發(fā)布時(shí)間：2018-04-22 02:41

  本文選題：羊腸道病毒 + CEV-JL14　； 參考：《吉林大學(xué)》2017年碩士論文

【摘要】：腸道病毒為小RNA病毒科腸道病毒屬成員。根據(jù)最新的病毒分類,腸道病毒屬包含9個(gè)腸道病毒種(A,B,C,D,E,F,G,H,J)和3個(gè)鼻病毒種(A,B,C),它們是引起人類和動物臨床上以神經(jīng)系統(tǒng)、呼吸系統(tǒng)和消化系統(tǒng)疫病的重要病原體。其中E和F種腸道病毒主要感染牛,G種腸道病毒主要感染豬,給畜牧業(yè)造成嚴(yán)重經(jīng)濟(jì)損失。羊腸道病毒感染為國內(nèi)外新發(fā)傳染病,有關(guān)該病的診斷、防治及病毒致病機(jī)理等方面缺乏研究。本實(shí)驗(yàn)室在國內(nèi)首次從腹瀉山羊體內(nèi)分離到一株20~30 nm的腸道病毒,命名為CEV-JL14。為了建立一種特異、敏感、快速與簡便檢測羊腸道病毒的診斷方法,以便為本病的流行病學(xué)研究提供技術(shù)手段,本研究應(yīng)用RT-PCR擴(kuò)增出CEV-JL14病毒的VP1基因序列,并將其克隆到原核表達(dá)載體,構(gòu)建表達(dá)GST-VP1重組蛋白的原核表達(dá)質(zhì)粒。以純化表達(dá)的CEV的結(jié)構(gòu)蛋白VP1,制備了針對VP1的單克隆抗體和兔源多克隆抗體,并以此建立了檢測CEV抗原的雙抗體夾心ELISA方法和研制檢測CEV抗原的試劑盒。特異性、敏感性、重復(fù)性和穩(wěn)定性試驗(yàn)結(jié)果顯示,建立的檢測CEV抗原的雙抗體夾心ELISA方法及其試劑盒具有特異、敏感、快速、簡便與穩(wěn)定等特點(diǎn)。試劑盒4℃條件下可保存180 d。應(yīng)用建立的夾心ELISA方法對吉林省和內(nèi)蒙古部分地區(qū)的777份羊糞進(jìn)行檢測,結(jié)果發(fā)現(xiàn)上述地區(qū)的羊群存在程度不同的腸道病毒感,羊群CEV的感染率為11.4%~100.0%。利用肽掃描技術(shù)對制備的單克隆抗體6A1進(jìn)行了抗原表位的篩選,確定羊腸道病毒單克隆抗體6A1的特異性表位為152TPPTDQDTYQWQT164。該結(jié)果為進(jìn)一步研究CEV所誘導(dǎo)的免疫應(yīng)答及病毒致病機(jī)理打下基礎(chǔ)。綜上所述,本研究制備了抗羊腸道病毒VP1的單克隆抗體和多克隆抗體,建立了特異敏感的檢測CEV抗原的雙抗體夾心ELISA方法和研發(fā)檢測羊腸道病毒抗原的試劑盒,發(fā)現(xiàn)吉林省和內(nèi)蒙古部分地區(qū)存在程度不同的的羊腸道病毒感染。同時(shí)確定羊腸道病毒單克隆抗體6A1的特異性表位。本研究結(jié)果為今后羊腸道病毒感染的診斷、檢疫及防制提供了有效的手段和流行病學(xué)理論依據(jù)。
[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，

本文編號：1785272