基于同源重组的猪肺炎支原体p97基因突变株的构建  

Construction of p97 mutant of Mesomycoplasma hyopneumoniae based on the homologous recombination system

作　　者：韦艳娜[1] 王吉英 谢欢 李志强[1,4] HASSAN Z.A.Ishag 谢星 徐彬 熊祺琰[1,5] 冯志新 邵国青[1] 于岩飞 WEI Yanna;WANG Jiying;XIE Huan;LI Zhiqiang;HASSAN ZAIshag;XIE Xing;XU Bin;XIONG Qiyan;FENG Zhixin;SHAO Guoqing;YU Yanfei(Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;School of Basic Medical Science,Hunan University of Medicine,Huaihua,Hunan 418000,China;College of Animal Science and Technology,Huaihua Vocational and Technical College,Huaihua,Hunan 418099 China;College of Veterinary Medicine,Hunan Agricultural University,Changsha 410125,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]江苏省农业科学院兽医研究所,江苏南京210014 [2]湖南医药学院基础医学院,湖南怀化418000 [3]怀化职业技术学院动物科技学院,湖南怀化418099 [4]湖南农业大学动物医学院,湖南长沙410125 [5]南京农业大学动物医学院,江苏南京210095

出　　处：《中国兽医学报》2025年第3期473-481,共9页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划资助项目(2022YFD1800903);国家自然科学基金面上资助项目(32172860);江苏省农业科技自主创新资金资助项目(CX[20]3090);江苏省科协青年科技人才托举工程资助项目(JSTJ-2023-XH031);湖南省教育厅科学研究资助项目(22B1055);湖南省自然科学基金资助项目(2022JJ50321)。

摘　　要：采用pGEM■-T载体作为骨架,通过质粒双酶切和连接以及全基因合成的方法,向载体质粒中插入猪肺炎支原体(Mesomycoplasma hyopneumoniae,Mhp)的oriC序列,获得pGEM■-Mhp-oriC-p97重组穿梭质粒,以实现质粒在Mhp中的复制;插入螺原体启动子及嘌呤霉素抗性基因用于重组单克隆的筛选;插入p97的上、下游同源臂用于同源重组的启动;插入大肠杆菌recA基因用于提高同源重组效率。然后,将pGEM■-Mhp-oriC-p97重组穿梭质粒通过电转化或化学转化递送入Mhp中,利用嘌呤霉素抗性基因及p97目的基因进行基因缺失株的筛选。结果显示,构建了一种能够同时在Mhp及大肠杆菌中复制的穿梭质粒pGEM■-Mhp-oriC-p97,该质粒的转化能实现嘌呤霉素基因、recA基因在Mhp中的表达及p97基因的变异,初步获得了p97基因突变株。结果表明,建立了一种利用同源重组原理实现Mhp基因编辑的工具,为Mhp致病机制研究工具的开发奠定了基础。The aim of this study is to establish an gene editing method of Mesomycoplasma hyopneumoniae(Mhp)based on the homologous recombination principle.The restriction enzyme digestion 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 upstream 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 recombination.The obtained shuttle plasmid was then delivered into Mhp by electro-transformation or chemical transformation.A shuttle plasmid,pGEM■-Mhp-oriC-p97,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 p 97 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 development of tools for studying the pathogenesis of Mhp.

关 键 词：猪肺炎支原体 基因突变株 p97基因 同源重组 

分 类 号：S852.62[农业科学—基础兽医学]