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Salmonella typhimurium X3337-pRST98lux and its application in living imaging

A technology of x3337-prst98lux and Salmonella, applied in the field of microorganisms, can solve the problems of inability to track bacterial invasion, proliferation and spread in real time, difficulty in quantification, low detection specificity, etc., and achieve the effect of stabilizing bioluminescent performance

Inactive Publication Date: 2013-10-16
SUZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] The present invention aims at the bottleneck that there is no suitable animal research model for Salmonella typhi and traditional methods cannot dynamically track the invasion, proliferation and dissemination of bacteria in the host body at each stage of infection in real time, and makes up for the low specificity and high background of fluorescent labeling in vivo detection , difficult to quantify and other shortcomings, and the previous plasmid-based bioluminescence detection requires additional screening pressure and substrates, and is difficult to quantify. A Salmonella typhimurium X3337- pR ST98 lux and its application in in vivo imaging

Method used

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  • Salmonella typhimurium X3337-pRST98lux and its application in living imaging
  • Salmonella typhimurium X3337-pRST98lux and its application in living imaging
  • Salmonella typhimurium X3337-pRST98lux and its application in living imaging

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Experimental program
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Embodiment 1

[0027] Specific construction and identification of strains:

[0028] 1. Material preparation

[0029] 1.1 Plasmids and strains

[0030] 1.1.1 Electroporation of plasmids and strains

[0031] Plasmid pBEN276 was kindly donated by Prof. Pierre Germon, Laboratory of Animal Infectious Diseases and Public Health Pathogen Research, French Institute of Natural Resources. 【Kevin Howe, Attila Karsi, Pierre Germon, et.al.Development of STable reporter sysTem cloning luxCDABE genes into chromosome of Salmonella enterica serotypes using Tn7transposon[J].BMC Microbiology2010,10:197】

[0032] Salmonella typhimurium (Salmonella typhimurium) X3337 was kindly provided by Professor ROY CURTISS III, School of Biological Sciences, Arizona State University, USA. [HIDENORI MATSUI, CHRI ST OPHER M. BACOT, WENDYA. Virulence Plasmid-Borne spvB ​​and spvC Genes Can Replace the 90-Kilobase Plasmid in Conferring Virulence to Salmonella enterica Serovar Typhimurium in Subcutaneously Inoculated Mice [J...

Embodiment 2

[0081] Application of Salmonella typhimurium (Salmonella typhimurium) X3337-pRST98lux in vivo infection model

[0082] 1. Bacterial culture

[0083] Select a single clone from the solid LB agar plate and inoculate it into LB liquid medium, and culture at 37°C and 200rpm for 14-16h until the logarithmic growth phase. Collect bacteria by centrifugation at 4000rpm, 4°C, 10min, wash twice with normal saline (4000rpm, 4°C, 10min), resuspend with appropriate amount of normal saline, measure OD 600 Calculate the bacterial concentration. Gradiently diluted with normal saline for use.

[0084] 2. Mice Grouping and Infection

[0085] All mice were given standard feed and free drinking water, and kept at room temperature (22°C). 54 mice were randomly divided into 3 groups: X3337-pRST98lux infection group and X3337lux infection group, 24 each, and normal saline control group, 6 mice. Intraperitoneal (i.p.) injection of 0.1 mL, 10 5 CFU bacteria, the control group was injected with t...

Embodiment 3

[0089] Application of Salmonella typhimurium (Salmonella typhimurium) X3337-pRST98lux in cell infection model

[0090] 3.1 Preparation of cell infection model

[0091] HeLa cells in the logarithmic growth phase were digested with trypsin, and washed twice in 10% wt FBS antibiotic-free RPMI1640 culture medium (centrifuged at 1000r / min for 10min). Adjust the cell concentration to 3.5×10 with cell culture medium 5 cells / mL, add 2 mL / well into 6-well cell culture plate, 5% CO 2 , Cultivate overnight at 37°C.

[0092] Collect the test bacteria cultured to the logarithmic growth phase, centrifuge at 4500r / min, 4°C for 5min, remove the supernatant, wash the bacteria twice with sterilized normal saline, and use antibiotic-free RPMI1640 culture medium containing 10%wt FBS After dilution, the multiplicity of infection (MOI) ratio of bacteria to cells was 100:1, respectively added to cell culture plates, and a control group without adding bacteria was set up. Centrifuge at 1000r / min,...

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Abstract

The invention relates to Salmonella typhimurium X3337 / pRST98lux and its application in living imaging. The above strain is preserved in China General Microbiological Culture Collection Center assigned by State Intellectual Property Office on Feb., 5, 2013, and has a preservation number of CGMCC No.7252. Lux operons compose a sexual expression luciferase and a substrate, so the bacterial luminescence can be maintained without applying a screened pressure or substrate; and the above joint mutant strain provides conveniences for constructing a cell model and an animal model, and deeply researching the pathogenic mechanisms of Salmonella typhi virulent plasmids, related medicine research and screening, the infectious disease control and the like.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, in particular to Salmonella typhimurium X3337-pR ST98 lux and its application to in vivo imaging. Background technique [0002] Salmonella (Salmonella) is a group of important pathogenic bacteria that endanger human and animal health. It is widely distributed in nature and is mainly transmitted by water and food. It can cause various diseases in humans and animals. Salmonellosis is a zoonotic disease of great significance in public health. In my country, food poisoning caused by Salmonella accounts for 70-80% of bacterial food poisoning, which can lead to nosocomial infection and fulminant food poisoning, with a high fatality rate. . Its pathogenic mechanism and prevention methods are currently research hotspots in the fields of medicine, food safety, agriculture and animal husbandry. Human typhoid fever caused by Salmonella typhi still has 21 million cases in developing countries every ...

Claims

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Application Information

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IPC IPC(8): C12N1/20A61K49/00C12Q1/02C12R1/42
Inventor 李嫄渊叶颖吴淑燕黄瑞
Owner SUZHOU UNIV
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