Construction method of helicobacter pylori cagA (cytotoxin-associated gene A) inactivated mutant strain

Abstract

The invention relates to the technical field of genetic engineering, in particular to a construction method of a helicobacter pylori cagA (cytotoxin-associated gene A) inactivated mutant strain. The method comprises following steps: (1), H. Pylori 11639 thalli growing on Columbia agar are scraped and added to an electrotransformation buffer solution, the electrotransformation buffer solution is diluted until concentration of H. Pylori 11639 thalli is 1012 / L, and a solution A is obtained; (2), 100 mu L of the solution A is added to an ice-cold 0.2 cm BioRed electrotransformation cup, and 50 mug of pHG1 targeting vectors are added; (3), the electrotransformation cup is placed on ice cubes below a solution extracting device; (4), an adequate quantity of SOC media are added to an SOC medium storage box, and a solution extracting tube extends into the electrotransformation cup; (5), a Columbia blood plate is fixed with a barrel, a coating brush is contacted with the surface of the Columbiablood plate, and starting time input to a controller is 10-15 min; (6), after coating of the Columbia blood plate is completed, the Columbia blood plate is taken down. With the application of the solution extracting device, errors caused by manual operation can be reduced, and efficiency is improved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for constructing a cagA gene inactivation mutant strain of Helicobacter pylori. Background technique [0002] Helicobacter pylori (H. pylori) is one of the main causes of gastritis, gastric ulcer and gastric cancer. The CagA protein (Cytotoxin-associated gene A antigen, CagA) encoded by the cag virulence island (cagpathogenicity is-land, PAI) in the H. pylori genome is the most important virulence factor of H. pylori. PAI is 40kb long and contains a multigene cluster of about 31 genes, and the cagA gene is located at its C-terminus. The type Ⅳ secretion system encoded by PAI injects the encoded CagA into gastric epithelial cells, and H. pylori can be divided into type Ⅰ and type Ⅱ according to the difference in the CagA protein coding region. The PAI island of type Ⅰ H. pylori bacteria encodes CagA protein and type Ⅳ secretion system, while the coding regio...

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Problems solved by technology

[0007] In the above step (4), it is necessary to place the electroporation cup on ice for 10 minutes, and then after adding SOC medium, it needs to be quickly spread on the Columbia blood plate; due to the long time on ice, and 10 minutes The time is not convenient to deal with other things, and it is easy to be distracted while waiting, so it often happens that the time on the ice is overtime

In addition, before spreading the electrotransformation buffer on the Columbia blood plate, it is necessary to quickly add the SOC medium, and mix the electroconversion buffer and the SOC medium evenly; due to the short duration, misoperation is prone to occur, which affects the Experimental results, so human steps should be minimized in this process

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