3) competently to obtain a single colony of recombinant bacteria expressing recombinant Cap protein E.coli-PCV2-FJ-Cap to prepare recombinant Cap protein according to the method of steps 3-7 in Example 1, namely : Escherichia coli E.coli-PCV2-FJ-Cap CGMCCNo.18235 was inoculated in 20ml LB medium, cultivated overnight at 37°C and 200rpm as the seed solution, and the next day the seeds were mixed at a ratio of 1:100 Inoculate 5L of LB medium containing Kanna resistance, culture at 37°C, 200rpm for 2-3h, when the OD600 of the bacterial solution reaches 0.6-0.8, add IPTG with a final concentration of 1mmol / L, and continue to induce culture at 37°C for 5h. The bacteria were centrifuged at 5000rpm for 10min to collect the bacteria, and the bacteria were resuspended in 500mL PBS, and th...

Embodiment 3

[0070]Example 3: Immunogenicity Research of Porcine Circovirus Type 2 Recombinant Cap Protein Subunit Vaccine

[0071] 1. Piglet safety test

[0072] 35 circoantigen-antibody double-negative piglets aged 2-3 weeks were screened for the safety test of circovirus type 2 recombinant Cap protein subunit vaccine. Grouping situation: 30 piglets were randomly selected and divided into 5 groups, 5 piglets per group, as the vaccine immunization group, each pig in immunization group 1 was immunized with 4ml of circovirus type 2 recombinant Cap protein subunit vaccine containing 5 μg / ml recombinant Cap protein In immunization group 2, each pig was immunized with circovirus type 2 recombinant Cap protein subunit vaccine 4ml containing 10 μg / ml recombinant Cap protein; in immunization group 3, each pig was immunized with circovirus type 2 recombinant vaccine containing 20 μg / ml recombinant Cap protein Cap protein subunit vaccine 4ml, each pig in immunization group 4 was immunized with cir...

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Abstract

The invention discloses expression preparation and application of porcine circovirus type 2 recombinant Cap protein. The sequence of the porcine circovirus type 2 recombinant Cap protein is the sequence 2 in a sequence table. The coding gene is a sequence 1 in the sequence table. The preparation method of the porcine circovirus type 2 Cap recombinant protein virion comprises the following steps: 1) transforming an escherichia coli BL21 (DE3) competence by expressing a porcine circovirus type 2 Cap recombinant protein recombinant vector to obtain recombinant bacteria for expressing the porcinecircovirus type 2 Cap recombinant protein virion; 2) performing induced expression on the recombinant bacteria, collecting thalli and crushing, and collecting inclusion bodies; 3) dissolving and renaturating the inclusion body to obtain the assembled porcine circovirus type 2 Cap recombinant protein virion. The porcine circovirus type 2 Cap recombinant protein is expressed by adopting an escherichia coli expression system, the protein expression quantity is large, the purification method is simple, the production cost is greatly reduced, and the large-batch production after promotion is facilitated.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the inclusion body expression, purification, renaturation and application of porcine circovirus type 2 recombinant Cap protein. Background technique [0002] Porcine circovirus (porcine circovirus, PCV) belongs to Circoviridae, Circovirus genus, has three serotypes PCV1, PCV2 and PCV3. The PCV virion has an icosahedral symmetrical structure with a diameter of about 17nm. It is the smallest known animal virus and has no envelope on the surface of the virus. Porcine circovirus disease is an infectious disease caused by porcine circovirus type 2. Clinically, it mainly manifests as poor growth and development, emaciation, reproductive impairment in sows, dyspnea, highly enlarged subcutaneous lymph nodes, hemorrhage, and necrosis. Divided into weaned piglet multisystem failure syndrome (PMWS), porcine dermatitis and nephrotic syndrome (PNDS), proliferative and necrotizing pneumonia (PNP), ...

Claims

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Application Information

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IPC IPC(8): C07K14/01C12N15/34C12N15/70C12N1/21A61K39/12A61P31/20C12R1/19
CPCA61K39/12A61K2039/5258A61K2039/552A61P31/20C07K14/005C12N15/70C12N2750/10022C12N2750/10023C12N2750/10034C12N2750/10052
Inventor 张文亮李鹏宇刘新月栗利芳侯旭张彤肖进齐鹏
Owner CHINA ANIMAL HUSBANDRY IND
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