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Method for efficient deletion of Riemerella anatipestifer gene

A duck plague Riemer's and gene technology, applied in the biological field, can solve the problems of low recombination efficiency, cumbersome steps, and long time-consuming, and achieve the effect of low cost, simple method, and high knockout efficiency

Active Publication Date: 2019-10-01
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has cumbersome steps, low recombination efficiency, and long time-consuming, which brings a lot of inconvenience to the research of Riemerella anatipestifer

Method used

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  • Method for efficient deletion of Riemerella anatipestifer gene
  • Method for efficient deletion of Riemerella anatipestifer gene
  • Method for efficient deletion of Riemerella anatipestifer gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1, the construction of recombinant fragment

[0023] 根据NCBI报道的RA ATCC11845株的RA0C_1193基因序列,具体序列如下:atgagccaaaccataaatacatcggaaagaaaagaccaaatcaaaagtgccatcatcacatttttgattagccttttggtattcttagctttgtatttttattcgtttactagagaacttccaaaggaggaagtcgtaagcacgatgctcatcaattttggagaccaaaacgagggtaacctgcctgaagaaccccaaaaccaagagggcagtctatccgccaccgaagcacctacaccaatacaagaaatacaacctacacctgtagaacctcagcccaaaaaagaagtggtaaaggagaaaattatcacagggcaaaataccaaaacaagtgctgaaaaggtggaaaaagtaacctctaaacctacaaaagaaagcacagcaaacacccaaaagaaagaaaccacgacacctaaaaaaagcagtaccactacacaaaataaaaccgtgaccaaccaaggtgatggcagaggtaacgaggctattggcaacctcatcagagggcgaggtgccaacaaaggagctcaaggaaactcccaaaacacttccggcaattcaggagacccactaggtggcgatagcaatggcgatagtaaaatcggcatagaccgaaagttaatcggatttatacctggaactatggggcgtggtggttcacagccgacccaccagtgttctgcctcaggcaccattagcatctcttatgtggtagataaagcgggcaatgtaatttcggctagacgaagtggcggtattacagacccttgtgccgtaaacacaaccatagagtgggtaaagaaatatgtaaaagcagaaagagctagcacttcttctacaggcacttacaaaatcactttctaa(SEQ ID NO.1)

[...

Embodiment 2

[0035] Embodiment 2, the preparation of recipient bacterium

[0036] Take out the RA ATCC11845 wild strain from -80°C and recover it on a blood plate. After it grows up, use an inoculation loop to pick a monoclonal colony and inoculate it into 10ml liquid GCB medium for overnight cultivation; after overnight cultivation, dilute the bacterial solution with medium to OD600=1, then add 5mM MgCl 2 Mix well and set aside.

Embodiment 3

[0037] Embodiment 3, natural transfer

[0038] Prepare two 1.5ml sterile centrifuge tubes, one as the experimental group and one as the control group, add 300μl (containing 5mM MgCl 2 ) the above bacterial solution, wherein 1 μg of the fusion fragment was added to the experimental group, and not added to the control group. Seal with sealant and incubate in a 37°C incubator for 30 minutes; take out the experimental group and control group incubated for 30 minutes, and add 700 μl of 5mM MgCl to them 2 GCB culture medium, that is, dilute to 1ml, seal with sealing glue, and incubate in a 37°C incubator for 7 hours; preparation of culture medium: 4 GCB solid medium containing erythromycin (1 μg / ml) (Erythromycin, Erm) ; Take out 100 μl of bacterial solution from the experimental group and the control group, respectively, and spread them on the erythromycin-resistant GCB solid medium with a disposable coating rod, and then place the medium in a 37°C incubator for 18 hours to form a...

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Abstract

The invention discloses an efficient riemerella anatipestifer gene deletion method. Particularly, a fusion fragment comprising an upstream fragment of a gene to be deleted, a resistant gene fragment and a downstream fragment of the gene to be deleted is naturally transferred into riemerella anatipestifer, and then resistant gene antibiotics are used for screening to obtain the riemerella anatipestifer with the gene deleted. The method is simple, the deleted gene constructs the fusion fragment and then can be directly and naturally exchanged with host bacteria, culture cycle is short, transformation rate is high, the riemerella anatipestifer gene can be efficiently deleted, and tools are provided for riemerella anatipestifer gene function and attenuated vaccine manufacture.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for efficiently deleting the Riemerella anatipestifer gene. Background technique [0002] Riemerella anatipestifer is a disease with a high incidence rate caused by Riemerella anatipestifer infecting ducklings. In addition to causing the death of ducklings, it can also lead to a reduction in feed conversion rate and an increase in treatment costs, causing huge losses to the duck industry. At present, the prevention and treatment of Riemerella anatipestifer mainly relies on inactivated vaccine prevention and antibiotic treatment. Since there are multiple serotypes of Riemerella anatipestifer, and there is no cross-protection between the serotypes, an inactivated vaccine can only target one serotype. At the same time, Riemerella anatipestifer is resistant to multiple antibiotics, so the effect of antibiotic treatment is not good and the environment is seriously p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90
Inventor 刘马峰张利王梦怡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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