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The rpsl mutant gene of Riemerella anatipestifer and its application

A duck plague Reemer's and mutant gene technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of affecting the traits of strains, restricting, polluting the environment, etc., and achieve the effect of reducing drug resistance and improving efficiency

Inactive Publication Date: 2019-08-06
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The traditional method will leave the resistance marker gene in the genome after each gene knockout, which has many adverse effects on subsequent research: First, for a transformation system, the number of applicable resistance marker genes is limited, and a The residue of the resistance marker gene means that it cannot be used again in the future genetic manipulation, which virtually limits the number of genetic manipulations; secondly, the insertion of foreign genes will increase the burden on cells, and may also cause gene mutations other than the target gene , affecting the traits of the strain; in addition, many marker genes are drug resistance genes. When making attenuated vaccines, the residue of this resistance gene will be unfavorable to animals, and the residue of these genes in the genome will bring resistance to the strain. Drug resistance, and this resistance may be transferred in parallel in the environment, which will seriously pollute the environment

Method used

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  • The rpsl mutant gene of Riemerella anatipestifer and its application
  • The rpsl mutant gene of Riemerella anatipestifer and its application
  • The rpsl mutant gene of Riemerella anatipestifer and its application

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Embodiment 1

[0029] Embodiment 1, screening of Riemerella anatipestifer ATCC11845 streptomycin-resistant mutant strain

[0030] Mix the Riemerella anatipestifer RA ATCC11845 strain cultured overnight on the blood plate in 200 μl of tryptone soybean broth (TSB) liquid medium, dilute to OD 600 = 1, then spread them all on the blood plate medium containing 100 μg / mL streptomycin, culture them overnight at 37°C, and select the grown monoclonal colonies to obtain the Riemerella anatipestifer mutant strain RA ATCCs. Using the genomic DNA of Riemerella anatipestifer mutant strain RA ATCCs as a template, the rpsL gene primer pair rpsL P1 and rpsL P2 were used for PCR amplification. The sequences of the rpsL P1 and rpsL P2 primer pairs are as follows:

[0031] rpsL P1: 5'-gggacgaaagttgttctatggaag-3' (SEQ ID NO. 1);

[0032] rpsL P2: 5'-ctgaatgcgatagacttcttaccg-3' (SEQ ID NO. 2);

[0033] The conditions of PCR amplification were pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, anne...

Embodiment 2

[0035] Example 2, Verification of Scarless Deletion Mutants

[0036] The rpsL fragment of the wild strain with the promoter was cloned into the shuttle plasmid pLMF03 (Genbank: KU997673), the specific method is as follows:

[0037]Using Riemerella anatipestifer RA ATCC11845 as a template, rpsl exp P1 and rpsl exp P2 as primers for PCR amplification, amplified to obtain the rpsL fragment of the wild strain with a promoter, the specific sequence is shown in SEQ ID NO.5 , where the primer sequences are as follows:

[0038] rpsl exp P1:5'-acgc gtcgac gtcggccatagcggatcaaaaataatttggttttcg-3' (SEQ ID NO.6), (the underline represents SalI);

[0039] rpsl exp P2: 5'-catg ccatgg catgttactttttagcatctttaggacgcttagcaccg-3' (SEQ ID NO.7), (the underline represents NcoI);

[0040] PCR amplification conditions were: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 55°C for 30s, extension at 72°C for 17s; cycle 30 times; extension at 72°C for 7min, and finally...

Embodiment 3

[0042] Example 3, Construction of the suicide vector pORS with seamless deletion

[0043] The shuttle plasmid pMM47.A was excised with PstI endonuclease after the replication origin of replication in Riemerella anatipestifer was ligated, and then the rpsL fragment (SEQ ID NO.5) of the wild strain with the promoter was used with SalI and The two restriction sites of NcoI were cut into the obtained vector to obtain the suicide vector, which was named pORS, and the plasmid map is as follows: figure 1 shown.

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Abstract

The invention discloses an rpsL mutant gene of riemerella anatipestifer and application thereof. A nucleotide sequence of the rpsL mutant gene is shown as SEQ ID NO.3, the riemerella anatipestifer containing the mutant gene is insensitive to streptomycin and can be used for constructing traceless deletion candidate strains of the riemerella anatipestifer, a target gene does not need to be replaced by a resistance gene by utilizing traceless deletion of the strains, therefore, the drug resistance of the deletion strains is greatly reduced, and the riemerella anatipestifer can be used for preparing gene deletion vaccines; meanwhile, a plurality of genes can be deleted in a genome, thereby improving the efficiency of gene function study and having good application prospect.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to the rpsL mutant gene of Riemerella anatipestifer, and also relates to the application of the gene in constructing a traceless deletion candidate strain of Riemerella anatipestifer and a method for constructing the traceless strain. Background technique [0002] Riemerella anatipestifer is a contagious disease caused by Riemerella anatipestifer (RA), also known as duck infectious serositis. It has become one of the most common bacterial diseases that endanger the duck industry in all countries in the world. The current prevention and control methods mainly include drug prevention and vaccine prevention. However, Riemerella anatipestifer is resistant to multiple drugs, and it is difficult to find sensitive drugs for this bacteria in many areas. On the other hand, there are multiple serotypes of Riemerella anatipestifer, and there is no cross-protection among the s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12N15/74
CPCC07K14/195C12N15/74C12N2800/101
Inventor 刘马峰王梦怡刘珈均程安春汪铭书贾仁勇朱德康陈舜杨乔吴英孙昆峰
Owner SICHUAN AGRI UNIV
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