Method for screening and identifying microbial cell surface antigen

A technology of microbial cells and surface antigens, applied in biological testing, material testing products, measuring devices, etc., can solve the problems of outer membrane protein difficulty, limited application, poor repeatability, etc., and achieve simple and fast operation steps, improve efficiency, and simplify experiments. effect of steps

Inactive Publication Date: 2015-05-20
SHANTOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In terms of antigen screening of microbial cell surface proteins, conventional immunoproteomics uses the method of isolating microbial cell surface proteins first, and then performing immunoblotting screening, but there are certain difficulties in extracting cell surface proteins, especially outer membrane proteins, and the separation of outer membrane Two-dimensional electrophoresis of proteins is cumbersome and repeatable, and cannot be displayed for low-copy proteins and proteins with extreme isoelectric points
Moreover, the current extraction and analysis of microbial cell surface proteins destroys the natural results of cell surface proteins and cannot reflect the process of immune response in vivo.
This limits its application to some extent

Method used

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  • Method for screening and identifying microbial cell surface antigen
  • Method for screening and identifying microbial cell surface antigen
  • Method for screening and identifying microbial cell surface antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] 1. Bacterial culture, counting and collection: Vibrio parahaemolyticus VPL4-90 was used as the microorganism to preserve the strains in this laboratory. VPL4-90 was inoculated in LB medium and cultured on a shaker at 28°C for 18h. After the cultivation, 2 mL of the bacterial liquid was taken to count the colonies on the plate. The rest of the culture solution was centrifuged at 5000rpm for 10min to collect the bacterial cells, and the bacterial cells were washed 3 times with physiological saline. Adjust its concentration to 10 with normal saline 9 CFU / mL.

[0022] 2. Preparation of anti-pathogen antiserum: add 0.025% formaldehyde by volume fraction to the prepared VPL4-90 cell suspension, act at 30°C for 4 hours to make inactivated pathogens, rotate at 5000rpm for 10min, wash with normal saline three times, and finally Then adjust its concentration to 10 with normal saline 9 CFU / mL, inject New Zealand white rabbits, each 1mL, inject normal saline in the control grou...

Embodiment 2

[0028] Example 2 was the same as Example 1, except that the bacteria were changed to Vibrio mimeticus ATCC33653 and Qing was added at a volume ratio of 1:250. The result is as follows

[0029] 1. The SDS-PAGE pattern of the immunoprecipitated cell surface proteins of Vibrio mimeticus. In the stained electrophoresis gel, there are 6 protein bands consistent with the silver-stained positive bands, numbered a~h respectively. The results of silver staining observation and staining mass spectrometry of Vibrio mimicus immunoprecipitation complex are shown in figure 2 -A and 2-B.

[0030] 2. The mass spectrometric identification results of the 6 protein bands are shown in Table 2. 8 proteins were identified by the 6 protein bands, 4 of which are outer membrane protein antigens on the cell surface of Vibrio mimicus, and 2 are inner membrane proteins of Vibrio mimicus. Membrane protein antigens, one is a protein translation-related factor that may be located on the surface of Vibrio...

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Abstract

The invention discloses a method for screening and identifying a microbial cell surface antigen, and relates to a method for conveniently and fast screening and identifying the microbial cell surface antigen. The method comprises the following steps: immuno-precipitating and separating a cell surface protein antibody by using microbial whole cells, capturing an antigen-antibody complex by using protein A/G agarose, and eluting to obtain the antigen-antibody complex, and analyzing an immuno-precipitated protein by using SDS-PAGE; analyzing the abundance of immunogen, the affinity of the antibody to the antigen and the determination of the immunogen, and determining the protein type of the antigen; and establishing an immunoprecipitation-based proteomic technology for performing high-flux screening on the protein with immunogenicity as candidate target position of multivalent vaccine. The possibility of the antigen as a vaccine protein can be determined according to the abundance of the immunogen and the affinity of the antibody to the antigen. The protein with the immunogenicity on the certain microbial cell surface can be fast determined, and the method is efficient, fast, economic and practical. The method has universality, and can be used for identifying both bacteria, fungus and other microbial immunogens.

Description

technical field [0001] The invention relates to a simple and quick method for screening and identifying protein antigens on the surface of microorganism cells. Background technique [0002] It has been proven that vaccination is an effective and economical way of immune intervention to prevent microbial diseases. Microbial cell surface proteins are located on the cell surface and directly contact the host immune system, which stimulates the body to produce specific antibodies that bind to corresponding microbial cell surface proteins, inhibit microbial infection through neutralization, or mediate phagocytosis of immune cells , can play a role in the initial stage of pathogen invasion. Therefore, microbial cell surface proteins are good candidate proteins for vaccines, and are also research hotspots. For example, scholars have found that Vibrio cell surface proteins OmpA and OmpU have good immunogenicity and immune protection, and can be used as candidate proteins for vacci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6851G01N33/56911G01N33/56961
Inventor 胡忠袁传飞伦镜盛张设熙
Owner SHANTOU UNIV
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