戊型肝炎病毒在家禽、家畜中的流行及其ORF3蛋白与人肝细胞蛋白相互作用的研究

发布时间：2018-01-05 07:38

本文关键词：戊型肝炎病毒在家禽、家畜中的流行及其ORF3蛋白与人肝细胞蛋白相互作用的研究　出处：《北京协和医学院》2011年博士论文　论文类型：学位论文

【摘要】：戊型肝炎病毒(Hepatitis E Virus, HEV)是戊型肝炎的病原体,是一种重要的传染因子。逐渐积累起来的研究结果显示戊型肝炎是一种人兽共患病,自然界存在HEV的动物宿主。我国是戊型肝炎的流行区,既有爆发流行也有散发病例,近年来散发病例报告数持续增加,可能与动物源性HEV的传播有关。了解HEV在我国动物中的流行情况,对于预防和控制戊型肝炎的流行、减少发病具有重要意义。在本研究中,我们从不同省份采集我国主要饲养动物的血清,对HEV在各种动物中的感染率、感染水平进行了调查分析,并对HEV基因型、基因亚型的分布进行了分析研究。为了进一步探索HEV感染、致病以及不同基因型别HEV宿主差异的分子机制,我们应用酵母双杂交系统对人肝cDNA文库进行筛选,筛选了与HEV ORF3蛋白有相互作用的蛋白分子,并对不同基因型HEV ORF3蛋白与肝细胞蛋白的相互作用进行比较；通过对这些蛋白分子的生物学信息分析,对ORF3蛋白的功能及其对宿主细胞生物过程可能产生的影响进行了分析、推测。 1.HEV在我国家畜、家禽中流行情况的调查从我国的27个省(市)共采集了猪、牛、羊、家兔、鸡和鸭血清6329份,应用EIA方法测定血清中HEV抗体和抗原。结果显示,猪、牛、羊和家兔的平均HEV抗体阳性率分别为82.2%、28.2%、10.4%和15.4%,猪HEV抗体阳性率明显高于牛、羊和家兔。鸡和鸭的抗体阳性率分别为3.58%和7.1%,二者都明显低于猪、牛羊和家兔。来源于不同省份的动物HEV抗体的阳性率有明显差异,猪、牛、羊和家兔HEV抗体阳性率范围分别为10.9%-100%、0～92.9%、0～48%和0-53.4%。大约1.9%的猪、0.8%的牛、1.6%的羊以及3.8%的兔血清样本为HEV抗原阳性。鸡和鸭的抗原阳性率分别为1.79%和1.43%。应用Nested RT-PCR在15份猪血清和22份不同品种的家兔的血清中检测到了HEV RNA,而牛、羊、鸡和鸭血清中未检测到HEV RNA。通过对ORF2区域304bp长的核苷酸序列进行比对和进化树分析,15株猪HEV均属于基因4型。在进化树上,这15株4型HEV被分在不同的四组,分别为4a、4d及两个新的亚型。综合分析表明,我国猪群中流行的HEV主要为基因4型HEV,包括4a,4b,4d,4f,4g以及本研究新发现的两个亚型。从家兔中分离到的22株HEV均属于兔HEV,在进化树上,兔HEV又进一步分成了3组,很可能代表三个基因亚型。本研究表明HEV在我国家畜、家禽中普遍流行,猪HEV感染率最高,我国流行的猪HEV主要为基因4型,包括4a,4b,4d,4f,4g以及两个新亚型。证实了兔HEV在我国不同地区不同品种的家兔中均有流行,支持兔HEV属于一个新基因型的观点。牛、羊、鸡和鸭血清样本中都检测到了HEV抗原,但未能检测出HEVRNA,仍需进一步研究。 2. HEV ORF3蛋白与肝细胞蛋白相互作用的研究本研究以基因1型和4型HEV ORF3蛋白为诱饵,通过酵母配对杂交实验对人肝cDNA文库进行筛选,筛选结果应用酵母杂交共转化实验进行验证。经过三次实验,筛选出了32种与ORF3蛋白有相互作用的肝细胞蛋白。其中4种蛋白(4/32,12.5%)已经有报告能够与ORF3蛋白结合。进一步应用哺乳动物细胞内化学发光免疫共沉淀实验对酵母杂交实验的筛选结果的可靠性进行评估,显示筛选结果的可信度比较高。生物信息学分析发现这32种相互作用蛋白在细胞核、细胞质、细胞膜、细胞外基质、以及线粒体、内涵体膜中均有分布,大多数蛋白定位于细胞的多个部位。这些蛋白分别参与细胞代谢、细胞免疫、细胞黏附等多个生物学过程。因此推测ORF3蛋白可能通过与这些蛋白相互作用,发挥多重作用。通过对蛋白功能富集聚类分析,发现分别有显著多数量的相互作用蛋白参与血液凝固、氧化应激和铁代谢过程。结合戊型肝炎临床表现和肝组织的病理特征进行分析,推测ORF3可能通过与宿主细胞蛋白的相互作用,影响凝血、脂质代谢以及铁代谢,而引发相应的病理过程。通过酵母杂交共转化实验分别检测基因1型HEV、4型HEV、兔HEV以及禽HEV ORF3蛋白与以基因4型和1型HEV ORF3为诱饵筛选到的这32种人肝细胞蛋白是否有相互作用。结果显示,基因1型、4型和兔HEV的ORF3蛋白都能够与这些蛋白结合,然而禽HEV的ORF3蛋白只与其中的13种蛋白有相互作用。因此,禽HEV对人肝细胞的影响作用可能不同于哺乳动物HEV,这种蛋白相互作用的差别是否与禽HEV与哺乳动物HEV宿主范围等的差别有关联,还需要进一步研究。这些发现为我们进一步研究ORF3蛋白的致病作用,以及研究HEV感染、复制和宿主限制性的分子机制提供了线索和方向。
[Abstract]:Hepatitis E Virus ( HEV ) is a pathogen of HEV , and it is an important infection factor . The research result shows that hepatitis E is a kind of animal host with HEV . In this study , we screened the infection rate and infection level of HEV in different provinces . In order to further explore the epidemic situation of HEV in our country , we screened the protein molecule interacting with HEV ORF3 protein and compared the interaction of HEV ORF3 protein with liver cell protein . 1 . Investigation of the prevalence of HEV in domestic animals and poultry in China The positive rates of anti HEV antibodies in pigs , cattle , sheep and rabbits were 82 . 2 % , 28 . 2 % , 10 . 4 % and 15.4 % , respectively . The positive rates of antibodies in pigs , cattle , sheep and rabbits were significantly lower than those in cattle , sheep and rabbits . The positive rates of antibodies in pigs , cattle , sheep and rabbits were respectively 10.9 % -100 % , 0 - 92.9 % , 0 - 48 % and 0 - 53.4 % . The positive rates of antibodies in pigs , cattle , sheep and rabbits were respectively 1 . 79 % and 1 . 43 % . HEV RNA was detected by Nested RT - PCR in serum of 15 pigs and 22 different breeds . HEV RNA was not detected in serum of cattle , sheep , chicken and duck . This study indicates that HEV is prevalent in domestic animals and poultry , and the prevalence of HEV infection is the highest . In our country , HEV is the most common type , including 4a , 4b , 4d , 4f , 4g and two new subtypes . It is confirmed that HEV is a new genotype in different regions of our country . HEV antigen is detected in the serum samples of cattle , sheep , chicken and duck . However , it is not possible to detect HEVRNA . 2 . Study on the Interaction of HEV ORF3 Protein with Hepatocytes Three experiments were carried out to select 32 liver cell proteins interacting with ORF3 protein . Four of these proteins ( 4 / 32 , 12.5 % ) had been reported to bind to ORF3 protein . Four of these proteins ( 4 / 32 , 12.5 % ) had been reported to bind to ORF3 protein . Bioinformatic analysis showed that the 32 interacting proteins were distributed in the nucleus , cytoplasm , cell membrane , extracellular matrix , and mitochondria and connotative body membranes . Most of the proteins were located in many parts of the cell . These proteins were involved in many biological processes such as cell metabolism , cellular immunity , cell adhesion and so on . The results showed that the ORF3 protein of HEV , 4 - type HEV , HEV and ORF3 protein could be combined with these proteins . These findings provide clues and directions for our further study of the pathogenic role of ORF3 protein , as well as to investigate the molecular mechanisms of HEV infection , replication and host restriction .

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R373.21;S852.65

【参考文献】