双抗体夹心ELISA检测羊肠道病毒抗原方法的建立与应用及病毒VP1蛋白抗原表位的筛选

发布时间：2018-04-22 02:41

  本文选题：羊肠道病毒 + CEV-JL14　； 参考：《吉林大学》2017年硕士论文

【摘要】：肠道病毒为小RNA病毒科肠道病毒属成员。根据最新的病毒分类,肠道病毒属包含9个肠道病毒种(A,B,C,D,E,F,G,H,J)和3个鼻病毒种(A,B,C),它们是引起人类和动物临床上以神经系统、呼吸系统和消化系统疫病的重要病原体。其中E和F种肠道病毒主要感染牛,G种肠道病毒主要感染猪,给畜牧业造成严重经济损失。羊肠道病毒感染为国内外新发传染病,有关该病的诊断、防治及病毒致病机理等方面缺乏研究。本实验室在国内首次从腹泻山羊体内分离到一株20~30 nm的肠道病毒,命名为CEV-JL14。为了建立一种特异、敏感、快速与简便检测羊肠道病毒的诊断方法,以便为本病的流行病学研究提供技术手段,本研究应用RT-PCR扩增出CEV-JL14病毒的VP1基因序列,并将其克隆到原核表达载体,构建表达GST-VP1重组蛋白的原核表达质粒。以纯化表达的CEV的结构蛋白VP1,制备了针对VP1的单克隆抗体和兔源多克隆抗体,并以此建立了检测CEV抗原的双抗体夹心ELISA方法和研制检测CEV抗原的试剂盒。特异性、敏感性、重复性和稳定性试验结果显示,建立的检测CEV抗原的双抗体夹心ELISA方法及其试剂盒具有特异、敏感、快速、简便与稳定等特点。试剂盒4℃条件下可保存180 d。应用建立的夹心ELISA方法对吉林省和内蒙古部分地区的777份羊粪进行检测,结果发现上述地区的羊群存在程度不同的肠道病毒感,羊群CEV的感染率为11.4%~100.0%。利用肽扫描技术对制备的单克隆抗体6A1进行了抗原表位的筛选,确定羊肠道病毒单克隆抗体6A1的特异性表位为152TPPTDQDTYQWQT164。该结果为进一步研究CEV所诱导的免疫应答及病毒致病机理打下基础。综上所述,本研究制备了抗羊肠道病毒VP1的单克隆抗体和多克隆抗体,建立了特异敏感的检测CEV抗原的双抗体夹心ELISA方法和研发检测羊肠道病毒抗原的试剂盒,发现吉林省和内蒙古部分地区存在程度不同的的羊肠道病毒感染。同时确定羊肠道病毒单克隆抗体6A1的特异性表位。本研究结果为今后羊肠道病毒感染的诊断、检疫及防制提供了有效的手段和流行病学理论依据。
[Abstract]:Enterovirus is a member of small RNA virus family. According to the latest classification of viruses, the genus Enterovirus consists of 9 species of enteroviruses, Astragalus (Astragalus) and 3 species of rhinovirus (Acanthovirus). They are the major pathogens causing diseases of nervous system, respiratory system and digestive system in humans and animals. Among them, E and F enterovirus mainly infects cattle and G enterovirus and mainly infects pigs, which causes serious economic loss to animal husbandry. Sheep enterovirus infection is a new infectious disease at home and abroad. A 20Nm enterovirus was first isolated from diarrhea goats in our laboratory and named CEV-JL14. In order to establish a specific, sensitive, rapid and simple diagnostic method for the detection of sheep enterovirus, and to provide a technical means for the epidemiological study of the disease, the VP1 gene sequence of CEV-JL14 virus was amplified by RT-PCR. It was cloned into prokaryotic expression vector and constructed prokaryotic expression plasmid expressing GST-VP1 recombinant protein. Monoclonal and rabbit polyclonal antibodies against CEV were prepared by purified CEV structural protein VP1. A sandwich ELISA method for detecting CEV antigen and a kit for detection of CEV antigen were developed. The results of specificity, sensitivity, reproducibility and stability test showed that the double antibody sandwich ELISA method for detecting CEV antigen and its kit had the characteristics of specificity, sensitivity, rapidity, simplicity and stability. The kit can be stored for 180 days at 4 鈩，

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