尼帕病毒截短G蛋白的原核表达及间接ELISA检测方法的建立  

Prokaryotic expression of truncated G protein of Nipah virus and establishment of indirect ELISA method

作　　者：王琼 莫若[1,2] 张颖 冯娜[2] 闫飞虎[2] 王铁成[2] 王开 赵永坤 WANG Qiong;MO Ruo;ZHANG Ying;FENG Na;YAN Fei-hu;WANG Tie-cheng;WANG Kai;ZHAO Yong-kun(College of Veterinary Medicine,Jilin Agricultural University,Changchun 130118,China;Changchun Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Changchun 130122,China;College of Wildlife and Protected Area,Northeast Forestry University,Harbin 150040,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]东北林业大学野生动物与自然保护地学院,黑龙江哈尔滨150040

出　　处：《中国兽医科学》2023年第10期1223-1231,共9页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2022YFC2305005)。

摘　　要：以截短的尼帕病毒(NiV)G基因原核表达产物为抗原建立检测尼帕病毒抗体的间接ELISA检测方法。对编码尼帕病毒G蛋白基因的部分序列进行扩增,将扩增获得的目的片段克隆至原核表达载体pET-30a(+),并转化至大肠杆菌BL21(DE3)。菌落PCR鉴定后,在最佳诱导条件下表达目的蛋白。用Ni-NTA柱纯化截短的G蛋白,并进行SDS-PAGE和Western-blot鉴定。以纯化的蛋白为抗原建立尼帕病毒抗体间接ELISA检测方法,并对该方法的特异性、灵敏性、稳定性进行验证。结果显示:成功构建重组质粒pET-30a(+)-NiV-G;表达的尼帕病毒截短G蛋白分子质量为50 ku,与预期值相符;截短的G蛋白能被His-tag抗体、抗尼帕病毒G蛋白粗提IgG识别。以该蛋白为包被抗原建立的间接ELISA检测方法检测已知阳性血清尼帕病毒抗体效价为1︰20480,裂谷热病毒、西尼罗病毒及克里米亚-刚果出血热病毒阳性马血清的检测结果均为阴性,且该方法批内、批间试验变异系数均小于10%。结果表明,成功构建了重组质粒pET-30a(+)-NiV-G,并诱导表达尼帕病毒截短G蛋白。以该蛋白建立的尼帕病毒抗体间接ELISA检测方法具有良好的特异性、灵敏性、稳定性,可用于尼帕病毒疫苗免疫效果的评价、尼帕病毒病的监测及尼帕病毒抗体检测试剂盒的研发。To develop an indirect ELISA for the detection of Nipah virus(NiV)antibodies using the prokaryotic expression product of truncated NiV G gene as the antigen,the gene sequence encoding Nipah virus G protein was partially amplified,the amplified target fragment was cloned into the prokaryotic expression vector pET-30a(+),and transformed into Escherichia coli BL21(DE3).After colony PCR identification,the target protein was induced expression under optimal induction conditions.Truncated G protein was purified using Ni-NTA column and tested by SDS-PAGE and Western-blot.Using purified proteins as antigens,an indirect ELISA method was established to detect NiV antibody level,and the specificity,sensitivity and stability of this method were tested.In results,recombinant plasmid pET-30a(+)-NiV-G was successfully constructed.The size of expressed NiV truncated G protein was 50 ku,which was consistent with the expected value.Truncated G protein can be recognized by His-tag antibody and crude IgG anti-NiV G protein.Using the indirect ELISA established with truncated G protein as the coating antigen,NiV antibody titer of a known positive serum was 1︰20480,the detection results of Rift Valley fever virus,West Nile virus and Crimean-Congco hemorrhagic fever virus positive horse serum were all negative,the coefficient of variation in both intra-batch and inter-batch experiments of this method was less than 10%.In conclusion,the recombinant plasmid pET-30a(+)-NiV-G was successfully constructed and the NiV truncated G protein was expressed.The indirect ELISA for detecting NiV antibodies based on this protein has good specificity,sensitivity and stability,and can be used to evaluate the immune efficacy of NiV vaccines,monitor Nipah virus disease,and develop NiV antibody detection kits.

关 键 词：尼帕病毒 间接ELISA 原核表达 截短G蛋白 

分 类 号：S852.659.5[农业科学—基础兽医学]