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A method for knocking out the flagellin gene of Vibrio anguillarum

A flagellin and gene knockout technology, applied in the field of microbial genetic engineering, can solve problems such as the difficulty of forming a unified technology, the difficulty of bacterial gene knockout technology, differences in morphological characteristics, internal structure and physiological characteristics, and achieve the goal of improving efficiency Effect

Inactive Publication Date: 2021-04-30
EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI +1
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Problems solved by technology

However, at present, there are many methods for bacterial gene knockout, and it is difficult to form a unified and effective technology; in addition, there are many types of bacteria, and their morphological characteristics, internal structures and physiological characteristics are quite different, so bacterial gene knockout technology more difficult

Method used

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  • A method for knocking out the flagellin gene of Vibrio anguillarum
  • A method for knocking out the flagellin gene of Vibrio anguillarum
  • A method for knocking out the flagellin gene of Vibrio anguillarum

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Embodiment 1

[0039] 1. Using overlapping PCR technology to amplify the FlaB fusion fragment

[0040] Using FlaB-MF1 / FlaB-MR1 as primers to amplify, obtain the 593bp fragment of the homology arm A of the upstream of FlaB; figure 1 ) (amplification program: 98°C 3min; 98°C 10sec, 56 / 58°C 20sec, 72°C 1min, 30cycles; 72°C 7min; TaKaRaPrimerSTAR Max DNAPolymerase), the result is as follows figure 1 shown. Gel recovery and purification were carried out for fragments A and B respectively.

[0041] Using the above fragments A and B as templates and FlaB-MF1 / FlaB-MR2 as primers, carry out overlapping PCR amplification (amplification program: 98°C 1min; 98°C 10sec, 68°C 20sec, 72°C 30sec, 7cycles; 98°C ℃10sec, 56 / 58℃20sec, 72℃1min, 30cycles, 72℃7min). The result is as figure 2 shown. The fusion AB fragment is 1138bp long ( figure 2 ), the AB fragment was purified by gel recovery DNA purification method.

[0042] 2. Construction and transformation of pLP12-FlaB recombinant suicide plasmid ...

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Abstract

The invention relates to a method for knocking out the flagellin gene of Vibrio anguillarum. The fusion fragments of the upstream and downstream homology arms are obtained by using overlapping PCR technology, the transformation of the recombinant plasmid is completed by the mixed incubation method, and the secondary homology Recombinant technology effectively knocked out the flagellin FlaB of Vibrio anguillarum. The invention is simple and easy to implement, greatly improves the efficiency of gene knockout, provides technical support for the gene knockout of Vibrio anguillarum, and also provides a new idea for the study of the biological function of flagellin.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering, in particular to a method for knocking out the flagellin gene of Vibrio anguillarum. Background technique [0002] Vibrio anguillarum belongs to Gram-negative bacteria, which widely exists in coastal water and silt in my country, and often adheres to the surface and intestines of marine organisms. It is a common type of pathogenic bacteria in aquaculture and can cause Symptoms such as shedding of intestinal mucosa of fish, liver necrosis, body surface ulceration, gill filament bleeding, and increased mucus. Research reports have pointed out that seawater fish such as large yellow croaker, perch, flounder, and turbot in my country are seriously infected by Vibrio anguillarum, and often lead to the occurrence of yellow gill disease in shrimp, so it is very harmful. Because eel vibriosis has the characteristics of high mortality, wide range of infection, and fast transmission, it often b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90C12N1/21C12R1/63
CPCC07K14/28C12N15/902
Inventor 高权新彭士明闵明华岳彦峰
Owner EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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