抗戊型肝炎病毒单克隆抗体识别表位的初步研究及戊型肝炎病毒基因1型和基因4型中和表位区域分子差异研究

发布时间：2019-07-04 13:46

【摘要】： 戊型肝炎病毒(HEV)是戊型肝炎的病原体,随着HEV流行病学研究的深入,发现HEV的4个主要基因型存在着明显的易感宿主差异:基因1型(HEV-1)和基因2型(HEV-2)仅分离于人类,能导致大规模的戊肝爆发流行,实验动物目前只成功感染非人灵长类,而基因3型(HEV-3)和基因4型(HEV-4)人畜共患,仅见于小规模流行和临床散发。我们将HEV分为两类:包括HEV-1/2的H(Human)类和包括HEV-3/4的Z(Zoonosis)类。首先我们通过Western blot、体外捕获PCR、ELISA阻断实验及合成的多肽库对实验室制备的多株抗HEV单抗的识别表位进行系统的研究,结果发现12株线性单抗识别的位都位于ORF2 aa408-458之间,17株构象型单抗识别表位都定位于ORF2 aa459-606之间,其中15株的识别表位位于天然病毒表面。现有研究结果表明HEV的主要中和表位区域集中于ORF2的aa459-606之间,并且也是主要介导HEV与嗜性细胞吸附的区域。本研究通过比较这两类HEV ORF2 aa368-606区段,发现存在4个类保守的差异位点,均位于HEV的主要中和表位区域,分别是Ser483Thr、Val492Met、Ser497Thr和Ala599Gly。以能形成类病毒颗粒的HEV 239(ORF2 aa368-606)为基础,对这四个位点进行定点替换突变,并以这15株能够捕获HEV-1和/或HEV-4的单克隆抗体比较各种突变体的免疫反应性,结果表明仅aa497的差异造成了这两类HEV中和表位构象的部分差异,提示aa497及其相关的病毒表面结构差异可能在H类和Z类HEV宿主选择中扮演重要角色。
文内图片：

图片说明：HEV基因组Fig1GenomicorganizationofHEV(A)ingenotype1-3and(B)ingenotype4
[Abstract]:Hepatitis E virus (HEV) is the pathogen of hepatitis E. with the deepening of HEV epidemiological research, it is found that there are obvious susceptible host differences in the four main genotypes of HEV: gene type 1 (HEV-1) and gene type 2 (HEV-2) are only isolated from human beings, which can lead to large-scale outbreak of hepatitis E. At present, experimental animals are only successfully infected with non-human primates. Gene 3 (HEV-3) and gene 4 (HEV-4) zoonosis are only found in small-scale epidemic and clinical dissemination. We divide HEV into two categories: the H (Human) class of HEV-1/2 and the Z (Zoonosis) class of HEV-3/4. Firstly, we systematically studied the recognition epitopes of several strains of anti-HEV monoclonal antibodies prepared in laboratory by Western blot, capture PCR,ELISA blocking experiment in vitro and the synthetic polypeptide library. The results showed that the recognition epitopes of 12 strains of linear monoclonal antibodies were located between ORF2 aa408-458 and 17 strains of constructed monoclonal antibodies were located between ORF2 aa459-606, and 15 of them were located on the surface of natural viruses. The existing results show that the main neutralizing epitope region of HEV is concentrated between the aa459-606 of ORF2, and it is also the region that mediates the adsorption of HEV with sex-like cells. In this study, by comparing the two types of HEV ORF2 aa368-606 regions, it was found that there were four conserved difference sites, all of which were located in the main neutralizing epitope regions of HEV, Ser483Thr,Val492Met,Ser497Thr and Ala599Gly., respectively. The site-directed substitution mutations of these four loci were carried out on the basis of HEV-239( ORF2 aa368-606), which can form viral particles, and the immunoreactivity of various mutants was compared with the 15 monoclonal antibodies that could capture HEV-1 and / or HEV-4. The results showed that only the difference of aa497 resulted in some differences in the conformational patterns of neutralizing epitopes between the two types of HEV. It is suggested that the surface structure difference of aa497 and its related viruses may play an important role in the host selection of class H and Z HEV.
【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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