本文選題：漢坦病毒 + 核蛋白��； 參考：《浙江大學(xué)》2007年碩士論文

【摘要】： 腎綜合征出血熱(HFRS)是由漢坦病毒(Hantavirus，HV)引起的一組急性病毒性傳染病，臨床上以發(fā)熱、出血及腎損害為主的一種自然疫源性疾病。全世界均有流行，我國(guó)是高發(fā)區(qū)，約占世界發(fā)病總數(shù)的90％以上，全國(guó)30個(gè)省市自治區(qū)均有本病的流行，近十年來(lái)，年報(bào)告發(fā)病人數(shù)4～6萬(wàn)人。 漢坦病毒在全世界廣泛分布，自1976年分離到該病毒以來(lái)，不斷有新的漢坦病毒被發(fā)現(xiàn)，到目前已有28個(gè)漢坦病毒血清型／基因型。我國(guó)至少存在2個(gè)血清型，即HTN型(姬鼠型)和SEO型(家鼠型)。小型嚙齒類動(dòng)物為本病毒的主要貯存宿主，不同的漢坦病毒有不同的貯存宿主，黑線姬鼠為姬鼠型病毒的主要宿主，常引起重型出血熱，病死率約為3～5％；褐家鼠為家鼠型病毒的主要宿主，常引起輕型出血熱，病死率約為1％。本病主要通過接觸帶病毒的宿主動(dòng)物及其排泄物受感染。 HV屬布尼亞病毒科(Bunyaviridae)，是一種有包膜病毒，其基因組為單股負(fù)鏈RNA，含大(L)、中(M)、小(S)3個(gè)基因片段，分別編碼多聚酶(polymerase)、包膜糖蛋白(glycoprotein)G1和G2、核蛋白(nucleocapsid protein，NP)。S基因長(zhǎng)全約1.6～1.8Kb，3'與5'端各有一個(gè)非編碼區(qū)，只有一個(gè)開放讀碼框架，編碼一個(gè)分子量在48～50kD的核蛋白。 盡管RT-PCR能檢測(cè)漢坦病毒特異性核酸，可用于HFRS的早期診斷，但由于急性期患者病毒血癥持續(xù)時(shí)間短、滴度低而難以檢出，故檢測(cè)特異性抗體的血清學(xué)檢查，仍是目前HFRS的主要確診方法。 目前血清學(xué)檢測(cè)所用抗原主要是由病毒感染細(xì)胞后收獲制備而成，由于漢坦病毒具有較強(qiáng)的傳染性，培養(yǎng)病毒需要在生物安全實(shí)驗(yàn)中進(jìn)行，加上病毒在細(xì)胞中繁殖滴度低，難以獲得大量的病毒抗原。研究發(fā)現(xiàn)HV病毒感染機(jī)體后，首先誘導(dǎo)產(chǎn)生較高水平的IgM抗體，隨后產(chǎn)生IgG抗體，而早期抗體主要是由NP誘導(dǎo)產(chǎn)生的。因此，許多國(guó)內(nèi)、外學(xué)者構(gòu)建了多種表達(dá)系統(tǒng)來(lái)表達(dá)NP，作為HFRS的血清學(xué)診斷抗原。 目前所用HFRS血清學(xué)試驗(yàn)，主要是基于病毒或表達(dá)核蛋白為抗原的IgM捕捉ELISA，此法有四個(gè)主要步驟，，操作煩瑣、費(fèi)時(shí)。因此，建立快速、簡(jiǎn)便、安全、敏感和特異的HFRS血清學(xué)診斷新方法有重要意義。 本研究以含有生產(chǎn)腎綜合征出血熱疫苗的Z10毒株全長(zhǎng)S基因的質(zhì)粒為模板，通過高保真PCR亞克隆S基因核蛋白編碼區(qū)，通過序列測(cè)定，分析它與國(guó)內(nèi)外漢坦病毒分離株的同源性，以確定它的代表性。構(gòu)建Z10株NP原核表達(dá)系統(tǒng)pET28a-Z10N-E.coli BL21DE3，表達(dá)重組核蛋白(rNP)。ELISA法檢測(cè)表達(dá)情況，通過離子交換法和Ni-NTA親和層析法提純r(jià)NP，SDS-PAGE了解rNP純化情況，Western blot檢測(cè)rNP的特異性免疫反應(yīng)。并以提純的重組NP為抗原，直接標(biāo)記辣根過氧化酶(HRP)，建立IgM直接捕捉ELISA。通過檢測(cè)95份HFRS患者血清(臨床表現(xiàn)典型，經(jīng)傳統(tǒng)的免疫熒光法證實(shí))及部分健康者血清，與HV為抗原的常規(guī)IgM捕捉ELISA法進(jìn)行比較，符合率為97.78％，兩種IgM捕捉ELISAs法的陽(yáng)性率分別為94.73％和92.63％，表明快速、簡(jiǎn)便、安全的rNP-IgM直接捕捉ELISA與HV-IgM間接捕捉ELISA對(duì)HFRS患者血清樣品具有相同的檢測(cè)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 .

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

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相關(guān)期刊論文 前3條

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