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Recombinant baculovirus expressing porcine circovirus type 3 truncated Cap protein and construction method and primers thereof

A technology of recombinant baculovirus and porcine circovirus, applied in the direction of virus, virus peptide, virus/phage, etc., to achieve good expression effect

Active Publication Date: 2019-01-15
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are few reports on PCV3 vaccines, and the expression of PCV3Cap protein by constructing recombinant baculovirus can lay the foundation for the development of PCV3 subunit vaccines

Method used

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  • Recombinant baculovirus expressing porcine circovirus type 3 truncated Cap protein and construction method and primers thereof
  • Recombinant baculovirus expressing porcine circovirus type 3 truncated Cap protein and construction method and primers thereof
  • Recombinant baculovirus expressing porcine circovirus type 3 truncated Cap protein and construction method and primers thereof

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Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0040] 1. Construction of recombinant baculovirus rBac-ΔCap expressing porcine circovirus type 3 truncated Cap gene:

[0041] The primer sequences designed for amplification are as follows:

[0042] Table 2 Required primers and enzyme cutting sites

[0043]

[0044] Note: The underlined part is each enzyme cutting site

[0045] 1. Construct pSK-sPCV3 containing the whole PCV3 genome:

[0046] Using the extracted PCV3 DNA as a template, use primers Xho I-3F and primers EcoR I-3R to amplify the whole genome of PCV3; recover the PCV3 whole genome gel and cut it with pBluescript SK(+) vector simultaneously with Xho I and EcoR I , after purification and recovery of the two digested products, they were ligated at a molar ratio of 3:1, and finally the recombinant plasmid pSK-sPCV3 was obtained. The recombinant plasmid was identified by double digestion with XhoI and EcoR I, and the digested product was electrophoresed with 0.7% agarose gel to obtain bands of about 2958bp and 20...

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Abstract

The invention belongs to the technical field of vaccine manufacturing. The invention discloses a recombinant baculovirus rBac-[delta]Cap expressing porcine circovirus type 3 truncated Cap protein, with the accession number of the recombinant baculovirus being CGMCC No. 15691. The invention also discloses primers used for constructing the recombinant baculovirus, and a constructing method of the recombinant baculovirus. The method comprises the following steps: 1, constructing pSK-SPCV3 containing PCV3 whole genome; 2. Constructing a transfer vector; 3. constructing a shuttle plasmid; 4. obtaining recombinant baculovirus, wherein the positive shuttle plasmid Bacmid-[delta]Cap transfects Sf9 cells to obtain the recombinant baculovirus rBac-[delta]Cap expressing the porcine circovirus type 3truncated Cap protein. The method can conveniently and quickly construct the recombinant baculovirus expressing the PCV3 truncated Cap protein, and can confirm that the expressed truncated Cap proteinhas good biological activity, and immunizes mice to find out that the recombinant baculovirus has good immunogenicity.

Description

technical field [0001] The invention relates to a construction method of a recombinant baculovirus expressing a porcine circovirus type 3 (PCV3) truncated Cap gene, which is used for making a vaccine. Background technique [0002] Porcine circovirus disease (PCVD) is one of the important diseases that endanger the swine industry at present, and PCV2 is the main pathogenic factor leading to this kind of disease. In 2016, Phan and other scholars reported a new type of porcine circovirus PCV3, which can cause porcine dermatitis and nephrotic syndrome (PDNS), porcine reproductive disorders, respiratory diseases, and digestive system inflammation [Phan TG, Giannitti F, Rossow S, et al. Detection of a novel circovirus PCV3in pigs with cardiac and multi-systemic inflammation [J]. Virology Journal, 2016, 13(1): 184.]. Through the analysis of PCV3 genome-wide genetic evolution, it was found that PCV3 is in a different evolutionary branch from the other two genotypes of PCV1 and PCV2...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866C12N15/66
CPCC12N15/86C07K14/005C12N2710/14043C12N2750/10022
Inventor 高崧程清如高清清王小波郇长超刘秀梵
Owner YANGZHOU UNIV
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