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Method for producing type 2 capsid protein grain vaccine of porcine circovirus by pichia pastoris expression system

A technology for porcine circovirus and capsid protein, applied in the biological field, can solve the problems of poor protein immunogenicity, affecting the large-scale production and application of PCV-2Cap protein, and high production cost, achieving high expression, low cost and good protection. Effect

Inactive Publication Date: 2013-06-26
BEIJING DAWEIJIA BIOTECH SHARE CO LTD
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  • Abstract
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Problems solved by technology

However, due to factors such as high production cost or poor protein immunogenicity, the large-scale production and application of PCV-2Cap protein have been seriously affected.

Method used

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  • Method for producing type 2 capsid protein grain vaccine of porcine circovirus by pichia pastoris expression system
  • Method for producing type 2 capsid protein grain vaccine of porcine circovirus by pichia pastoris expression system
  • Method for producing type 2 capsid protein grain vaccine of porcine circovirus by pichia pastoris expression system

Examples

Experimental program
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Effect test

example 2

[0034] 2. Efficacy detection of the recombinant PCV2 Cap capsid protein particle-like vaccine of Example 2:

[0035] Granule-like vaccines containing different doses of recombinant Cap protein were prepared according to the emulsification method in Example 1 (respectively comprising recombinant capsid protein 0.5 μg / head portion, 1 μg / head portion, 2 μg / head portion, 5 μg / head portion, 10 μg / head portion) To screen 10-14 day-old pigs free of major pathogens such as porcine circovirus, swine fever virus, porcine parvovirus, and porcine blue-ear disease virus. The qualified piglets were randomly divided into 7 groups, 10 pigs in each group, 5 groups were given particle-like vaccines containing different amounts of recombinant Cap protein, 1 group was used as the challenge control group, and the other group was used as the negative control group. The vaccine group was immunized twice with an interval of 2 weeks, and the challenge group was injected with the same dose of PBS, 1ml ...

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Abstract

The invention provides a method for producing type 2 capsid protein grain vaccine of porcine circovirus by a pichia pastoris expression system. The method comprises the following steps of: based on a PCV2-TJ strain gene sequence in GenBank as a template, obtaining a mature coding sequence of PCV-2 capsid protein Cap by amplification by a normal PCR method; connecting the gene with a capsid protein secreting type expression carrier pP-secSUMO3 to construct a pP-secSUMO3-Cap recombinant plasmid and amplifying by a heat stock method to convert escherichia coli JMS115; purifying the positive recombinant plasmid and electrically converting pichia pastoris strain GS115 by an electric converter of BioRad company; sieving high copy strains according to concentration gradient of antibiotic ZeocinTm; and respectively extracting genomic DNAs of the high copy strains as the template to perform PCR reaction to identify positive clone. The piglet challenge test verifies that the grain vaccine has good immunoprotection effect.

Description

technical field [0001] The invention provides a method for producing porcine circovirus type 2 (PCV-2) particle-like vaccine using a Pichia pastoris expression system, which belongs to the field of biotechnology and aims to strengthen the immune protection of pigs against porcine circovirus type II Effect and reduce the economic loss of the pig farm. technical background [0002] Porcine circovirus II (PCV-2) belongs to the genus circovirus of the Circoviridae family (Circoviridae). The PCV-2 genome is 1767bp or 1768bp in length and contains 11 open reading frames (ORF) , wherein ORF1 and Cap are the two largest open reading frames, encoding replication-associated protein (Rep protein) and capsid protein (Cap protein), respectively. Cap protein is the main structural protein, has good immunogenicity, and can be used as a candidate protein for vaccines. At present, there have been a large number of relevant reports at home and abroad. Luoyang Pulaike has successfully expres...

Claims

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Application Information

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IPC IPC(8): A61K39/12A61P31/20C12N15/81C07K14/01C12R1/84
Inventor 文贤子张志刚杜恩岐廖峰
Owner BEIJING DAWEIJIA BIOTECH SHARE CO LTD
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