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Porcine circovirus II type capsid protein gene, construction of expression vector and efficient expression method of proteins of porcine circovirus II type capsid protein gene

A technology of capsid protein gene and porcine circovirus, applied in viral peptides, genetic engineering, plant genetic improvement, etc., can solve the problems of high production cost and limited application range, and achieve low cost, simple and stable method The effect of efficient expression

Active Publication Date: 2012-08-15
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although domestic PCV2 inactivated vaccines are already on the market, the production cost is still too high due to the limitation of virus content and other factors. As a result, the domestic circovirus inactivated vaccine is one of the most expensive swine disease vaccines. Expensive price of inactivated virus vaccine will limit its application

Method used

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  • Porcine circovirus II type capsid protein gene, construction of expression vector and efficient expression method of proteins of porcine circovirus II type capsid protein gene
  • Porcine circovirus II type capsid protein gene, construction of expression vector and efficient expression method of proteins of porcine circovirus II type capsid protein gene
  • Porcine circovirus II type capsid protein gene, construction of expression vector and efficient expression method of proteins of porcine circovirus II type capsid protein gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Synthesis of transformed porcine circovirus type II capsid protein gene

[0049] The porcine circovirus type II capsid protein gene PCV2-rCap was artificially synthesized without changing the natural amino acid sequence after DNA analysis and RNA structure prediction. The nucleotide sequence of the porcine circovirus type II capsid protein gene is the nucleotide sequence shown in SEQ ID NO.1.

Embodiment 2

[0050] Small amount preparation of embodiment 2 porcine circovirus type II capsid protein

[0051] 1. Construction of porcine circovirus type II capsid protein engineering yeast strain

[0052] 1) Materials and methods

[0053] Pichia pastoris GS115 and pPIC9K expression plasmid were purchased from Invitrogen, USA. DNA polymerase, restriction endonuclease EcoRI, NotI, SacI were purchased from TaKaRa Company, and T4 DNA ligase was purchased from NEB Company. For the specific preparation methods of BMGY, BMMY, and YPD medium, see the Pichia operation manual of Invitrogen Company. Plasmid extraction kits and PCR product recovery kits were purchased from Axgen. The primary antibody was porcine circovirus-positive serum, which was self-made, and the secondary antibody was rabbit anti-pig IgG-HRP antibody, which was purchased from Sigma;

[0054] 2) Artificially synthesized porcine circovirus type II capsid protein gene

[0055] A) The 5' end of the porcine circovirus type II c...

Embodiment 3

[0065] Example 3 Large-scale preparation of porcine circovirus type II capsid protein

[0066] 1. Materials:

[0067] Porcine circovirus type II capsid protein strain: pPIC9K-rCap-GS115 (prepared in Example 2);

[0068] Instruments: shaker, electrophoresis apparatus;

[0069] Medium: For the specific preparation methods of YPD, BMGY, and BMMY medium, see the Pichia operation manual of Invitrogen Company;

[0070] 2. Method

[0071] Induce expression at a ratio of 1:100, that is, use a sterilized toothpick to pick several G418-resistant single colonies pPIC9K-rCap-GS115 grown on the G418+YPD plate to activate in 100mL BMGY, and culture at 26°C with shaking at 260 rpm 24h, to OD 600 =4, the cells are in the logarithmic growth phase, and then directly add the activated cultured bacteria and BMGY medium to BMMY and add methanol to induce expression. In order to ensure sufficient dissolved oxygen, the amount of methanol added should be controlled, that is, every 24h Add methan...

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Abstract

The invention discloses a porcine circovirus II type capsid protein gene, construction of an expression vector and an efficient expression method of proteins of the porcine circovirus II type capsid protein gene. A sequence of the modified porcine circovirus II type capsid protein gene disclosed by the invention is shown as SEQ ID NO.1. The invention also discloses a preparation method of proteins of the porcine circovirus II type capsid protein gene and particularly relates to the modification of the porcine circovirus II type capsid protein gene, gene cloning and steps including the modification of operation methods such as efficient expression in pichia pastoris, protein capture time and method, protein content and antigenicity detection and the expansion of cultural conditions. The method disclosed by the invention is simple and practicable and low in cost, and realizes the efficient expression of the porcine circovirus II type capsid protein in pichia pastoris, in which the expression index in a test tube or a shake flask is greater than 218 mg / L, thus providing a foundation for the porcine circovirus II type antibody detection and the subunit vaccine development.

Description

technical field [0001] The invention relates to the transformation of porcine circovirus type II capsid protein gene, the construction of expression vector, the high-efficiency expression in Pichia pastoris and the preparation method of the protein. The invention belongs to the technical fields of animal genetic engineering and animal virology. Background technique [0002] German scholar Tischer et al. detected a small spherical virus and papovavirus-like virions morphologically similar to picornaviruses from the contamination virus of PK-15 cells in 1974 (Tischer et al.Characterization of papovavirus and picornavirus- like particles in permanent pig kidney cell lines.Zbl Bakt Hyg I Abt Orig A.1974,226:153-167), and in 1982 it was named porcine circovirus (Porcine circovirus, PCV) (Tischer et al.A very small porcine virus with circular single stranded DNA. Nature, 1982, 295: 64-66.). In 1991, a disease called postweaning multisystemic wasting syndrome (PMWS) was discovere...

Claims

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Application Information

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IPC IPC(8): C12N15/34C12N15/63C12N15/81C12N1/15C12N1/19C12N1/21C12N5/10C07K14/01
Inventor 蔡雪辉涂亚斌武嘉男
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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