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Recombinant Bordetella bronchiseptica strain expressing ORF2 gene fragment of porcine circovirus type 2, vaccine and application

A technology of Bordetella blood and circovirus, applied in application, antiviral agents, genetic engineering, etc., can solve biological safety problems and other problems, and achieve the effects of convenient immune operation, good immune protection, and broad market application prospects

Inactive Publication Date: 2011-09-28
HUAZHONG AGRICULTURAL UNIVERSITY +1
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Problems solved by technology

Most of the studies reported so far generally use expression plasmids carrying resistance genes, which are not accepted by people due to biosafety issues

Method used

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  • Recombinant Bordetella bronchiseptica strain expressing ORF2 gene fragment of porcine circovirus type 2, vaccine and application
  • Recombinant Bordetella bronchiseptica strain expressing ORF2 gene fragment of porcine circovirus type 2, vaccine and application
  • Recombinant Bordetella bronchiseptica strain expressing ORF2 gene fragment of porcine circovirus type 2, vaccine and application

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Embodiment 1

[0034] 1. Primer design and PCR amplification

[0035] According to the reported whole genome sequence of Bordetella bronchiseptica RB50 (refer to the gene sequence of GenBank accession number AF315119), two pairs of primers were designed (all primers were synthesized by Shanghai Sangon Bioengineering Technology Co., Ltd.) to amplify the upstream of aroA gene respectively. For the homology arm and the downstream homology arm, the amplified fragment sizes are 1388bp and 1383bp, respectively. KpnI and SalI restriction sites are designed at both ends of the upstream arm, and HindIII and SacI restriction sites are respectively designed at both ends of the downstream arm. According to the reported whole genome sequence of porcine circovirus type 2 WH strain (refer to the gene sequence of GenBank accession number FJ598044), a pair of primers were designed to amplify the ORF2 gene, the size of the amplified fragment was 582bp, and SalI and HindIII enzymes were designed at both ends c...

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Abstract

The invention discloses the construction of a recombinant Bordetella bronchiseptica strain which does not contain a resistance marker and is used for expressing an ORF2 gene fragment of porcine circovirus type 2, the preparation of a vaccine and application, and belongs to the field of genetic engineering. The recombinant Bordetella bronchiseptica strain QH0814delta aroA / ORF2 which does not contain the resistance marker and is used for expressing the ORF2 gene fragment of the porcine circovirus type 2 is obtained. In the strain, a 5-enolpyruvyl-shikimate-3-phosphate synthase (aroA) gene of Bordetella bronchiseptica is deleted; and the strain contains the ORF2 gene fragment of the porcine circovirus type 2. The invention discloses the preparation of the recombinant strain and the vaccine and application of the recombinant strain and the vaccine to the preparation of porcine circovirus type 2 and porcine Bordetella bronchiseptica vaccines. The genetic engineering vaccine can stimulate pigs to produce the protective immune reaction of resisting the porcine circovirus type 2 and porcine Bordetella bronchiseptica wild strains, and can effectively prevent the porcine circovirus type 2 and porcine Bordetella bronchiseptica from infecting the pigs.

Description

technical field [0001] The invention belongs to the technical field of animal bacterial genetic engineering, and in particular relates to the construction, vaccine preparation and application of a recombinant Bordetella bronchiseptica live vaccine strain expressing porcine type 2 circovirus ORF2 gene fragments without a resistance marker. Background technique [0002] Bordetella Bronchiseptica (Bb) is a Gram-negative bacterium with a wide host range, and most mammals and birds can be infected. This bacterium is one of the main pathogenic bacteria that cause atrophic rhinitis (Atropic Rhinitis, AR) in pigs. Bb alone often causes recessive infection in pigs. The clinical manifestations of diseased pigs are slow growth and fattening delay, which greatly reduces feed remuneration and Production efficiency; and pig atrophic rhinitis caused by the joint action of Bb and toxin-producing Pasteurella multocida (Pm) is clinically characterized by rhinitis, turbinate atrophy and decrea...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/34A61K39/295A61P31/20A61P31/04C12R1/01A61K39/02A61K39/12
Inventor 吴斌夏欣何启盖汤细彪陈焕春金梅林李伦勇陈冬焕余腾吴磊刘文静胡睿铭
Owner HUAZHONG AGRICULTURAL UNIVERSITY
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