Construction method and application of pseudomonas aeruginosa mutant strain

A technology for Pseudomonas aeruginosa and mutant bacteria, which is applied in chemical instruments and methods, microorganism-based methods, biochemical equipment and methods, etc., and can solve the problem of not yet obtained attenuated strains of Pseudomonas aeruginosa exotoxin A.

Pending Publication Date: 2016-06-08
BENHEALTH BIOPHARMACEUTIC SHENZHEN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because it is impossible or very difficult to achieve site-directed mutation on the genome of the bacterium, an attenuated strain of Pseudomonas aeruginosa exotoxin A has not yet been obtained

Method used

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  • Construction method and application of pseudomonas aeruginosa mutant strain
  • Construction method and application of pseudomonas aeruginosa mutant strain
  • Construction method and application of pseudomonas aeruginosa mutant strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1. Preparation of Pseudomonas aeruginosa exotoxin A protein Glu578Asp mutant and Glu578 deletion mutant

[0065] According to the description of the first step to the sixth step above, a mutant strain of Pseudomonas aeruginosa exotoxin A protein Glu578Asp was prepared, wherein the triparental hybridization was carried out according to the following steps:

[0066] (1) Donor bacteria Escherichia coli (toxA) and auxiliary bacteria Escherichia coli HB101 (pRK2013) (gifted by Dr. Wang Hailong, State Key Laboratory of Microbial Technology, Shandong University) were activated by streaking on LB plate solid medium containing Kan20μg / mL, Cultivate for 16-18h, then pick a single colony into 3ml LB liquid medium of Kan15μg / mL, culture overnight at 37°C, and grow to logarithmic phase.

[0067] (2) Receptor bacterium Pseudomonas aeruginosa PAO1 (purchased from the American type culture collection (American type culture collection), preservation number is ATCC15692) was acti...

Embodiment 2

[0072] Embodiment 2. toxicity test

[0073] (1) Cytotoxicity test

[0074] The cytotoxicity of recombinant PEA (Glu578Asp mutant protein) and parental PEA protein was determined by MTT assay. Recombinant PEA and parental PEA protein were isolated and purified from Pseudomonas aeruginosa exotoxin A protein Glu578Asp mutant strain and Pseudomonas aeruginosa POA1 respectively. The recombinant PEA and parental PEA proteins stored at -80°C were melted, the protein content was measured, and the protein concentration was determined for the next step of the cytotoxicity experiment. There were three gradients in the experiment, namely 250 μg / well, 325 μg / well, and 500 μg / well. Passage L929 cells in a 96-well plate. When the cell density reaches 70% fullness, change the DMEM medium containing 2% serum, and add DMEM-dissolved gradients of recombinant PEA or parental PEA protein to treat the adherent L929 cells , and treated L929 cells with the same concentration of BSA as a blank cont...

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Abstract

The invention relates to a pseudomonas aeruginosa mutant strain genetic engineering mutant strain, in particular to a pseudomonas aeruginosa mutant strain exotoxin A attenuated strain, and further relates to a preparation method of the mutant strain and application of the mutant strain or the attenuated strain in preparing vaccines or adjuvant therapy drugs.

Description

technical field [0001] This application relates to genetically engineered mutant bacteria, especially Pseudomonas aeruginosa genetically engineered mutant bacteria, especially Pseudomonas aeruginosa exotoxin A attenuated strain, also relates to its preparation method and the mutant bacteria or attenuated strain in the preparation of vaccines Or the application in adjuvant medicine. Background technique [0002] Pseudomonas aeruginosa (Pseudomonas aeruginosa, Pa), also known as Pseudomonas aeruginosa, is an aerobic Gram-negative bacterium with no spores, several pili and a single terminal flagella, and is widely distributed in soil, water, and air. , normal human skin, respiratory tract and intestinal tract. The bacterium is an opportunistic pathogenic bacterium that can cause disease only under certain conditions. Due to the reduced body resistance, patients who are severely burned or suffer from metabolic diseases, blood diseases, malignant tumors, and patients after surg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C07K14/21C12N15/74A61K39/104A61P31/04A61P35/00A61P37/02C12R1/385
Inventor 苏静符军陈敏文
Owner BENHEALTH BIOPHARMACEUTIC SHENZHEN CO LTD
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