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PhoP deleted pasteurella multocida attenuated strain of birds, as well as construction method and application thereof

A Pasteurella, construction method technology, applied in the field of genetic engineering, can solve the problems of incomplete coverage of virus strains, high production cost, strong virulence and other problems

Inactive Publication Date: 2015-04-29
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, lipopolysaccharide is the main virulence factor and immunogen of Pasteurella multocida, but among the 16 LPS serotypes, the structure of LPS and its effect on pathogenicity vary, making it a candidate for vaccine development target becomes difficult
All of the above vaccines have obvious defects: such as lack of stability and safety, the attenuated vaccines have the risk of strong virulence; most of the immunization routes are injections, the production cost is relatively high, and adjuvant components need to be added or processed by special processes; Covering strains is incomplete, and cannot achieve a good preventive effect
In Pasteurella multocida, a globally regulated gene phoP The research has not been reported, and the existing technology has not yet incorporated it into the known or potential virulence-related genes, and there is no report on the phoP Whether transformation can obtain attenuated strain

Method used

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  • PhoP deleted pasteurella multocida attenuated strain of birds, as well as construction method and application thereof
  • PhoP deleted pasteurella multocida attenuated strain of birds, as well as construction method and application thereof
  • PhoP deleted pasteurella multocida attenuated strain of birds, as well as construction method and application thereof

Examples

Experimental program
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Embodiment 1

[0066] Embodiment 1 Pasteurella multocida phoP Functional identification of genes

[0067] 1. Primer design (for functional identification)

[0068] According to bioinformatics analysis, the amino acid sequence of PhoP protein of Salmonella typhimurium UK-1 strain was compared with the published whole genome sequence of Pasteurella avium multocida Pm 70 to obtain two potential Pasteurella multocida phoP gene sequence, named phoP1 and phoP2 . phoP1 The nucleotide sequence is shown in SEQ ID No.5; phoP2 The nucleotide sequence of is shown in SEQ ID No.1.

[0069] According to sequence SEQ ID No.5, and sequence SEQ ID No.1, design primers respectively ( phoP1 -F / phoP1 -R and phoP2 -F / phoP2 -R, see Table 1), to virulent strains of Pasteurella avuli Pasteurlla multocida The 0818 genome was used as a template to amplify potential phoP1 and phoP2 gene, and introduced at both ends of the product Nco I and Bam H Ⅰ Restriction site. Primers were synthesized ...

Embodiment 2

[0081] Example 2 Pasteurella multocida phoP Construction of gene deletion strains

[0082] 1. Primer design (for the construction and identification of mutant strains)

[0083] According to the results in Example 1, with reference to the whole genome sequence of Pasteurella avium multocida Pm 70, two pairs of primers (Dp-1F / Dp-1R, Dp-2F / Dp-2R) were designed from Pasteurella avium multocida Amplified separately in Pm 0818 phoP The upstream and downstream segments of the gene: up- phoP (upper arm) and down- phoP (lower arm), the sizes of the amplified fragments are 402bp and 435bp respectively, and the end of the upper arm and the front end of the lower arm contain 18bp of the same repetitive sequence, which is convenient for the fusion of the upper and lower homology arms. Design a pair of primers ( can -F / can -R) Amplify the kanamycin gene fragment from pYA4372 plasmid DNA ( can ). Three pairs of primers were designed for the identification of mutant strains, one p...

Embodiment 3

[0094] Example 3. Pasteurella multocida P. multocia 0818 Δ phoP Study on Biological Characteristics of Gene Deletion Strains

[0095] 1. Identification of biochemical characteristics of gene deletion strains

[0096] The gene deletion strains were respectively inserted into biochemical tubes such as glucose, maltose, lactose, sucrose, rhamnose, and mannitol to verify the carbon source metabolism ability, and the H 2 S test, MR test and VP test to observe its biochemical reaction characteristics.

[0097] experiment result shows: Pasteurella multocida 0818 Δ phoP All of them and their parental strains can utilize glucose, mannitol and sucrose; they cannot utilize lactose, rhamnose and maltose. h 2 S test, MR test and VP test were all negative.

[0098] 2. Determination of growth characteristics of gene deletion strains

[0099] Inoculate the wild strain and the gene-deficient strain into the brain-heart infusion medium, shake well and divide into multiple test tubes,...

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Abstract

The invention discloses a phoP deleted pasteurella multocida attenuated strain of birds, as well as a construction method and application thereof, and belongs to the technical field of biological engineering. The attenuated strain has a classification name of Pasteurella multocida subsp.multocida str pm0818 delta phoP, is preserved in the China Center for Type Culture Collection, has a preservation number of CCTCC NO: M2014522, and is preserved on Oct. 29, 2014. The invention further provides a method for preparing the attenuated strain. According to the method, a phoP gene is knocked out based on a suicide plasmid mediated homologous recombination method to prepare the attenuated strain. The attenuated strain has relatively low toxicity, and can be used for studying a living vaccine for preventing fowl cholera. The invention discloses application of the attenuated strain on vaccines.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to deletion phoP The attenuated strain of Pasteurella multocida and its construction method and application. Background technique [0002] Pasteurella multocida ( Pasteurella multocida ) Gram-negative short bacilli capable of causing disease in a variety of animals. People can also be infected with the bacteria when they are scratched by cats and dogs. Pasteurella multocida is widely prevalent in the world and can infect a variety of animals through the respiratory tract or oral cavity, causing significant economic losses to aquaculture and animal husbandry. Organization as a class of important animal infectious diseases. [0003] Pasteurella multocida is widely prevalent in my country. Fowl cholera caused by the infection of poultry by this bacterium occurs from time to time among chickens and ducks in the vast rural areas, causing an acute course characterize...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74A61K39/102A61P31/04C12R1/01
Inventor 孔庆科肖康鹏刘青赵新新杨雪
Owner SICHUAN AGRI UNIV
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