水貂阿留申病毒串联表位重组抗原的表达及血清学初步评价  

Expression and its serological preliminary evaluation on tandem-epitope recombinant antigens of Aleutian disease virus

作　　者：秦飞燕 孙杰 马凡舒 王洋[1] 柏玲 张蕾[1] 闫喜军[1] QIN Fei-yan;SUN Jie;MA Fan-shu;WANG Yang;BAI Ling;ZHANG Lei;YAN X-jun(Institute of Special Animal and Plant Science of CAAS,State Key Labora ory of Special EconomicAnimal Molecular Biology,Key Laboratory of Special Animal Epidemic Disea e,Changchun130122,China;Key Laboratory of Animal Im munology,Ministry of Agricult re/Henan Provincial Key Laboratory of Animal Im munology/Henan Academy of Agricultural Sciences,Zhengzhou 450002,China)

机构地区：[1]中国农业科学院特产研究所特种经济动物分子生物学国家重点实验室/农业部经济动物疫病重点实验室,吉林长春130122 [2]河南省农业科学院动物免疫学重点实验室/农业部动物免疫学重点实验室/河南省动物免疫学重点实验室,河南郑州450002

出　　处：《中国兽医学报》2019年第11期2135-2139,2151,共6页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划资助项目(2016YFD0501000)

摘　　要：为探究水貂阿留申病毒(ADV)VP2主要抗原表位在水貂阿留申病血清学诊断中的作用,本试验将VP2蛋白主要抗原区的表位基因用柔性肽串联连接,将其克隆到表达载体pET-30a(+)中进行原核表达,并对诱导条件进行优化。利用Ni-NTA His-Bind纯化系统对融合蛋白进行纯化,经SDS-PAGE和Western blot鉴定表达产物,并将融合蛋白作为检测抗原进行CIEP试验。结果显示,融合蛋白在IPTG终浓度为1mmol/L、37℃诱导表达4h,蛋白表达量最高,其大小约为30000,以可溶性表达形式为主;融合蛋白能与6×His单克隆抗体及ADV多克隆抗体发生特异性反应,说明该蛋白具有较好的反应原性;且该蛋白作为对流免疫电泳技术(CIEP)检测抗原与ADV阳性血清之间产生了明显的沉淀线,证实了该蛋白作为CIEP检测抗原的可能性。以纯化后的重组蛋白为抗原初步建立的间接ELISA方法具有较高的准确性。结果表明该蛋白具有良好的血清学检测价值,为水貂阿留申病血清学诊断方法的建立奠定了基础。In order to study the role of main antigen epitopes of Aleutian virus structural protein VP2in serological diagnosis of Aleutian disease,the epitopes in the main antigen region of VP2 protein were connected in tandem with a flexible peptide to synthesize a multi-epitope recombination gene.The multi-epitope gene was cloned into pET-30a(+)to express fusion protein,and optimize the inducing conditions.The recombinant protein was purified using a Ni-NTA His-Bind purification system and identified by SDS-PAGE and Western blot.The fusion protein was used as a diagnostic antigen for the CIEP assay.The SDA-PAGE analysis showed that the optimum conditions of induction were 1mmol/L IPTG,at 37℃for 4h.The molecular weight of the recombinant protein was about 30000,which was mainly soluble expression.The Western blot analysis indicated that the recombinant protein could be recognized by anti-His monoclonal antibody and the ADV polyclonal antibody,confirming that the recombinant protein has a strong reactivity.Moreover,the protein produced a distinct precipitation line between the CIEP detection antigen and the leech Aleutian virus-positive serum,confirming the possibility of the protein as a CIEP detection antigen.Indirect ELISA,which was established primarily by purified recombinant protein,has high accuracy.The results indicated that the fusion protein had good serological detection value,which laid a foundation for the serological diagnosis of mink Aleutian disease.

关 键 词：阿留申病毒 VP2蛋白 表位基因 原核表达 CIEP ELISA 

分 类 号：S852.65[农业科学—基础兽医学]