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Salmonella enteritidis strain with ssrAB gene deletion and construction method thereof

A gene deletion strain, Salmonella Enteritidis technology, applied in the field of Salmonella Enteritidis ssrAB gene deletion strain and its construction, can solve the problem of lack of effective methods for the prevention and control of pathogenic bacteria

Inactive Publication Date: 2015-11-04
史记生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no effective method for the prevention and treatment of this pathogen, so the pathogenic factor of this pathogen needs to be further studied

Method used

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  • Salmonella enteritidis strain with ssrAB gene deletion and construction method thereof
  • Salmonella enteritidis strain with ssrAB gene deletion and construction method thereof
  • Salmonella enteritidis strain with ssrAB gene deletion and construction method thereof

Examples

Experimental program
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Effect test

example 1

[0053] A gene-deleted Salmonella enteritidis, the construction method is as follows:

[0054] 1) Selection of the experimental strain G9-2012: the strains with stronger invasive ability were selected from the strains isolated from pet dogs in Anhui area, and the strain G9-2012 was sensitive to ampicillin through the determination of the drug resistance of the strains, and its minimum bactericidal concentration was less than After continuous culture, a G9-2012 strain resistant to streptomycin and sensitive to ampicillin was obtained. Drug-resistant Salmonella Enteritidis G9-2012 was used as the parent strain.

[0055] 2) Proliferation of G9-2012 and extraction of total genomic DNA: Inoculate the Salmonella enteritidis G9-2012 screened in 1) in liquid LB medium, culture at 37°C until its OD 600 When the value reaches above 3 (12h), take 3mL of the culture, place it in an Eppendorf centrifuge tube, centrifuge at 12830g for 5min, discard the supernatant, add 1.5mL PBS buffer, cen...

example 2

[0077] Study on Growth Characteristics of Salmonella Enteritidis Deletion Strain G9-2012(ΔssrAB)

[0078] After recovering the deletion strain G9-2012 (ΔssrAB) prepared in Example 1 and the parent type G9-2012 strain (WT), the four-section line was placed on the LB solid medium plate to purify the strain, cultured overnight at 37°C, and a single colony was picked , respectively inoculated into LB liquid medium, cultivated at 37°C, and after 2 hours of cultivation, every 1 hour, take 0.1mL of the bacterial liquid of the deletion strain and the wild-type strain, and perform plate counting. The total number of colonies (CFU×106 ) is the ordinate, and the culture time (h) is the abscissa, and the counting results of the deletion strain G9-2012 (ΔssrAB) and the parental strain G9-2012 (WT) were plotted into a generative curve to analyze and identify the growth characteristics of the deletion strain. As shown in the figure below, there is no significant difference in growth character...

example 3

[0080] Determination of Biofilm of Salmonella Enteritidis Deletion Strain G9-2012(ΔssrAB)

[0081] After recovering the deletion strain G9-2012 (ΔssrAB) prepared in Example 1 and the parent type G9-2012 strain (WT), the four-section line was placed on the LB solid medium plate to purify the strain, cultured overnight at 37°C, and a single colony was picked , respectively inoculated into LB liquid medium, cultured overnight at 37°C, inoculated into small finger-shaped test tubes, inoculated with 0.8mL LB medium in each test tube, cultured at 37°C, and discarded the bacterial solution after 18 hours of culture , add 1mL distilled water to wash 3 times, stain with 0.2% crystal violet for 5min, discard crystal violet, rinse 3 times under running water, take pictures after natural air-drying, add 1mL DMSO to dissolve for 2h, and detect at 570nm under UV-Vis spectrophotometer Absorbance A 570 The results were plotted as shown in the figure below. According to statistical analysis, ...

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Abstract

The invention discloses a salmonella enteritidis strain with ssrAB gene deletion. The strain is obtained after knockout of altogether 915 base pairs, from amino acid located at site 689 of ssrA gene to amino acid located at site 63 of ssrB gene. The invention further discloses a construction method for the salmonella enteritidis strain with ssrAB gene deletion. The construction method comprises the following steps: (1) acquiring SN and SC genes of ssrAB and connecting upstream and downstream homologous arms through overlap PCR; (2) constructing recombinant suicide plasmid pWM91-ssrAB; (3) carrying out solid phase joint of bacteria; and (4) screening and acquiring G9-2012 (delta ssrAB). The invention has the following beneficial effects: the G9-2012 (delta ssrAB) strain has a decreased colonization rate and obviously weakened virulence in vivo, can be used for development of attenuated live vaccine or live vector vaccine against salmonella enteritidis, and thus reduces the rate of infection of human beings or animals by salmonella enteritidis and guarantees health of the mankind.

Description

technical field [0001] The invention relates to a Salmonella enteritidis ssrAB gene deletion strain and a construction method thereof, and belongs to the technical field of gene deletion strain research. Background technique [0002] Salmonella enteritidis (Salmonella enteritidis) is an important foodborne zoonotic pathogen, which not only causes human gastroenteritis and other diseases, but also is the main pathogen that causes contamination of poultry eggs and related products. Salmonella is a typical source of food contamination, and it can be easily isolated and detected in contaminated food or water. According to statistics, more than one million people are infected by Salmonella typhimurium or Salmonella enteritidis every year, and less than 1% of these infected people receive timely and effective treatment. Food poisoning caused by Salmonella enteritidis has occurred in all countries in the world and the number is increasing, which has become an important issue of co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12R1/42
Inventor 李郁陈传荣曹堃孙裴魏建忠罗聪玉
Owner 史记生物技术有限公司
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