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Method for constructing ompA gene-deleted riemrellaanatipestifer CH3 attenuated strain

A technology of Riemerella anatipestifer and a construction method, which is applied in the field of construction of a Riemerella anatipestifer CH3 attenuated strain lacking the ompA gene, can solve the problem that the total amino acid transport rate is decreased, the coverage of the virus strain is incomplete, and the complete protection cannot be provided. Injection absorption and other problems

Inactive Publication Date: 2018-06-29
安徽安龙基因科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

And these inactivated vaccines have defects: as the widely used oil emulsion fire extinguishing vaccines cannot provide complete protection (protection rate of about 70%) and there are problems such as unsatisfactory injection absorption and incomplete strain coverage. Riemerella anatipestifer covering multiple serotypes has become an urgent need for duck industry in my country
Mutant strains lacking the OmpA protein have been reported to have unstable outer membrane function, decreased overall amino acid transport, inability to transport peptides, and loss of F factor-mediated cell engagement

Method used

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  • Method for constructing ompA gene-deleted riemrellaanatipestifer CH3 attenuated strain
  • Method for constructing ompA gene-deleted riemrellaanatipestifer CH3 attenuated strain
  • Method for constructing ompA gene-deleted riemrellaanatipestifer CH3 attenuated strain

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Embodiment 1

[0119] The first step: preparation of Riemerella anatipestifer CH3 attenuated strain:

[0120] OmpA, as an adhesin or invasin, is related to the virulence of bacteria. Hu et al. obtained the ompA gene deletion strain Th4ΔompA of the serotype 2 Riemerella anatipestifer Th4 strain through homologous recombination. The results of the study showed that the virulence of the Th4ΔompA strain significantly lower than that of the parental strain Th4, and the adhesion and invasion ability to Vero cells was also significantly lower than that of the parental strain Th4, indicating that OmpA is a very important virulence factor in Riemerella anatipestifer, and may be an adhesion factor ;

[0121] Step 2: Construction of R. anatipestifer CH3 mutant strain, design primers according to ompA upstream and downstream homology arms of R. anatipestifer CH3 whose gene function has been determined

[0122] ompA-L F: TTGGATCCTGTATTTCAATGATTGGTATAGTAATT

[0123] ompA-L R: CTTGGCCATCCAATTCTCTTATTATCA...

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Abstract

The invention discloses a method for constructing an ompA gene-deleted riemrella anatipestifer CH3 attenuated strain. The method specifically comprises the following steps: culturing a riemrella anatipestifer CH3 strain and genome; cloning a homologous arm of riemrella anatipestifer CH3 strain ompA gene and a resistance spec gene fragment; constructing a recombinant plasmid pT-ompA-LSR; constructing a suicidal recombinant plasmid pDS-ompA-LSR; and transferring the suicidal recombinant plasmid pDS-ompA-LSR into BW19851 Escherichia coli. The method provided by the invention is very meaningful for a molecular pathogenic mechanism of riemrella anatipestifer (RA), and lays a foundation for future research and development of seedlings.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for constructing an attenuated strain of Riemerella anatipestifer CH3 lacking the ompA gene. Background technique [0002] Riemerella anatipestifer, also known as duck infectious serositis, is a contagious disease caused by Riemerella anatipestifer (RA), which mainly affects ducks, turkeys, geese and other poultry. . At least 21 serotypes of Riemerella anatipestifer have been identified so far, and the cross-immune protection between serotypes is poor. At present, little is known about the molecular pathogenic mechanism of RA. In addition, the complete genome sequences of 5 strains of Riemerella anatipestifer have been submitted to GenBank, and the analysis results show that there are about 2000 predicted proteins encoded by Riemerella anatipestifer, but the understanding of the biological functions of these proteins is not yet known. just started. OmpA p...

Claims

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Application Information

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IPC IPC(8): C12N1/21A61K39/02A61P31/04C12R1/01
CPCC07K14/195A61K39/0208A61K2039/522
Inventor 韦玉军李航崔俊生苏军吴远航
Owner 安徽安龙基因科技有限公司
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