猪繁殖与呼吸综合征病毒核衣壳蛋白的原核可溶性表达和间接ELISA抗体检测方法的初步建立  

Soluble prokaryotic expression of nucleocapside protein encoding gene of porcine reproductive and respiratory syndrome virus and the primary establishment of indirect ELISA for the detection of serum antibodies against PRRSV

作　　者：王俊[1,2] 代军飞 马炳 丁耀忠[1] 欧云文[1] 刘永生 赵乐辉[2] 张永光 张杰[1] WANG Jun1,2, DAI Jun-fei1, MA Bing1, DING Yao-zhong1, OU Yun-wen1, LIU Yong-sheng1, ZHAO Le-hui2, ZHANG Yong-guang1,3,ZHANG Jie1(1. State Key Laboratory of Veterinary Etiological Biology/National Foot-and-Mouth Disease Reference Laboratory/Lanzhou Veterinary Research Institute ,Chinese Academy of Agricultural Sciences ,Lanzhou 730046,China;2. College of Agricultural Sciences ,Eastern Liaoning University,Dandong 118003, China; 3. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses , Yangzhou 225009, Chin)

机构地区：[1]中国农业科学院,兰州兽医研究所,家畜疫病病原生物学国家重点实验室/国家口蹄疫参考实验室,甘肃兰州730046 [2]辽东学院农学院,辽宁丹东118003 [3]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医科学》2018年第4期419-427,共9页Chinese Veterinary Science

基　　金：国家国际科技合作项目(2012DFG31890);中波农业科技中心课题;中国农业科学院农业科技创新工程项目

摘　　要：猪繁殖与呼吸综合征病毒（porcine reproductive and respiratory syndrome virus，PRRSV）的核衣壳蛋白N是一种含量高、免疫原性强、最早刺激机体产生特异性抗体的主要结构蛋白，研制针对N蛋白抗体的检测方法对进一步实现PRRS的流行病学监测和诊断具有重要意义。本研究以带有MBP标签的pMAL—C4X作为表达载体。实现了N蛋白在大肠杆菌中的可溶性表达，每升菌液经纯化可得约20mg的融合N蛋白。Western—blot结果表明．融合N蛋白能被抗MBP的单抗和PRRSVN蛋白的家兔多抗特异识别。以融合N蛋白为包被抗原建立的间接ELISA方法不与猪圆环病毒2型（PCV2）、古典猪瘟病毒（CSFV）、伪狂犬病病毒（PRV）、猪细小病毒（PPV）的阳性猪血清发生交叉反应，但与1型PRRSV阳性猪血清有较强反应性。田间样品检测结果表明。本研究建立的间接ELISA抗体检测方法与IDEXX同类试剂盒的总符合率为90％左右。本研究以MBP作为促溶标签，实现了PRRSVN蛋白在大肠杆菌内的高效可溶性表达，重组蛋白具有良好的免疫反应性．这为深入研究N蛋白功能以及开发PRRS相关诊断制剂奠定了基础。The nucleocapsid（N） protein of porcine reproductive and respiratory syndrome virus （PRRSV）is one of the major structural proteins and the most abundant component of the virion. N protein contains important immunogenic epitopeswhich can elicit the immune system to produce non-neutralized antibody as early as one week after infection. It is critical to generate the recombinant N protein with natural properties for PRRS epidemiological survey and diagnosis. Maltose-Binding Protein （MBP）can work as molecular chaperone to facilitate the correct folding of the proteins and promote the exogenous gene to be expressed in soluble forms in prokaryotic system. In present study,the soluble expression of PRRSV N protein was achieved in E. coli BL21（DE3）via using PMAL-C4X with MBP-tag as the vector. The gen eral output of MBP-fused N protein was around 18 mg in a liter of LB culture. Western-blot analysis indicated that the fused N protein had strong specific reaction to the murine monoclonal antibody against MBP and the rabbit serum against type I PRRSV N protein. Furthermore,the indirect ELISA was established based on MBP-fused N protein served as the coated-antigen. The newly-established ELISA showed no cross-reactions with the porcine serum against porcine circovirus type 2（PCV2）,classical swine fever virus（CSFV）,pseudorabies virus（PRV） and porcine parvovirus（PPV）.However,there was strong im- munoreaction with type i PRRSV serum. The general diagnostic conincidence rate was approximately 90% between the newly-established ELISA and IDEXX PRRS X3 Kit for PRRSV antibody detection according to the results of 36 sera collected from the pig farms, ln conclusion,PRRSV N protein was highly expressed in E. Coli in soluble forms under the help of MBP-tag. TheMBP-fused N protein possesses strong immunoreac- tion which laid foundation for further exploring N protein function and developing PRRS diagnostic reagents.

关 键 词：猪繁殖与呼吸综合征病毒 核衣壳蛋白 原核可溶性表达 间接ELISA 抗体检测 

分 类 号：S852.651[农业科学—基础兽医学]