BTV16重组VP2蛋白原核表达、多克隆抗体制备及间接ELISA检测方法的建立与初步应用  

Establishment and preliminary application of prokaryotic expression of BTV16recombinant VP2protein,preparation of polyclonal antibody and indirect ELISA detection method

作　　者：张明珠 王鹏 田佳鑫 陈士刚 鲍君铎 仇相书 鲁会军[1,2] 李昌 ZHANG Mingzhu;WANG Peng;TIAN Jiaxin;CHEN Shigang;BAO Junduo;QIU Xiangshu;LU Huijun;LI Chang(College of Animal Medicine,Jilin Agricultural University,Changchun 130118,China;Changchun Veterinary Research Institute,Chinese Academy of AgriculturalSciences,Changchun 130122,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

出　　处：《中国兽医学报》2024年第12期2549-2555,共7页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划资助项目(2017YFD0501803)。

摘　　要：蓝舌病毒(BTV)是我国法定的多种动物共患的二类动物疫病,对反刍动物养殖业的危害极大,BTV共有29个血清型,BTV16是我国目前流行的主要血清型之一。BTV感染后主要表现为隐性感染,因此建立ELISA检测方法对流行病学的检测极为重要。本研究通过原核表达系统进行BTV16VP2蛋白的表达,使用BALB/c小鼠进行多克隆抗体的制备。建立并优化了以VP2蛋白为包被抗原的间接ELISA检测方法。对广西壮族自治区临床样品进行检测,并与商品化试剂盒进行符合率分析。结果表明,成功表达并纯化出BTV16VP2蛋白,制备的多克隆抗体具有良好的免疫原性。ELISA检测方法具有良好的特异性,对赤羽病病毒(AKAV)、口蹄疫病毒(FMDV)和盖塔病毒(GETV)等反刍动物疫病无交叉反应,阴、阳性的临界值为0.314,批间和批内的变异系数(Cv)均小于5%,具有良好的重复性,对79份广西壮族自治区的样品进行检测,阳性率为92.4%,与商品化试剂盒比较符合率为98.7%。本研究成功建立了一种BTV16间接ELISA检测方法,用于牛临床样品的检测。Bluetongue virus(BTV)is classified as a categoryⅡanimal epidemic disease in China,infecting multiple species and posing significant threats to the ruminant breeding industry.There are 29serotypes of BTV,with BTV16being one of the major serotypes currently prevalent in China.Bluetongue virus infection mainly manifests as a latent infection,making the establishment of ELISA assays crucial for epidemiological detection.In this study,the expression of the BTV16VP2 protein was achieved using aprokaryotic expression system,and polyclonal antibodies were prepared using BALB/c mice.An indirect ELISA assay using VP2protein as the encapsulated antigen was established and optimized.Clinical samples from the Guangxi Zhuang Autonomous Region were tested and analyzed for compliance with commercial kits.The results showed that the BTV16 VP2protein was successfully expressed and purified,and the prepared polyclonal antibody exhibited good immunogenicity.The ELISA assay had good specificity,with no cross-reactivity against ruminant diseases such as AKAV,FMDV and GETV.The critical values for negativity and positivity were determined to be 0.314,and the coefficients of variation(Cv)between batches and within batches were both less than 5%,indicating good reproducibility.The ELISA assay revealed apositive rate of 92.4%for 79samples from the Guangxi Zhuang Autonomous Region,with a compliance rate of 98.7%when compared to the commercialized kit.In conclusion,this study successfully established an indirect ELISA method for BTV16,facilitating the detection of bovine clinical samples.

关 键 词：原核表达 多克隆抗体 间接ELISA BTV 

分 类 号：S852.65[农业科学—基础兽医学]