绵羊肺炎支原体p60蛋白N端与C端的表达及其免疫学特性研究  

Expression of N-terminal Domain and C-terminal Domain of Mycoplasma ovipneumoniae p60 Protein and Initial Identification on Their Immunologic Activity

作　　者：麻昌姣 郑佳琪[1] 黄海碧[1] 王晓晖[1] 白帆[1] 郝永清[1] 

机构地区：[1]内蒙古农业大学兽医学院微生物与免疫实验室,呼和浩特010018

出　　处：《中国畜牧兽医》2017年第2期371-376,共6页China Animal Husbandry & Veterinary Medicine

基　　金：内蒙古自治区科技计划"舍饲肉羊疫病动态防控关键技术研究"(201502070);国家科技支撑计划"重点牧区"生产生态生活"保障技术集成与示范"(2012BAD13B00)

摘　　要：为分析绵羊肺炎支原体(Mycoplasma ovipneumoniae,MO)膜表面脂蛋白p60的免疫学特性,本研究分别扩增p60蛋白N端与C端的基因,并通过重叠延伸PCR将C端基因序列中的TGA(1 459-1 461bp)定点突变为同义密码子TGG。将扩增的基因片段分别插入到pET-32a(+)表达载体上,重组质粒分别转化至大肠杆菌BL21(DE3)感受态细胞中进行诱导表达,得到分子质量约57ku的p60-N蛋白和分子质量约为30.5ku的p60-C蛋白;再对纯化后的重组蛋白进行Western blotting分析,结果表明重组蛋白均可与MO阳性血清发生特异性免疫学反应,表明表达的重组蛋白p60-N和p60-C均有良好的反应原性。本研究为筛选MO主要的免疫原性蛋白和建立特异的血清学诊断方法提供了依据。In order to analyze the immunological characteristics of lipid-associated membrane protein p60 of Mycoplasma ovipneumoniae,we cloned the N-terminal part and C-terminal part of p60 gene by PCR separately.The overlap-extension PCR was used to mutagenate nucleotides TGA（1 459-1 461bp）to TGG.Then the PCR product was inserted to the pET-32a（＋）plasmid for expression in E.coli BL21（DE3）.The purified recombinant protein was analyzed by SDSPAGE,and the recombinant N-terminal domain of p60 protein was successfully expressed with a mass of molecule of 57 ku,and a molecular of 30.5ku of the N-terminal domain of p60 protein.Western blotting revealed that the two kinds of proteins could specifically react with positive serum,which confirmed that the recombinant N-terminal domain of p60 protein and the recombinant C-terminal domain of p60 protein both had a good reactionogenicity.This study could provide an effective reference data for screening important immunogenicity protein and establishment available diagnosis methods.

关 键 词：绵羊肺炎支原体 p60蛋白 表达 

分 类 号：S852.62[农业科学—基础兽医学]