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A method for rapidly complementing the deleted gene of Riemerella anatipestifer

A technique for Riemerella anatipestifer and Bacillus anatipestifer, applied in the biological field, can solve problems such as time-consuming, cumbersome steps, inconvenient research on Riemerella anatipestifer, etc., and achieve the effects of low cost and short cultivation period

Active Publication Date: 2019-12-03
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method needs to use Escherichia coli as the donor bacteria, and the steps are cumbersome and time-consuming, which brings a lot of inconvenience to the research of Riemerella anatipestifer

Method used

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  • A method for rapidly complementing the deleted gene of Riemerella anatipestifer
  • A method for rapidly complementing the deleted gene of Riemerella anatipestifer
  • A method for rapidly complementing the deleted gene of Riemerella anatipestifer

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Embodiment 1

[0023] Preparation of the shuttle plasmid: First, take out the shuttle plasmid pLMF03 ( figure 1 , SEQ ID NO.7) host bacterium DH5α (preserved in this laboratory) on the LB plate, after it grows up, use the inoculation loop to pick the monoclonal colony and inoculate it into 4ml liquid LB medium and cultivate it overnight; then use the plasmid The mini-extraction kit (purchased from Tiangen) was used to extract the plasmid from the bacterial solution cultivated overnight, and measure the concentration.

[0024] Preparation of recipient bacteria: First, take out the wild strain of Riemerella anatipestifer ATCC11845 (preserved in our laboratory) from -80°C and resuscitate it on a blood plate. After it grows up, use an inoculation loop to pick a monoclonal colony and inoculate it to 10ml Liquid GCB medium was cultured overnight; it was adjusted to OD600=1, and 5 mM MgCl was added 2 Mix well and set aside.

[0025] Natural transfer: Take two 1.5ml sterile centrifuge tubes, one a...

Embodiment 2

[0033] Example 2. Replenishment of Riemerella anatipestifer RA ATCCΔRA0C-1193

[0034] Construction of recombinant plasmid: RAOC-1193 gene (about 861bp) of RA ATCC11845 strain was searched on NCBI, and the sequence is shown in SEQ ID NO.4: then design the primers for amplifying the target gene, the specific primers are as follows:

[0035] p1: 5'-catgccatggatgagccaaaccataaatac-3' (SEQ ID NO.5), the underline indicates NcoI;

[0036] p2: 5'-ctagtctagattagaaagtgattttgtaagtgcctg-3' (SEQ ID NO.6) underlined represents XbaI;

[0037] Then, the Riemerella anatipestifer ATCC11845 genome was used as a template, and p1 and p2 were used as primers to amplify the target gene fragment. The PCR amplification procedure was as follows: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 55°C for 30s, and extension at 72°C for 30s , cycled 30 times; then extended at 72°C for 10 min; finally stored at 16°C; amplified products were subjected to agarose gel electrophore...

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Abstract

The invention relates to a method for quickly complementing the missing gene of Riemerella anatipestifer, specifically: constructing a recombinant shuttle plasmid containing a target gene, and then adding MgCl 2 of activated Riemerella anatipestifer bacteria, incubate at 37°C for 30 minutes, and then add MgCl 2 Incubate the GCB culture medium for 7 hours, spread it on the solid GCB culture medium containing the corresponding resistance, and cultivate it in a 37°C incubator for 18-24 hours until a single clone grows, and then the Riemerella anatipestifer anaplerotic strain is obtained; The method is simple. After recombination, the vector does not need to be transferred into the donor bacteria. It can directly enter the host bacteria and exert relevant gene functions. The culture cycle is short, and a complementing strain clone can be isolated in 18 hours under normal circumstances. The cost is low and it is the best method for duck plague. Tools provided for functional validation of Riemerella genes.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for quickly replenishing the missing gene of Riemerella anatipestifer. Background technique [0002] At present, the function of the gene is mainly studied by knocking out the gene of Riemerella anatipestifer, and then the pathogenic mechanism of Riemerella anatipestifer is studied. Knocking out a certain gene in a bacterium usually causes its phenotype to change, but some phenotypic changes may be caused by the knocked out gene affecting the transcription and expression of other genes. Therefore, when we knock out a gene, we also need to complement the gene to determine the function of the gene. For bacteria such as Escherichia coli, the recombinant plasmid can be directly introduced into bacterial cells by the calcium chloride transformation method. However, the requirements of Riemerella anatipestifer are relatively strict. At present, recombinant plasmids a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/74
CPCC12N15/74Y02A50/30
Inventor 刘马峰张利黄月程安春汪铭书贾仁勇朱德康陈舜孙昆峰杨乔吴英
Owner SICHUAN AGRI UNIV
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