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Porcine circovirus 2 type recombinant subunit vaccine and preparation method thereof

A technology of porcine circovirus and subunit vaccine, which is applied in the fields of biochemical equipment and methods, antiviral agents, viral antigen components, etc.

Active Publication Date: 2012-12-12
河南后羿生物工程股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no registered or domestically produced PCV2 commercial vaccine in my country at present, and there is an urgent need for a safe and highly protective vaccine against PCV2 clinically.

Method used

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  • Porcine circovirus 2 type recombinant subunit vaccine and preparation method thereof
  • Porcine circovirus 2 type recombinant subunit vaccine and preparation method thereof
  • Porcine circovirus 2 type recombinant subunit vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] A porcine circovirus type 2 recombinant subunit vaccine, mainly composed of porcine circovirus type 2 fusion protein rYSORF and Freund's incomplete adjuvant, the Freund's adjuvant added in the fusion protein rYSORF is incomplete Freund's adjuvant Adjuvant, first adjust the purified fusion protein to 1 mg / ml, and add it under stirring according to the volume ratio of Freund's incomplete adjuvant to fusion protein at 1:1.

Embodiment 2

[0065] A preparation method of porcine circovirus type 2 recombinant subunit vaccine, the specific steps of the method are as follows:

[0066] SOEing PCR Synthesis of Artificial Gene YSORF

[0067] According to literature reports, there are two main open reading frames in the PCV2 virus genome: ORF1 and ORF2. Among them, ORF1 encodes a protein related to replication, which has weak immune protection, and its immunodominant region is around 180-220aa; while ORF2 encodes the main structural protein of the virus, which is the main immunoprotective protein of PCV2, and its immunodominant region is at 60~210aa. The immunodominant regions of ORF1 and ORF2 proteins were cut out, and the flexible peptide (G 4 S) 5 Connect the two immunodominant regions to form the amino acid sequence of the fusion protein rYSORF, and then use the bioinformatics software Vector NTI to optimize the codons of the sequence to obtain the sequence of the artificial gene YSORF, and at the same time add B...

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Abstract

The present invention relates to a porcine circovirus 2 type recombinant subunit vaccine and a preparation method thereof. According to the present invention, an ORF1 gene dominant antigen epitope region and an ORF2 gene dominant antigen epitope region of porcine circovirus 2 type are reasonably combined; bioinformatics and a SOEing PCR technology are adopted to optimize codons to obtain an artificial gene sequence YSORF; the artificial gene sequence YSORF is constructed to a prokaryotic expression vector; escherichia coli is adopted to express recombinant fusion protein; and the resulting recombinant fusion protein is subjected to moderate purification, and then is mixed with an adjuvant to prepare a subunit vaccine containing an ORF1 protein fragment and an ORF2 protein fragment. With the present invention, the ORF1 gene dominant antigen epitope region and the ORF2 gene dominant antigen epitope region of the porcine circovirus 2 type are combined in a series connection manner creatively by using the flexible peptide, such that the number of the epitope is increased, and correct spatial conformation of the fusion protein is easily formed; the codons of the ORF1 and the ORF2 are optimized so as to increase an expression amount of the fusion protein and reduce production cost. The vaccine of the present invention has advantages of high yield, low cost, strong protection efficacy, and the like. In addition, the preparation method of the present invention is suitable for large-scale production.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to a porcine circovirus type 2 recombinant subunit vaccine, a preparation method of the vaccine, and a method for constructing artificial gene YSORF and fusion protein rYSORF. technical background [0002] Porcine circovirus disease (PCV) is an infectious disease of pigs caused by porcine circovirus type 2 (PCV2), and it is another important disease newly discovered after porcine reproductive and respiratory syndrome (PRRS). Since Clark et al first isolated porcine circovirus type 2 (PCV2) in 1997, the disease has caused huge economic losses to the swine industry all over the world. At present, how to effectively prevent and control PCV has become the focus of the global pig industry. [0003] So far, four commercialized PCV2 vaccines have been released in the world, among which the PCV2 vaccine developed by Merial Animal Health It is a whole virus inactivated vaccine, suitable fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/12A61P31/20C12N15/62C07K19/00C12R1/93
Inventor 吴红云郑鸣徐进李厚伟
Owner 河南后羿生物工程股份有限公司
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