本文選題：EV71病毒　切入點(diǎn)：人源基因工程抗體　出處：《中國(guó)疾病預(yù)防控制中心》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：腸道病毒71型(Enterovirus 71, EV71)屬于小RNA病毒科腸道病毒A屬,主要引起嬰幼兒的手足口病,重癥者出現(xiàn)病毒性腦炎、腦膜炎、肺水腫、肺出血,個(gè)別病例進(jìn)展快速,迅速導(dǎo)致死亡。EV71病毒從1969年在美國(guó)加尼福尼亞最早被發(fā)現(xiàn)以來(lái),至今一共引起了10多次的世界范圍的爆發(fā)和流行,其中又有四次大的流行。從2008年安徽阜陽(yáng)爆發(fā)EV71以來(lái),衛(wèi)生部發(fā)布《手足口病預(yù)防控制指南》并將手足口病納入丙類傳染病來(lái)管理。在丙類傳染病中手足口病發(fā)病數(shù)位于第三,而死亡數(shù)為第一位。對(duì)EV71的感染臨床上主要采用廣譜抗病毒藥物以及對(duì)癥治療,但目前沒有針對(duì)EV71特效抗病毒藥物,疫苗正處于研制階段。所以研究EV71作為一種微生物致病原所引發(fā)的機(jī)體免疫應(yīng)答,對(duì)明確發(fā)病機(jī)制、制定預(yù)防診治策略具有重要意義。 在EV71抗體的研究方面,目前僅有鼠源的單克隆中和性抗體的報(bào)道,而鼠源抗體本身就存在很多缺陷,從而限制了其很多的應(yīng)用。本文采用了pHAL14系統(tǒng),構(gòu)建了人源抗EV71病毒sc-Fv單鏈抗體基困庫(kù),并利用原核表達(dá)純化的VP1蛋白篩選獲得了1株抗EV71病毒特異性人源sc-Fv單克隆抗體,并將其分別構(gòu)建表達(dá)成為單鏈-Fc和IgG形式的全抗體,為進(jìn)一步研究病毒免疫和抗體結(jié)合表位奠定了基礎(chǔ)。本研究以噬菌體表面展示技術(shù)為平臺(tái),篩選人源抗EV71病毒抗體,實(shí)驗(yàn)有以下兩部分： 一,人源抗EV71病毒scFV(?)笨菌體抗體庫(kù)的構(gòu)建與篩選 采集9位EV71引起的手足口病患兒恢復(fù)期外周全血,分離淋巴細(xì)胞,然后提取總RNA并反轉(zhuǎn)錄成cDNA,接著用特異性的PCR引物經(jīng)過(guò)兩輪擴(kuò)增特異性輕鏈和重鏈可變區(qū)基因,在對(duì)PCR產(chǎn)物鑒定和純化后,用輕鏈基因與噬菌粒pHAL14構(gòu)建了輕鏈庫(kù),隨后將重鏈基因克隆入輕鏈庫(kù)中構(gòu)建scFv噬菌體抗體庫(kù)。最后使用輔助噬菌體對(duì)scFv噬菌體抗體庫(kù)進(jìn)行包裝,檢測(cè)庫(kù)容量,結(jié)果表明我們所構(gòu)建scFV(?)噬菌體抗體庫(kù)的容量為4.0×108,輕鏈插入率和重鏈插入率均為100%,滿足篩庫(kù)的需要。 在成功構(gòu)建scFv噬菌體抗體庫(kù)的基礎(chǔ)上,用原核表達(dá)純化的VP1蛋白對(duì)噬菌體抗體庫(kù)進(jìn)行富集篩選,利用ELISA、IFA以及Western Blot對(duì)所獲人源單克隆抗體的功能特性進(jìn)行鑒定,并通過(guò)序列測(cè)定確定所獲抗體的基因序列,然后與Genebank報(bào)道的抗體序列進(jìn)行比較,根據(jù)抗體基因序列推斷出其氨基酸序列,與vbase database中抗體序列信息比較,確定其CDR區(qū)。結(jié)果獲得1株scFv抗體,一系列功能試驗(yàn)均證明該抗體是特異針對(duì)EV71 VP1蛋白。 二,人源抗EV71病毒全抗體表達(dá)及功能鑒定 在原核表達(dá)scFv抗體的基礎(chǔ)上,分別利用哺乳動(dòng)物瞬時(shí)表達(dá)系統(tǒng)和昆蟲桿狀病毒載體技術(shù)平臺(tái)實(shí)現(xiàn)了全抗體的真核分泌表達(dá)。將該EV71抗體的輕鏈和重鏈的可變區(qū)基因插入桿狀病毒載體pCMX2.51和pAc-L-CH3R,然后將構(gòu)建好的重組質(zhì)粒轉(zhuǎn)染293T細(xì)胞和與桿狀病毒重組后轉(zhuǎn)染昆蟲細(xì)胞,通過(guò)IFA檢測(cè)轉(zhuǎn)染效果,接著對(duì)昆蟲細(xì)胞表達(dá)上清進(jìn)行純化。利用SDS-PAGE、ELISA、IFA、Western Blot等方法檢測(cè)純化的抗EV71病毒IgG全抗的功能活性,結(jié)果表明該人源單克隆抗體特異性針對(duì)EV71病毒VP1蛋白。在和A型以及兩株C4亞型EV71病毒進(jìn)行中和試驗(yàn),結(jié)果顯示該抗體沒有中和活性。 綜上所述,本研究運(yùn)用噬菌體抗體庫(kù)技術(shù),成功構(gòu)建了人源抗EV71病毒scFv噬菌體抗體庫(kù),篩選獲得了1株特異性針對(duì)EV71病毒VP1蛋白的人源單鏈抗體。將該抗體構(gòu)建為IgG全抗體,經(jīng)IFA、Western Blot檢測(cè)顯示具有功能活性,與A型和C4亞型的病毒中和試驗(yàn)結(jié)果顯示沒有中和活性。
[Abstract]:Enterovirus 71 (Enterovirus 71, EV71) belongs to the small RNA virus, enterovirus A species, the main cause of infant hand foot and mouth disease, severe viral encephalitis, meningitis, pulmonary edema, pulmonary hemorrhage, individual cases progress fast, quickly lead to death from the.EV71 virus found in the United States since 1969 California first, so far a total of 10 times caused worldwide outbreaks and epidemics, which have four big popular. Since the 2008 Anhui outbreak in Fuyang EV71, the Ministry of Health issued the HFMD prevention and control guidelines > and included HFMD to class C infectious diseases. In class C infectious diseases the incidence of HFMD the number is third, and the number of deaths was first. Of EV71 infection on the main use of broad-spectrum antiviral drugs and symptomatic treatment, but currently no EV71 effects of antiviral drugs, vaccines are in the development stage. It is of great significance to study the immune response of the organism caused by EV71 as a pathogenic microorganism. It is of great significance to clear the pathogenesis and formulate the strategies for the prevention and treatment of the organism.
In the study of EV71 antibody, reported only murine monoclonal antibody, and the antibody itself has many defects, which limits the many applications. This paper uses the pHAL14 system, the construction of human anti EV71 virus sc-Fv antibody gene library, and prokaryotic expression purification VP1 protein were screened 1 strains of anti EV71 virus specific human sc-Fv monoclonal antibody, and its expression were constructed as the antibody of single stranded -Fc and IgG, for further research on virus immunity and antibody binding epitopes of the foundation. Based on the technology platform of phage displaying, screening of anti EV71 virus antibody the source has the following two parts:
Construction and screening of human anti EV71 virus scFV (?) clumsy cell antibody library
Collected 9 EV71 caused HFMD patients recovery of peripheral blood lymphocytes, and then total RNA extraction and reverse transcription into cDNA, then using PCR specific primers amplified by gene specific light chain and heavy chain variable regions in two rounds, identification and purification of PCR products, with a light chain gene and apoptosis the fungus grain of pHAL14 light chain library was constructed, then the heavy chain gene was cloned into scFv to construct phage antibody light chain library. Finally using the helper phage packaging of scFv phage antibody library, detection of storage capacity, the result showed that the construction of scFV (?) phage antibody library containing 4 * 108 light. The rate of chain insertion and heavy chain insertion rate was 100%, to meet the needs of library screening.
In the foundation of the success of the scFv phage antibody library, using prokaryotic expression and purification of VP1 protein was enriched by ELISA screening of phage antibody library, IFA and Western Blot were used to identify the functional properties of the human monoclonal antibody, and the sequence was determined by antibody gene sequence, then antibody sequence and Genebank the reports were compared according to the antibody gene sequences to infer its amino acid sequence, compared with the antibody sequence information of vbase database, to determine the area of CDR. Results 1 strains of scFv antibody were obtained, a series of function tests showed that the antibody was specific for EV71 VP1 protein.
Two, full antibody expression and functional identification of human anti EV71 virus
Based on the prokaryotic expression of scFv antibodies, respectively using mammalian transient expression vector system technology platform and realizes the baculovirus eukaryotic expression secretion of all antibodies. The VL gene of the EV71 antibody heavy chain and inserted into the baculovirus vector pCMX2.51 and pAc-L-CH3R, and then transfected with the constructed recombinant insect cells. The plasmid was transfected into 293T cells and the recombinant baculovirus and detected by IFA after transfection, then the supernatant was purified in insect cells. By using SDS-PAGE, ELISA, IFA, Blot and other functional activity of Western method for detection of purified anti EV71 virus IgG antibody, the results show that the human monoclonal antibodies specific for EV71 virus VP1 protein in type A and two strains of C4 subtype EV71 virus neutralization test results showed that the antibody has no neutralizing activity.
In summary, this study using phage antibody library technique, successfully constructed human anti EV71 virus scFv phage antibody library, 1 strains were screened for the EV71 virus specific VP1 protein of human scFv antibody. The construction of IgG antibody by IFA, Western, Blot showed that the functional activity, and A and the C4 subtype of the virus neutralization test results showed no neutralizing activity.

【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392.1

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本文編號(hào)：1580663