牛乳腺炎无乳链球菌β溶血素基因cylE缺失突变株的构建  

Construction of a β-hemolysin gene deletion mutant strain of bovine mastitis Streptococcus agalactiae

作　　者：布日额 吴金花 锡林高娃 孙立杰 BU Ri-e;WU Jin-hua;XI Lin gao-wa;SUN Li-jie(Research Institute for Pathogens in Milk,Inner Mongolia University for Nationalities,Tongliao,Inner Mongolia,China028043)

机构地区：[1]内蒙古民族大学生命科学学院内蒙古自治区乳源性致病菌防控工程技术研究中心内蒙古民族大学乳源性致病菌研究所

出　　处：《中国病原生物学杂志》2019年第7期773-779,共7页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.31760725);内蒙古自治区乳源性致病菌防控工程技术研究中心开放课题(No.MDK2018001,MDK2018002)

摘　　要：目的构建牛乳腺炎无乳链球菌cylE基因缺失突变菌株,为进一步开展其致病机制与免疫机制研究奠定实验基础。 方法参考GenBank上公布的无乳链球菌cylE基因上、下游序列(AF157015.2),利用primer 5.0生物信息软件设计扩增引物,经PCR扩增出cylE基因上游序列L、下游序列R,并根据pACYC184质粒氯霉素抗性基因cat r序列设计引物,扩增cat r基因片段C,依次将L、R和C片段克隆至温敏型自杀质粒pSET4S中,构建cylE基因缺失重组质粒pSET4S-LCR,通过电转化技术将其转化至无乳链球菌,通过温度和抗生素抗性筛选cylE缺失突变株。 结果经PCR和溶血表型鉴定结果表明,成功获得1株牛乳腺炎无乳链球菌β溶血素cylE基因缺失突变菌株。 结论为进一步研究其致病机制与免疫机制奠定了良好的研究基础。Objectives To construct a mutant strain of Streptococcus agalactiae with the cylEgene deleted from cattle with mastitis and to lay an experimental foundation for further research on its pathogenesis and immune mechanism. Methods Based on the upstream and downstream sequences of the cylE gene of Streptococcus agalactiae published in GenBank(AF157015.2),the bioinformatic software primer 5.0was used to design and amplify primers.The L and R upstream and downstream sequences of the cylEgene were amplified using PCR.The primers were designed according to the cat r sequence of the chloramphenicol resistance gene of the pACYC184plasmid,and CATR gene fragment C was amplified. The L,R,and C fragments were each cloned in the thermosensitive plasmid.In the suicide plasmid pSET4S,a cylEgene deletion recombinant plasmid pSET4S-LCR was constructed.The plasmid was transformed into Streptococcus agalactiae via electrotransformation.CylEdeletion mutants were screened for temperature and antibiotic resistance. Results PCR results and determination of the hemolytic phenotype indicated that a mutant strain of Streptococcus agalactiae with theβ-hemolysin gene cylEdeleted was successfully obtained.Determination of the hemolytic phenotype indicated that the cylEgene deletion mutant strain grew on rabbit blood agar plates and that the hemolytic phenotype was absent around the colony,while the control wild-type strain had an obviousβ-hemolytic phenotype. Conclusion This study has laid a good foundation for further study of the pathogenesis and immune mechanism of S.agalactiae.

关 键 词：无乳链球菌 溶血素 基因敲除 

分 类 号：R383.15[医药卫生—医学寄生虫学]