摘要
本研究以贵州羊传染性胸膜肺炎支原体分离株(GM01株)基因组DNA为模板,通过PCR技术扩增目的基因RPS11,并克隆到pET28a(+)载体,成功构建了重组质粒pET28a-RPS11,将其转化至大肠杆菌Rosetta感受态细胞中诱导表达重组RPS11蛋白并建立了羊传染性胸膜肺炎间接ELISA检测方法。SDS-PAGE分析表明,重组蛋白分子量为19 kDa,纯度为85%,具有良好的抗原性及特异性;检测方法经条件优化,确定了抗原包被浓度为1μg/mL,血清稀释度为1∶400,酶标二抗工作浓度为1∶4000,底物显色时间为10min。用建立的间接ELISA检测方法与羊传染性胸膜肺炎间接血凝检测试剂分别对临床180份血清进行检测,阳性率分别为35.0%和29.44%,总符合率达91.11%。本研究成功完成了RPS11蛋白的重组表达,制备出特异性较好的抗体,建立的间接ELISA检测方法为羊传染性胸膜肺炎的血清学检测诊断及流行病学调查提供了有效的技术手段。
In this study,RPS11 gene of Mycoplasma contagious pleuropneumonia strain GM01 was amplified in PCR and cloned into pET28 a(+)vector.The recombinant plasmid was transformed into E.coli Rosetta for prokaryotic expression.The recombinant(MW-19 kDa)was purified and confirmed in SDS-PAGE.The RPS11 protein was used as coating antigen to develop an indirect ELISA.The reaction conditions were optimized and determined to be 1μg/mL coating antigen,1:100 diluted antiserum,1:4000 diluted second antibody,and 10 min for the substrate development.The clinical 180 serum samples were tested using this indirect ELISA and the reference indirect hemagglutination test.As a result,positive rates were 35.0%(63/180)in indirect ELISA and 29.44%in indirect hemagglutination.In conclusion,the indirect ELISA developed here using the recombinant RPS11 provided an effective tool for serological detection,diagnosis and epidemiological investigation of infectious pleuropneumonia in sheep and goats.
作者
王璇
吴玙彤
潘淑惠
黄波
高明琴
文正常
WANG Xuan;WU Yu-tong;PAN Shu-hui;HUANG Bo;GAO Ming-qin;WEN Zheng-chang(Institute of Animal Husbandry and Veterinary Medicine of Guizhou Academy of Agricultural Sciences,Gufyang 550005,China)
出处
《中国动物传染病学报》
CAS
北大核心
2020年第1期39-44,共6页
Chinese Journal of Animal Infectious Diseases
基金
贵州省科技计划项目(黔科合NZ[2013]3025)
