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Vibrio harveyi homologous recombination gene knockout method based on hydrochloric acid stimulation

A technology of Vibrio harveylius and gene knockout, which is applied in the field of microorganisms, can solve the problems of hindering and restricting genetics, and achieve the effects of not being prone to polar effects, simple operation, and accurate functional analysis

Pending Publication Date: 2019-12-20
SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the traditional method of using Escherichia coli (Escherichia coli) host bacteria to carry exogenous genes, and conjugating transfer with recipient bacteria to realize exogenous vectors into cells, and further homologous recombination for gene knockout methods, in Harvey Vibrio is hampered by the inability of most strains to successfully conjugate transfer, which limits the study of the genetics of this bacterium

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  • Vibrio harveyi homologous recombination gene knockout method based on hydrochloric acid stimulation
  • Vibrio harveyi homologous recombination gene knockout method based on hydrochloric acid stimulation
  • Vibrio harveyi homologous recombination gene knockout method based on hydrochloric acid stimulation

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Embodiment 1

[0058] Stimulation treatments of different hydrochloric acid concentrations and different time periods were performed on the early logarithmic recipient bacteria liquid, indicating that hydrochloric acid stimulation improved the conjugative transfer efficiency of Vibrio harveyi (Table 1).

[0059] 1. Preparation of donor bacteria E.coli GEB883

[0060] (1) Streak the E.coli GEB883 containing the expression plasmid pSCT32 preserved in glycerol on an LB solid plate containing 20 μg / mL chloramphenicol and 0.3 mM DAP, and culture overnight at 37°C;

[0061] (2) Pick the monoclonal colonies formed by the activated cells in (1), inoculate them in LB liquid medium supplemented with 20 μg / mL chloramphenicol and 0.3 mM DAP, and cultivate them at 37°C and 200 rpm until the early logarithmic OD600nm =0.3~0.7;

[0062] 2. Preparation of recipient bacteria Vibrio harveyli

[0063] (1) Configure an LBS solid plate to activate Vibrio harvey for overnight culture at 28°C;

[0064] (2) Pick...

Embodiment 2

[0080] In view of the results of Example 1, the gene hsdR (type I restriction enzyme, R subunit) was taken as an example to illustrate a step based on the hydrochloric acid stimulation method to realize homologous recombination gene knockout of Vibrio harveii (the specific operation process is as follows: figure 1 shown).

[0081] The hsdR gene (its nucleotide sequence is as the 534th to 3788th base of SEQ ID NO.1) ORF has a full length of 3255bp, and can encode the type I restriction modification system R subunit HsdR consisting of 1084 amino acids. HsdR is one of the host specificity determinants (hsd) of the type I restriction modification system, responsible for the restriction subunit (R) of unmethylated and mismethylated nucleotide sequences. Bacterial horizontal gene transfer has certain regulatory effects.

[0082] The hsdR gene knockout strain obtained in this study provides a premise for in-depth research on the function of the hsdR gene, and the elucidation of the ge...

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Abstract

The invention discloses a vibrio harveyi homologous recombination gene knockout method based on hydrochloric acid stimulation. Vibrio harveyi serves as receptor bacteria and is subjected to conjugal transfer with donor bacteria, gene knockout of the vibrio harveyi is achieved through homologous recombination, and the vibrio harveyi is vibrio harveyi subjected to hydrochloric acid stimulation treatment. According to the vibrio harveyi homologous recombination gene knockout method based on hydrochloric acid stimulation, on the technical basis of a traditional conjugal transfer method, the hydrochloric acid stimulation treatment is carried out on conjugated receptor bacteria vibrio harveyi, so that the conjugal transfer efficiency of the vibrio harveyi develops from nothing and generates qualitative change, and the method is applied to successfully carry out homologous recombination gene knockout on the vibrio harveyi. The method has very important significance for future vibrio harveyi gene engineering transformation, research of pathogenic mechanisms of the vibrio harveyi and the like.

Description

technical field [0001] The invention belongs to the field of microorganisms, and in particular relates to a method for knocking out Vibrio harvelius homologous recombination genes based on hydrochloric acid stimulation. Background technique [0002] Vibrio harveyi (Vibrio harveyi) is an important conditional pathogenic bacteria of mariculture organisms, which has caused great harm to the mariculture industry. In recent years, in the investigation of the disease of southern cultured marine fish, it was found that about 90% of the vibriosis of marine fish was caused by Vibrio harvei, and the damage range and degree of Vibrio harvei had gradually replaced Vibrio alginolyticus and traumatic bacteria. Vibrio, becoming the main pathogenic Vibrio. Therefore, it is imminent to elucidate its pathogenic mechanism and prepare related vaccines for immune prevention and control. [0003] The study of pathogenic mechanism and the development of attenuated live vaccines usually involve g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/90C12R1/63
CPCC07K14/28C12N15/902
Inventor 邓益琴冯娟郭志勋程长洪马红玲
Owner SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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