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ExPEC (extraintestinal pathogenic escherichia coli) double-gene deleted strain and vaccine prepared from same

A double-gene, deletion strain technology, applied in the field of microorganisms, can solve the problems of inconvenient immunization pathways and poor immunization effects

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

[0005] Although traditional inactivated vaccines and subunit vaccines have been used all the time, they are gradually replaced by attenuated live vaccines due to poor immune effects and inconvenient immunization routes.

Method used

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  • ExPEC (extraintestinal pathogenic escherichia coli) double-gene deleted strain and vaccine prepared from same
  • ExPEC (extraintestinal pathogenic escherichia coli) double-gene deleted strain and vaccine prepared from same
  • ExPEC (extraintestinal pathogenic escherichia coli) double-gene deleted strain and vaccine prepared from same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 Construction of ExPEC double gene deletion strain DE205BΔrelA / spoT

[0019] In the present invention, the ExPEC double-gene deletion strain DE205BΔrelA / spoT is derived from the wild-type extraintestinal pathogenic Escherichia coli virulent strain, and the strain name is DE205B, which is isolated from ducks suffering from colibacillosis (ZhuGe X, Wang S, FanH, Pan Z, Ren J, Yi L, Meng Q, Yang X, Lu C, and Dai J.Characterization and Functional Analysis of AatB, a Novel Autotransporter Adhesin and VirulenceFactor of Avian Pathogenic Escherichia coli[J].Infection and Immunity.July2013 81:7 2437-2447). The present invention mainly adopts the method of Red homologous recombination to knock out the relA gene and the spoT gene in the virulent extra-intestinal Escherichia coli strain DE205B one by one to construct an ExPEC double-gene-deleted extra-intestinal pathogenic Escherichia coli.

[0020] The deleted ribosomal protein relA gene sequence is shown in SEQ ID NO.1...

Embodiment 2

[0032] Example 2: Effect of double deletion of relA and spoT genes on the pathogenicity of extra-enteric pathogenic Escherichia coli

[0033] In order to verify the attenuation effect of the deletion strain of the present invention on extraintestinal pathogenic E. The effect of strain and deletion strain on the lethality of animals. And test the effect of wild strain and deletion strain on the ability to infect DF-1 cells, as well as the detection of bacterial colonization levels in various viscera when infecting chicks and ducklings. The results showed that the double deletion of relA and spoT genes significantly reduced the pathogenicity of extra-intestinal Escherichia coli, and the deletion strain DE205BΔrelA / spoT was an attenuated strain.

[0034] 1. Chick embryo lethal test

[0035] Chicken embryo lethality test was used to determine the pathogenicity of wild-type DE205B and deletion strain DE205BΔrelA / spoT. The strains were cultured to mid-late logarithmic period, wash...

Embodiment 3

[0068] Example 3 Parenteral Pathogenic Escherichia coli Double Gene Deletion Vaccine

[0069] Identify the extra-intestinal pathogenic Escherichia coli double-gene deletion strain DE205BΔrelA / spoT, inoculate each generation on LB agar medium to observe the colony color, and use the genes of extra-intestinal pathogenic Escherichia coli to detect and identify the deletion Genetic stability of bacteria. After sub-passaging, it was found that DE205BΔrelA / spoT maintained the phenotypic characteristics of gene deletion, and the identified gene was still missing, and heredity was stable. The double-gene deletion strain DE205BΔrelA / spoT was cultured 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 every 100mL of deionized water, fully melt, sterilize at 121°C for 30min, and store for l...

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Abstract

The invention discloses an ExPEC (extraintestinal pathogenic escherichia coli) double-gene deleted strain and a vaccine prepared from the same. A construction method of the ExPEC double-gene deleted strain comprises the following steps: knocking out a relA gene and a spoT gene in an ExPEC virulent strain DE205B by a Red homologous recombination method to obtain an ExPEC relA and SpoT double-gene deleted strain DE205B[delta]relA / spoT, the strain is preserved in the China Center for Type Culture Collection, and the preservation number of the strain is CCTCC NO: M 2020610. The deleted strain has good biological safety and immunogenicity, is relatively weak in toxicity, and can be used for preparing the attenuated vaccine and preventing ExPEC infection. The attenuated vaccine prepared from the ExPEC double-gene deleted strain has an immune protective effect on ExPEC infection, and has a good toxin attacking immune protective effect.

Description

technical field [0001] The invention belongs to the field of microorganisms, and in particular relates to an ExPEC double-gene deletion strain and a vaccine prepared therefrom. Background technique [0002] Extraintestinal pathogenic Escherichia coli (ExPEC) can cause extraintestinal infections in humans or animals, such as Urinary Tract Infections (UTIs) in humans or mammals. UTIs are a type of disease that seriously endangers human health. Acute cystitis and pyelonephritis are caused by ExPEC; ExPEC can cause meningitis and sepsis in newborns, which seriously endanger the development of the central nervous system of newborns, and severe patients will die acutely or be mentally retarded for life; Infect poultry and cause multi-system mixed infection of poultry, including myocarditis, sepsis, perihepatitis and air sacculitis, etc. Highly pathogenic ExPEC can cause acute death of poultry hosts, and the disease has become an important bacterial pathogen that endangers the poul...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/54C12N15/55A61K39/108A61P31/04C12R1/19
Inventor 诸葛祥凯戴建君姜敏周洲王忠星
Owner NANTONG UNIVERSITY
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