牛支原体NOX2的原核表达及黏附特性  

Prokaryotic Expression of NOX2 of Mycoplasma bovis and Its Adherence Characterization

作　　者：包世俊[1] 朱彩宏 邢小勇[1] 丁小琴[1] 温峰琴[1] 武小椿 薛惠文[1] BAO Shijun;ZHU Caihong;XING Xiaoyong;DING Xiaoqin;WEN Fengqin;WU Xiaochun;XUE Huiwen(College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China)

机构地区：[1]甘肃农业大学动物医学院,兰州730070

出　　处：《畜牧兽医学报》2020年第11期2895-2902,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：甘肃农业大学学科建设基金项目(GAU-XKJS-2018-062);甘肃省教育厅产业支撑引导项目(2019C-03);国家肉牛/牦牛产业技术体系项目(CARS-37)。

摘　　要：旨在探究牛支原体(Mycoplasma bovis,Mb)NADH氧化酶NOX2的生物学功能,本研究参照GenBank中Mb湖北分离株(Mb Hubei-1 strain)nox2基因序列设计引物,应用PCR扩增获得Mb临洮分离株的nox2基因,在测序及序列优化的基础上,构建原核表达载体pET-nox2,并在大肠杆菌Rosetta(DE3)中诱导表达,进而对表达产物rMbNOX2的酶促活性、免疫原性,NOX2在Mb内的分布,rMbNOX2抗血清的补体介导体外杀菌活性和对Mb黏附宿主细胞的抑制活性进行了分析。结果表明,Mb临洮株nox2基因全长1350 bp,与GenBank中已知序列的Mb nox2基因序列比较,除Mb JF4278株相似性为97.93%外,其余均为99.93%。SDS-PAGE结果显示,优化的nox2基因在大肠杆菌中成功表达,重组蛋白rMbNOX2相对分子质量约为67 ku,且具有良好的酶促活性;ELISA与Western blot结果显示,rMbNOX2具有良好的免疫原性,且Mb NOX2在细胞浆中的分布多于细胞膜;补体介导的体外杀菌试验及黏附抑制试验证实,rMbNOX2抗血清具有明显的补体介导杀支原体活性,并可有效抑制Mb对宿主细胞的黏附。本研究为深入探讨Mb NOX2生物学功能奠定了基础。To explore the biological function of NADH oxidase NOX2 from Mycoplasma bovis(Mb),according to the nox2 gene sequence of Mb strain Hubei(GenBank.CP002513.1),the primers were designed and the nox2 gene of Mb strain Lintao was amplified by PCR.Based on sequencing and gene optimization,the prokaryotic expression vector pET-nox2 was constructed,and was expressed in Escherichia coli Rosetta(DE3).Subsequently,the analysis of the enzymatic activity and the immunogenicity of recombinant proteins rMbNOX2 were completed.And then the subcellular localization of NOX2,complement-dependent bactericidal activity of anti-rMbNOX2 serum,and as well as inhibition effect of anti-rMbNOX2 serum to Mb adhering to host cells was determined.The results showed that the CDS sequence of the nox2 gene of Mb Lintao strain was 1350 bp,and showed 99.93%homology with nox2 gene of all Mb except Mb JF4278 strain in GenBank.The result of SDS-PAGE displayed the optimized nox2 was successfully expressed in E.coli.The recombinant protein rMbNOX2 was about 67 ku.Enzyme activity analysis showed that the purified rMbNOX2 had good enzymatic activity.The results of ELISA and Western blot showed that the rMbNOX2 has excellent immunogenicity,and the Mb NOX2 distribute both in the cell membrane and cytoplasm,but it’s more distributed in the cytoplasm.Complement-dependent mycoplasmacidal assay and adherence inhibition assay confirmed anti-rMbNOX2 serum has distinct complement-dependent mycoplasmacidal activity and can also effectively inhibit the adherence of Mb to host cells.The results of this study lay a foundation for further study on the biological function of Mb NOX2.

关 键 词：牛支原体 nox2基因 原核表达 功能分析 

分 类 号：S852.62[农业科学—基础兽医学]