Construction of p97 mutant of Mesomycoplasma hyopneumoniae based on the homologous recombination system
10.16303/j.cnki.1005-4545.2025.03.11
- VernacularTitle:基于同源重组的猪肺炎支原体p97基因突变株的构建
- Author:
Yanna WEI
1
;
Jiying WANG
;
Huan XIE
;
Zhiqiang LI
;
Z.A.Ishag HASSAN
;
Xing XIE
;
Bin XU
;
Qiyan XIONG
;
Zhixin FENG
;
Guoqing SHAO
;
Yanfei YU
Author Information
1. 江苏省农业科学院兽医研究所,江苏南京 210014
- Publication Type:Journal Article
- Keywords:
Mesomycoplasma hyopneumoniae;
mutant;
p97 gene;
homologous recombination
- From:
Chinese Journal of Veterinary Science
2025;45(3):473-481
- CountryChina
- Language:Chinese
-
Abstract:
The aim of this study is to establish an gene editing method of Mesomycoplasma hyo-pneumoniae(Mhp)based on the homologous recombination principle.The restriction enzyme di-gestion and ligation method combined with gene synthesis were used to construct a shuttle plasmid to achieve replication in both Mhp and Escherichia coli(E.coli).The pGEM?-T vector was used as the skeleton.The oriC sequence of Mhp which can achieve the replication of the plasmid in Mhp was inserted into the vector.Sequences of the Spiroplasma promoter and puromycin resistance gene were then inserted into the above constructed plasmid to screen recombinant clones.The up-stream and downstream homologous arms of p97 were constructed to initiate homologous recombination.The recA gene of E.coli is inserted to improve the efficiency of homologous recom-bination.The obtained shuttle plasmid was then delivered into Mhp by electro-transformation or chemical transformation.A shuttle plasmid,pGEM?-Mhp-oriC-p 97,which can replicate in both Mhp and E.coli was constructed.With the transformation of this plasmid,the carried puromycin gene and recA gene can be expressed,the p97 gene can be edited.Finally,the genetically unstable p97 gene mutant was initially obtained.In this study,a tool for Mhp gene editing based on the principle of homologous recombination was established,which laid a foundation for the develop-ment of tools for studying the pathogenesis of Mhp.
