绵羊肺炎支原体Hsp70蛋白C末端基因真核表达载体的构建  

Construction of an eukaryotic expression vector for the Hsp70 C-terminal protein gene of Mycoplasma ovipneumoniae

作　　者：杨源[1,2] 王慧 王军[1,2] 张云丹[1,2] 文明 周碧君 程振涛[1,2] 岳筠 张双翔[3] YANG Yuan;WANG Hui;WANG Jun;ZHANG Yundan;WEN Ming;ZHOU Bijun;CHENG Zhentao;YUE Jun;ZHANG Shuangxiang(College of Animal Science,Guizhou University,Guiyang 550025,China;Key Laboratory of Animal Health and Veterinary Public Health of Guizhou Province,Guiyang 550025;uizhou Animal Disease Prevention and Control Center,Guiyang 550008,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]贵州省动物疫病与兽医公共卫生重点实验室,贵州贵阳550025 [3]贵州省动物疫病预防控制中心,贵州贵阳550008

出　　处：《畜牧与兽医》2018年第12期59-63,共5页Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金项目(31660723);省州合作项目(黔科合NY字2011-3105号);贵州省科技计划项目(黔科合支撑[2018] 2271号);贵州省研究生工作站计划项目(GZZ2017002)

摘　　要：为探讨绵羊肺炎支原体(Mo) Hsp70蛋白作为核酸疫苗的可能性,在对Mo贵州分离株Hsp70蛋白基因进行生物信息学分析的基础上,选择Mo Hsp70蛋白C末端基因,将其与载体pcDNA 3.1 (+)进行连接构建重组质粒,通过脂质体转染至CHO细胞,采用RT-PCR和间接免疫荧光方法评价转染效果和真核细胞表达效果。结果显示:Mo贵州分离株Hsp70蛋白的C末端是具有优势抗原表位的区域,通过PCR方法成功扩增出567 bpHsp70蛋白的C末端基因,并成功构建pc DNA 3.1-Hsp70重组质粒,在转染细胞中PCR扩增出预期大小的目的基因,可以观察到特异性的绿色荧光,提示本研究成功构建的重组质粒能在真核细胞中良好表达,为后续Mo Hsp70蛋白抗原性和免疫佐剂特性的研究奠定基础。In order to study the possibility of Hsp70 C-terminal protein of Mycoplasma ovipneumoniae as a target gene of genetic vaccine,the target gene was chosen based on bioinformation and amplified by PCR,and the target gene was ligated with the vector pc DNA 3.1(+).The recombinant plasmid was identified by PCR,double digestion and sequencing,and then transfected into CHO cells by lipo-transfection.The transfection conditions were verified by using PCR,RT-PCR and indirect immunofluorescence.The results showed that the C-terminus of the Hsp70 protein of the Guizhou strain was a predominant epitope region.The target gene was successfully amplified by PCR and the recombinant plasmid pc DNA 3.1-Hsp70 was successfully constructed in the transfection group.The specific green fluorescence was observed by IFA.The target gene of the desired fragment size was be amplified in the corresponding cell by PCR or RT-PCR,indicating that the constructed recombinant plasmid expressed antigenic Hsp70 C-terminal protein in mammalian cells.

关 键 词：绵羊肺炎支原体 HSP70蛋白 核酸疫苗 真核表达 

分 类 号：S858.26[农业科学—临床兽医学]