骆驼科动物中东呼吸综合征冠状病毒抗体间接ELISA检测方法的建立  

Development of an indirect ELISA for detection of the camelids Middle East respiratory syndrome coronavirus-specific antibodies

作　　者：王振山 韩秋雪 王馨悦 毕津豪 李恩涛 莫若 赵永坤[2] 冯娜[2] 王铁成 杨松涛 迟航 夏咸柱[1,2] WANG Zhen-shan;HAN Qiu-xue;WANG Xin-yue;BI Jin-hao;LI En-tao;MO Ruo;ZHAO Yong-kun;FENG Na;WANG Tie-cheng;YANG Song-tao;CHI Hang;XIA Xian-zhu(College of Veterinary Medicine,Jilin Agricultural University,Changchun 130118,China;Changchun Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Changchun 130122,China;Comparative Medicine Center,Peking Union Medical College,Beijing 100021,China;College of Veterinary Medicine,Northeast Agricultural University,Harbin 150036,China)

机构地区：[1]吉林农业大学动物医学院,吉林长春130118 [2]中国农业科学院长春兽医研究所,吉林长春130122 [3]北京协和医学院比较医学中心,北京100021 [4]东北农业大学动物医学学院,黑龙江哈尔滨150036

出　　处：《中国兽医科学》2022年第10期1230-1237,共8页Chinese Veterinary Science

基　　金：国家自然科学基金青年科学基金项目(31902306)

摘　　要：通过PCR扩增中东呼吸综合征冠状病毒(MERS-CoV)RBD基因片段,将其克隆至真核表达载体pCAGGS中,转染哺乳动物细胞Expi293F,并利用带有StrepTrap标签的亲和柱纯化制备MERS-CoV RBD蛋白,利用SDS-PAGE和Western-blot鉴定纯化后的RBD蛋白。以RBD蛋白为包被抗原,建立骆驼科动物MERS-CoV抗体间接ELISA检测方法。结果显示,MERS-CoV RBD蛋白在Expi293F细胞中以分泌形式表达,纯化后RBD蛋白纯度大于95%;成功建立了骆驼科动物MERS-CoV抗体间接ELISA检测方法。将其初步应用于骆驼、羊驼血清抗体的检测,检测结果与已知血清结果相符合。基于真核表达纯化的高纯度MERSCoV RBD蛋白,建立了骆驼科动物MERS-CoV抗体间接ELISA检测方法,为骆驼、羊驼等骆驼科动物的MERSCoV疫苗免疫效果评价、疫病监测等相关研究奠定了基础。The fragment of Middle East respiratory syndrome coronavirus(MERS-Co V)receptor binding domain(RBD)was amplified with PCR and the RBD protein gene was cloned into the eukaryotic expression vector p CAGGS and transfected into the Expi293F suspension cells.MERS-Co V RBD protein were purified using Strep Trap HP,then identified by SDS-PAGE and Western-blot.Using RBD protein as the coating antigen,an indirect ELISA method for the detection of MERS-Co V antibodies in camelid was established.The MERS-Co V RBD protein was expressed in a secreted form in Expi293F cells,with the purity higher than95%.An indirect ELISA detection method based on the detection of MERS-Co V antibodies in camelid was established.And it was preliminarily applied to the detection of serum antibodies of camel and alpaca,which were consistent with the results of the known immune serum.In conclusion,eukaryotic expression MERS-Co V RBD protein was successfully purified,an indirect ELISA assay for the detection of the camelids MERS-Co V-specific antibodies was developed.The current study was expected to lay the foundation for related researches on evaluating immune effect of vaccines,as well as epidemic monitoring of Camelidae family such as camels and alpacas.

关 键 词：中东呼吸综合征冠状病毒 真核表达 RBD蛋白 间接ELISA检测 

分 类 号：S852.65[农业科学—基础兽医学]