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Method for producing porcine circovirus type II recombinant capsid protein subunit vaccine by utilizing silkworm bioreactor and products thereof

A porcine circovirus and subunit vaccine technology, applied in biochemical equipment and methods, botanical equipment and methods, viral peptides, etc., can solve the limitations of large-scale production and application of PCV2Cap protein, ineffective modification, foreign protein Low efficiency and other problems, to achieve the effect of enriching silkworm resources, high modification efficiency, and easy separation and purification

Active Publication Date: 2012-12-12
PU LIKE BIO ENG +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, commercially available insect cells and cell culture media are expensive, and large-scale cell culture requires the purchase of special cell culture bioreactors, which requires a large investment, and the production process is easily polluted by environmental conditions, and cell culture can obtain recombinant protein products Post-translational modification cannot be carried out efficiently, and the efficiency of cell culture to produce foreign proteins is relatively low
These all limit the large-scale production and application of PCV2 Cap protein in insect cells

Method used

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  • Method for producing porcine circovirus type II recombinant capsid protein subunit vaccine by utilizing silkworm bioreactor and products thereof
  • Method for producing porcine circovirus type II recombinant capsid protein subunit vaccine by utilizing silkworm bioreactor and products thereof
  • Method for producing porcine circovirus type II recombinant capsid protein subunit vaccine by utilizing silkworm bioreactor and products thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Example 1 Bombyx mori baculovirus expression system expresses recombinant porcine circovirus type 2 capsid protein

[0041] 1) Cloning the gene sequence ORF2 of porcine circovirus type 2 capsid protein, and connecting it with the baculovirus transfer vector to obtain the recombinant transfer vector;

[0042] The DNA of PCV2-SH strain (preservation number: CGMCC No. 2389) was extracted as a template for amplifying the Cap protein gene, and stored at -20°C for future use.

[0043] According to the complete PCV2 gene sequence in GenBank, a pair of primers were designed to amplify the PCV2 ORF2 open reading frame gene sequence. Add BamH I and Xhol I restriction sites (underlined) to the 5' ends of the upstream and downstream primers, respectively.

[0044] Upstream primer: 5′-CGC GGATCC ATGACGTATCCAAGGAGGC-3′;

[0045] Downstream primer: 5′-CCG CTCGAG GGGTTTAAGTGGGGGGTCT-3';

[0046]The PCV2 ORF2 gene fragment was amplified by PCR with the designed primers, and the b...

Embodiment 2

[0068] Efficacy detection of embodiment 2 recombinant PCV2 Cap protein subunit vaccine:

[0069] Screen pigs negative for major pathogens such as porcine circovirus, swine fever virus, porcine blue ear disease virus, and porcine parvovirus at the age of 10 to 14 days. Qualified piglets were randomly divided into 9 groups, 10 in each group, and 7 groups were given subunit vaccines containing different amounts of recombinant Cap protein (comprising 0.2 μg / head, 0.5 μg / head, and 1 μg of recombinant capsid protein respectively). / head portion, 2 μg / head portion, 5 μg / head portion, 10 μg / head portion and 15 μg / head portion), one group was used as a challenge control, and the other group was used as a negative control. Among them, group 1 was given a subunit vaccine containing 0.2 μg / head of recombinant Cap protein; group 2 was given a subunit vaccine containing 0.5 μg / head of recombinant Cap protein; group 3 was given 1 μg / head of recombinant Cap protein subunit vaccine; group 4 w...

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Abstract

The invention provides a method for producing porcine circovirus type II capsid protein by utilizing a silkworm bioreactor, and belongs to the field of biotechnology. In the method, by taking a bombyx nuclear polyhedrosis virus as a vector, porcine circovirus type II capsid protein genes are integrated into a polyhedrosis promoter of the bombyx nuclear polyhedrosis virus so as to express target protein by a mode of the homologous recombination of a homologous arm of the nuclear polyhedrosis virus in a transfer vector and bombyx nuclear polyhedrosis virus (BmNPV) genes; a recombinant bombyx nuclear polyhedrosis virus containing target protein genes is obtained by a plaque sieving method, and the target protein is expressed in large scale by using a bombyx bioreactor so as to prepare a subunit vaccine containing recombinant porcine circovirus type II capsid protein; and piglet infection experiments verify that the subunit vaccine has the excellent immune protective effect. The method has the characteristics of high expression efficiency, good activity of the target protein, low production cost and the like, and is suitable for large-scale production.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for producing a porcine circovirus type 2 recombinant capsid protein subunit vaccine by using a silkworm bioreactor and a product thereof. Background technique [0002] Porcine circovirus type 2 (Porcine circovirus II, PCV2) belongs to circoviridae (circoviridae) circovirus genus (circovirus), porcine circovirus type 2 (PCV2) infection can damage the immune system of pigs, causing weaned piglets Porcine circovirus disease (PCVD) such as systemic wasting syndrome (PMWS). The open reading frame 2 (ORF2) of porcine circovirus type 2 (PCV2) encodes the viral capsid protein (Cap), which is about 30KDa in size. There is an antigen expression site on the Cap protein, which is immunogenic, so it becomes the basis of PCV2-specific serological detection methods, subunit vaccines, and nucleic acid vaccines. [0003] Patent CN101180406A discloses a method for producing PCV2 Cap protein ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/12C12N15/34C12N15/866C12N7/01C07K14/01A61P31/20
Inventor 张许科孙进忠乔荣岑
Owner PU LIKE BIO ENG
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