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Salmonella enteritidis double knockout attenuated mutant and preparation as well as application thereof

A Salmonella enteritidis, gene knockout technology, applied in the field of microbiology, can solve the problem of bacterial drug resistance and drug residues

Inactive Publication Date: 2013-12-11
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The drug control method is often used in China, which has caused a series of problems such as bacterial drug resistance and drug residues

Method used

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  • Salmonella enteritidis double knockout attenuated mutant and preparation as well as application thereof
  • Salmonella enteritidis double knockout attenuated mutant and preparation as well as application thereof
  • Salmonella enteritidis double knockout attenuated mutant and preparation as well as application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Construction of C50041 Gene Mutant Strain

[0078] Source of C50041: China Medical Microbiology Culture Collection Management Center, preservation number CMCC50041

[0079] 1. Identification of C50041:

[0080] 1.1. Bacteria identification

[0081] 1.1.1 Staining Microscopic Examination After Gram staining, observe the shape of the bacteria and measure the size of the bacteria under an ordinary optical microscope. The results of Gram staining showed that microscopic examination could observe scattered arrangement of red short bacilli, the size of which was about 1 μm×4 μm.

[0082] 1.1.2 Biochemical test

[0083] Pick a purely cultured single colony and inoculate it into a biochemical identification tube, and observe the results after incubating at 37°C for 24 hours.

[0084] 2. Construction of C50041 gene mutant strain

[0085] 2.1 Target gene sequence determination

[0086] 2.1.1 Amplification of target genes

[0087] According to the Salmonella genome sequence...

Embodiment 2

[0143] Safety test: Lethality test of 1-day-old Hy-Line white chicks

[0144] Comparison of the virulence of the △C50041 (crp, spiC / Km) mutant strain prepared in Example 1 and the wild strain C50041 (CMCC: 50041)

[0145] Bacterial culture: Bacteria were cultured on LB plates at 37°C for 24 hours, picked 10 plump single colonies and streaked on the whole plate, cultured at 37°C for 5 hours, scraped the bacteria on LB plates with sterilized PBS Next, the bacterial concentration was adjusted to 10 9 CFU / ml.

[0146] One-day-old Hy-Line white chicks were randomly divided into 10 groups, 5 groups for each strain, and 14 chickens in each group. Set the concentration to 10 9 The bacterial solution of CFU / ml was diluted into 5 dilutions with sterilized PBS, and each chicken was intramuscularly injected with 0.1ml bacterial suspension, and the death situation was observed within two weeks, and the mortality rate was calculated. The test results showed that the virulence of the ΔC5...

Embodiment 3

[0149] C50041 and △C50041 (crp, ΔspiC / Km) were treated with 10 7 Bacterial amount of CFU was inoculated by intramuscular injection, and the experimental chickens were slaughtered in each group on 1d, 4d, 7d, 10d, 13d, 16d, 20d, 24d, and 28d after infection, and the spleen and part of the liver were aseptically collected, and the bacteria were counted. Changes in the number of bacteria in chicken liver Figure 16 , the change rule of the number of bacteria in the spleen see Figure 17 . The wild-type C50041 infection group all died 6 days after inoculation, but within a detectable time point, the amount of bacteria carried in the liver and spleen was significantly higher than that of the mutant bacteria, and the mutant bacteria were finally completely cleared in the liver and spleen, and in the liver In , mutants were undetectable at day 10 post-infection and in spleen at day 13.

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Abstract

The invention discloses a salmonella enteritidis double knockout attenuated mutant and preparation as well as application thereof. The salmonella enteritidis double knockout attenuated mutant is obtained by knocking out crp gene and spiC gene of salmonella enteritidis C50041. The invention further discloses a preparation method as well as application of the salmonella enteritidis double knockout attenuated mutant. Further attenuation of the salmonella enteritidis attenuated mutant is realized. A foundation is laid for researching salmonella enteritidis attenuated live vaccines and live vector vaccines.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a double gene knockout attenuated mutant strain of Salmonella enteritidis and its preparation and application. Background technique [0002] Salmonella is an important pathogen of food-borne zoonotic bacterial diseases. Dominant, in poultry, S. Enteritidis and S. Typhimurium are considered the two most important serotypes. Salmonella can cause asymptomatic occult infection in poultry, but in young chicks less than two weeks old, it has the potential to cause outbreaks of high lethality and systemic infection. The contamination of poultry products by Salmonella Enteritidis has long been the main source of contamination in the human food chain. Salmonella in the cecum of chickens is an important hazard because it can cause horizontal infections, contaminate eggshells via feces, contaminate meat products during slaughter, and possibly contaminate ovaries. [0003] The drug control m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/09A61K39/112A61P31/04C12R1/42
Inventor 焦新安耿士忠李宝利潘志明陈祥孙林
Owner YANGZHOU UNIV
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