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APEC (avian pathogenic E .coli) double-gene rfaH and hfq deleted strain and attenuated vaccine

An attenuated vaccine and dual-gene technology, applied in the field of microorganisms, can solve the problems of inconvenient immunization route and poor immunization effect, and achieve broad market application prospects, good immune protection and biological safety, and the effect of reducing pathogenicity.

Inactive Publication Date: 2021-04-20
NANTONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although traditional inactivated vaccines and subunit vaccines have been used, they are gradually replaced by attenuated live vaccines due to poor immune effects and inconvenient immunization routes.

Method used

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  • APEC (avian pathogenic E .coli) double-gene rfaH and hfq deleted strain and attenuated vaccine
  • APEC (avian pathogenic E .coli) double-gene rfaH and hfq deleted strain and attenuated vaccine
  • APEC (avian pathogenic E .coli) double-gene rfaH and hfq deleted strain and attenuated vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1 Construction of APEC double gene rfaH and hfq deletion strain (avian pathogenic Escherichia coli rfaH and hfq double gene deletion strain)

[0017] In the present invention, the rfaH and hfq double gene deletion strain of avian pathogenic Escherichia coli is derived from the wild-type avian pathogenic Escherichia coli virulent strain, and the strain name is FY26, which is isolated from chickens suffering from colibacillosis. (Zhuge X, Sun Y, Jiang M, Wang J, Tang F, Xue F, Ren J, Zhu W, Dai J. Acetate metabolic requirement of avianpathogenic Escherichia coli promotes its intracellular proliferation within macrophage. Veterinary research. 2019 Dec 1; 50(1 ): 31.) The present invention mainly adopts the method of Red homologous recombination to knock out the rfaH and hfq double genes in the said avian pathogenic E. bacilli.

[0018] The missing rfaH gene sequence is shown in SEQ ID NO.1.

[0019] The deleted hfq gene sequence is shown in SEQ ID NO.2.

[0020]...

Embodiment 2

[0031] Example 2: The effect of deletion of rfaH and hfq genes on the pathogenicity of avian pathogenic Escherichia coli

[0032] In order to verify the attenuation effect of the deletion strain of the present invention on avian pathogenic Escherichia coli infection, through a variety of animal infection tests, including chicken embryo infection test, chick infection test and duckling infection test, etc., the wild strain and the deletion strain were determined to chicken. The effect of lethal dose. The effect of the wild strain and the deletion strain on the ability to infect DF-1 cells was tested, as well as the level of bacterial colonization in various viscera when infected chicks were tested. The results showed that the deletion of rfaH and hfq genes significantly reduced the pathogenicity of avian pathogenic Escherichia coli, and the deletion strain FY26ΔrfaH / hfq was an attenuated strain.

[0033] 1 Chick embryo lethal test

[0034] Chicken embryo lethality test was us...

Embodiment 3

[0062] Embodiment 3: Poultry pathogenic Escherichia coli attenuated vaccine (avian pathogenic Escherichia coli rfaH and hfq double gene deletion vaccine)

[0063]The obtained avian pathogenic Escherichia coli rfaH and hfq double gene deletion strain FY26ΔrfaH / hfq was identified, and each generation was inoculated on LB agar medium to observe the colony color, and the gene of avian pathogenic Escherichia coli was used to detect and identify Loss of genetic stability in bacteria. After sub-passaging, it was found that FY26ΔrfaH / hfq maintained the phenotypic characteristics of gene deletion, and the identified gene was still missing, and heredity was stable. The rfaH and hfq double gene deletion strain FY26ΔrfaH / hfq was cultivated on solid medium, and a single colony was picked and cultured in liquid medium until the concentration of viable bacteria reached 5.0×10 9 CFU / mL. The preparation method of the gelatin protectant is as follows: add 40g of sucrose and 9g of gelatin to e...

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Abstract

The invention belongs to the field of microorganisms, and discloses an APEC (avian pathogenic E .coli) double-gene rfaH and hfq deleted strain and an attenuated vaccine. A construction method of the APEC double-gene deletion strain is a Red homologous recombination method, rfaH and hfq double genes are knocked out to obtain APEC FY26[delta]rfaH / hfq, and the APEC FY26[delta]rfaH / hfq is preserved in China Center for Type Culture Collection, and the preservation number of the strain is CCTCC NO: M 2020612. The deleted strain has good biological safety and immunogenicity, is low in toxicity, and can be used for preparing the APEC attenuated vaccine to prevent APEC infection. The attenuated vaccine prepared from the APEC FY26[delta]rfaH / hfq has immune protection against APEC infection, and has good toxicity attacking immune protection effect.

Description

technical field [0001] The invention belongs to the field of microorganisms, and in particular relates to an APEC double-gene rfaH and hfq deletion strain and an attenuated vaccine prepared therefrom. Background technique [0002] Avian pathogenic Escherichia coli (Avian pathogenic E.coli, APEC) is an important bacterial pathogen that endangers the poultry industry, and it belongs to extraenteric pathogenic Escherichia coli (ExPEC). Avian pathogenic Escherichia coli mainly infects poultry through the respiratory tract, leading to multi-system mixed infection of poultry, and even acute death of poultry, causing serious losses to the poultry industry and posing a threat to food safety. The pathogenic subtypes of extraintestinal Escherichia coli are named as urethral pathogenic Escherichia coli (UPEC), sepsis Escherichia coli (SEPEC), neonatal meninges coli (NMEC) and avian pathogenic Escherichia coli. The evolution relationship between avian pathogenic Escherichia coli and c...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70A61K39/108A61P31/04C12R1/19
Inventor 诸葛祥凯戴建君姜敏周洲王忠星
Owner NANTONG UNIVERSITY
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