IBRV重组gD蛋白的真核表达与间接ELISA检测方法的建立  

Eukaryotic Expression of IBRV Recombinant gD Protein and Development of an Indirect ELISA Assay

作　　者：郭宇 杨波 兰德松[3] 郁茵 杨冬梅 王旭红 云涛 牧仁 李雷斌 孙雨[6] Guo Yu;Yang Bo;Lan Desong;Yu Yin;Yang Dongmei;Wang Xuhong;Yun Tao;Mu Ren;Li Leibin;Sun Yu(Inner Mongolia Autonomous Region Animal Disease Control Center,Hohhot,Inner Mongolia 010051,China;Ordos Animal Disease Prevention and Control Center of Inner Mongolia Autonomous Region,Ordos,Inner Mongolia 017000,China;Liaoning Animal Epidemic Disease Prevention and Control Center,Shenyang,Liaoning 110164,China;Miyun District Animal Disease Control Center,Beijing 101500,China;College of Animal Science and Technology,Guangxi University,Nanning,Guangxi 530004,China;China Animal Disease Control Center,Beijing 102600,China)

机构地区：[1]内蒙古自治区动物疫病预防控制中心,内蒙古呼和浩特010051 [2]鄂尔多斯市动物疫病预防控制中心,内蒙古鄂尔多斯017000 [3]辽宁省动物疫病预防控制中心,辽宁沈阳110164 [4]北京市密云区动物疫病预防控制中心,北京101500 [5]广西大学动物科学技术学院,广西南宁530004 [6]中国动物疫病预防控制中心,北京102600

出　　处：《中国动物检疫》2023年第2期139-145,共7页China Animal Health Inspection

基　　金：农业农村部跨境动物疫病防控机制建设项目(2130114-FK);现代农业产业体系建设专项(CARS39)。

摘　　要：通过优化牛传染性鼻气管炎病毒(IBRV)gD基因序列为Sf9细胞偏好密码子,转座形成穿梭载体,将质粒瞬时转染Sf9昆虫细胞,利用昆虫杆状病毒表达系统表达纯化重组IBRV gD蛋白;以制备的重组IBRV gD蛋白为包被抗原,建立检测IBRV血清中和抗体的间接ELISA方法,并通过比对检测已知血清中和抗体效价的牛血清,验证所建立的间接ELISA方法的敏感性、特异性与重复性等指标。结果显示:本研究建立的间接ELISA方法对相关的牛病毒血清抗体无交叉反应,敏感性可达1:512,组内和组间变异系数均低于10%;使用建立的间接ELISA检测方法结合血清中和试验,对480份临床血清样品进行检测,发现两者敏感性符合率为98.18%,特异性符合率为93.33%,总符合率为96.67%。结果表明,本研究建立的检测IBRV血清中和抗体的间接ELISA方法具有良好的特异性、敏感性和重复性,可开发为临床适应的检测试剂盒。The sequence of infectious bovine rhinotracheitis virus(IBRV)gD gene was optimized as Sf9 cell preferred codon,transposed to form a shuttle vector,the plasmid was transiently transfected into Sf9 insect cells,then the recombinant IBRV gD protein was expressed and purified by insect baculovirus expression system;the prepared recombinant IBRV gD protein was used as the coating antigen to establish an indirect ELISA assay for serum neutralizing antibodies against IBRV.Bovine serum with a known neutralizing antibody titer was compared and detected to verify the sensitivity,specificity and repeatability of the established assay.The results showed that the established assay failed to crossly react with relevant serum antibodies against bovine viral diseases,its sensitivity could reach 1:512,and the intra-group and inter-group coefficients of variation(CV)were both less than 10%;480 clinical serum samples were detected by the established assay in combination with serum neutralization test,it was found that the coincidence rates of the sensitivity and specificity of the two methods were 98.18%and 93.33%,respectively,and the total one was 96.67%.In conclusion,the assay established in the study,with good specificity,sensitivity and repeatability,could be used as a clinically applicable detection kit.

关 键 词：重组IBRV gD蛋白 杆状病毒 真核表达 蛋白纯化 间接ELISA 

分 类 号：S852.65[农业科学—基础兽医学]