汉坦病毒N蛋白的重组表达及其IgM直接捕捉ELISA的建立和应用

发布时间：2018-04-25 23:40

本文选题：汉坦病毒 + 核蛋白　；参考：《浙江大学》2007年硕士论文

【摘要】： 肾综合征出血热(HFRS)是由汉坦病毒(Hantavirus，HV)引起的一组急性病毒性传染病，临床上以发热、出血及肾损害为主的一种自然疫源性疾病。全世界均有流行，我国是高发区，约占世界发病总数的90％以上，全国30个省市自治区均有本病的流行，近十年来，年报告发病人数4～6万人。汉坦病毒在全世界广泛分布，自1976年分离到该病毒以来，不断有新的汉坦病毒被发现，到目前已有28个汉坦病毒血清型／基因型。我国至少存在2个血清型，即HTN型(姬鼠型)和SEO型(家鼠型)。小型啮齿类动物为本病毒的主要贮存宿主，不同的汉坦病毒有不同的贮存宿主，黑线姬鼠为姬鼠型病毒的主要宿主，常引起重型出血热，病死率约为3～5％；褐家鼠为家鼠型病毒的主要宿主，常引起轻型出血热，病死率约为1％。本病主要通过接触带病毒的宿主动物及其排泄物受感染。 HV属布尼亚病毒科(Bunyaviridae)，是一种有包膜病毒，其基因组为单股负链RNA，含大(L)、中(M)、小(S)3个基因片段，分别编码多聚酶(polymerase)、包膜糖蛋白(glycoprotein)G1和G2、核蛋白(nucleocapsid protein，NP)。S基因长全约1.6～1.8Kb，3'与5'端各有一个非编码区，只有一个开放读码框架，编码一个分子量在48～50kD的核蛋白。尽管RT-PCR能检测汉坦病毒特异性核酸，可用于HFRS的早期诊断，但由于急性期患者病毒血症持续时间短、滴度低而难以检出，故检测特异性抗体的血清学检查，仍是目前HFRS的主要确诊方法。目前血清学检测所用抗原主要是由病毒感染细胞后收获制备而成，由于汉坦病毒具有较强的传染性，培养病毒需要在生物安全实验中进行，加上病毒在细胞中繁殖滴度低，难以获得大量的病毒抗原。研究发现HV病毒感染机体后，首先诱导产生较高水平的IgM抗体，随后产生IgG抗体，而早期抗体主要是由NP诱导产生的。因此，许多国内、外学者构建了多种表达系统来表达NP，作为HFRS的血清学诊断抗原。目前所用HFRS血清学试验，主要是基于病毒或表达核蛋白为抗原的IgM捕捉ELISA，此法有四个主要步骤，，操作烦琐、费时。因此，建立快速、简便、安全、敏感和特异的HFRS血清学诊断新方法有重要意义。本研究以含有生产肾综合征出血热疫苗的Z10毒株全长S基因的质粒为模板，通过高保真PCR亚克隆S基因核蛋白编码区，通过序列测定，分析它与国内外汉坦病毒分离株的同源性，以确定它的代表性。构建Z10株NP原核表达系统pET28a-Z10N-E.coli BL21DE3，表达重组核蛋白(rNP)。ELISA法检测表达情况，通过离子交换法和Ni-NTA亲和层析法提纯rNP，SDS-PAGE了解rNP纯化情况，Western blot检测rNP的特异性免疫反应。并以提纯的重组NP为抗原，直接标记辣根过氧化酶(HRP)，建立IgM直接捕捉ELISA。通过检测95份HFRS患者血清(临床表现典型，经传统的免疫荧光法证实)及部分健康者血清，与HV为抗原的常规IgM捕捉ELISA法进行比较，符合率为97.78％，两种IgM捕捉ELISAs法的阳性率分别为94.73％和92.63％，表明快速、简便、安全的rNP-IgM直接捕捉ELISA与HV-IgM间接捕捉ELISA对HFRS患者血清样品具有相同的检测效果。
[Abstract]:Hemorrhagic fever with renal syndrome is a group of acute viral infections caused by Hantaviruses . It is a natural epidemic disease characterized by fever , hemorrhage and kidney damage . There are epidemic in the world . Our country is a high - incidence area , accounting for more than 90 % of the world ' s disease , and 30 provinces and autonomous regions of the country have the epidemic of this disease . In recent ten years , the number of diseases reported has been 460,000 .

Hantaviruses are widely distributed throughout the world . Since the virus has been isolated from 1976 to the virus , it has been discovered that there are 28 serotypes / genotypes of Hantaviruses . There are at least 2 serotypes in China , namely ( Apodemus type ) and SE O ( family mouse type ) . Small rodents are the main storage hosts of the virus , and different Hantaviruses have different storage hosts . The Apodemus Apodemus is the main host of the Apodemus , which often causes severe hemorrhagic fever , with a mortality rate of about 3 - 5 % .
Rattus norvegicus is the main host of murine type virus , which often causes light hemorrhagic fever with a mortality rate of about 1 % . This disease is mainly affected by contact with the infected host animal and its excretion .

HV belongs to Bunyaviridae , a enveloped virus whose genome is a single strand of minus - strand RNA , containing large ( L ) , medium ( M ) , small ( S ) 3 gene fragments , encoding polymerase , glycoprotein G1 and G2 , nucleoprotein ( NP ) , respectively . The S gene has a length of about 1.6 - 1.8Kb , each of the 3 ' and 5 ' ends has a non - coding region , and only one open reading frame is used for coding a nuclear protein having a molecular weight of 48 - 50 kD .

Although RT - PCR can detect the specific nucleic acid of Hantavirus , it can be used in the early diagnosis of the disease , but because of the short duration of virosis in the acute stage and low titer , the serological examination of the specific antibody is still the main diagnosis method .

At present , the antigen used for serological detection is mainly prepared from virus - infected cells , and the virus needs to be carried out in the biological safety experiment due to the strong infectivity of Hantaviruses , and the virus antigen is difficult to obtain after the virus is infected by the virus .

This method has four main steps , such as complicated operation and time - consuming . Therefore , it is important to establish a rapid , simple , safe , sensitive and specific diagnostic new method for the serologic diagnosis .

The recombinant NP was used to purify rNP . The positive rate of ELISA was 97.78 % . The positive rates of two IgM capture ELISA were 94.73 % and 92.63 % , respectively .

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

【参考文献】