人源抗EV71病毒基因工程抗体的研究

发布时间：2018-03-07 19:27

本文选题：EV71病毒　切入点：人源基因工程抗体　出处：《中国疾病预防控制中心》2011年硕士论文　论文类型：学位论文

【摘要】：肠道病毒71型(Enterovirus 71, EV71)属于小RNA病毒科肠道病毒A属,主要引起婴幼儿的手足口病,重症者出现病毒性脑炎、脑膜炎、肺水肿、肺出血,个别病例进展快速,迅速导致死亡。EV71病毒从1969年在美国加尼福尼亚最早被发现以来,至今一共引起了10多次的世界范围的爆发和流行,其中又有四次大的流行。从2008年安徽阜阳爆发EV71以来,卫生部发布《手足口病预防控制指南》并将手足口病纳入丙类传染病来管理。在丙类传染病中手足口病发病数位于第三,而死亡数为第一位。对EV71的感染临床上主要采用广谱抗病毒药物以及对症治疗,但目前没有针对EV71特效抗病毒药物,疫苗正处于研制阶段。所以研究EV71作为一种微生物致病原所引发的机体免疫应答,对明确发病机制、制定预防诊治策略具有重要意义。在EV71抗体的研究方面,目前仅有鼠源的单克隆中和性抗体的报道,而鼠源抗体本身就存在很多缺陷,从而限制了其很多的应用。本文采用了pHAL14系统,构建了人源抗EV71病毒sc-Fv单链抗体基困库,并利用原核表达纯化的VP1蛋白筛选获得了1株抗EV71病毒特异性人源sc-Fv单克隆抗体,并将其分别构建表达成为单链-Fc和IgG形式的全抗体,为进一步研究病毒免疫和抗体结合表位奠定了基础。本研究以噬菌体表面展示技术为平台,筛选人源抗EV71病毒抗体,实验有以下两部分：一,人源抗EV71病毒scFV(?)笨菌体抗体库的构建与筛选采集9位EV71引起的手足口病患儿恢复期外周全血,分离淋巴细胞,然后提取总RNA并反转录成cDNA,接着用特异性的PCR引物经过两轮扩增特异性轻链和重链可变区基因,在对PCR产物鉴定和纯化后,用轻链基因与噬菌粒pHAL14构建了轻链库,随后将重链基因克隆入轻链库中构建scFv噬菌体抗体库。最后使用辅助噬菌体对scFv噬菌体抗体库进行包装,检测库容量,结果表明我们所构建scFV(?)噬菌体抗体库的容量为4.0×108,轻链插入率和重链插入率均为100%,满足筛库的需要。在成功构建scFv噬菌体抗体库的基础上,用原核表达纯化的VP1蛋白对噬菌体抗体库进行富集筛选,利用ELISA、IFA以及Western Blot对所获人源单克隆抗体的功能特性进行鉴定,并通过序列测定确定所获抗体的基因序列,然后与Genebank报道的抗体序列进行比较,根据抗体基因序列推断出其氨基酸序列,与vbase database中抗体序列信息比较,确定其CDR区。结果获得1株scFv抗体,一系列功能试验均证明该抗体是特异针对EV71 VP1蛋白。二,人源抗EV71病毒全抗体表达及功能鉴定在原核表达scFv抗体的基础上,分别利用哺乳动物瞬时表达系统和昆虫杆状病毒载体技术平台实现了全抗体的真核分泌表达。将该EV71抗体的轻链和重链的可变区基因插入杆状病毒载体pCMX2.51和pAc-L-CH3R,然后将构建好的重组质粒转染293T细胞和与杆状病毒重组后转染昆虫细胞,通过IFA检测转染效果,接着对昆虫细胞表达上清进行纯化。利用SDS-PAGE、ELISA、IFA、Western Blot等方法检测纯化的抗EV71病毒IgG全抗的功能活性,结果表明该人源单克隆抗体特异性针对EV71病毒VP1蛋白。在和A型以及两株C4亚型EV71病毒进行中和试验,结果显示该抗体没有中和活性。综上所述,本研究运用噬菌体抗体库技术,成功构建了人源抗EV71病毒scFv噬菌体抗体库,筛选获得了1株特异性针对EV71病毒VP1蛋白的人源单链抗体。将该抗体构建为IgG全抗体,经IFA、Western Blot检测显示具有功能活性,与A型和C4亚型的病毒中和试验结果显示没有中和活性。
[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.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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