【作者】 伏丽萍；

【导师】 赵宏坤； 相文华；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2004， 硕士

【摘要】 戊型肝炎病毒(Hepatitis E virus ,HEV)过去曾被认为是一种经肠道传播的非甲-非乙型肝炎病毒,在流行普遍的发展中国家在公共卫生方面受到广泛的关注。HEV主要感染青少年,虽然全球成年人HEV的感染率较低,但是对怀孕妇女的病死率却高达20%。戊型肝炎病毒是一种无包膜的正义单链RNA病毒,主要经粪便—口传播,在人群中可以通过饮用HEV污染的水引起暴发流行。HEV基因组全长约为7.5kb,它具有3个互相重叠的开放阅读框架即ORF1、ORF2和ORF3,并且5‘-端有27个核苷酸的非编码区,3’-端为非结构区。ORF1起于核苷酸第28位,延伸5079bp,终止于核苷酸第5107位,长约5kb,它主要编码与HEV复制相关的非结构蛋白,其中存在几个相对保守区域,分别编码甲基转移酶,蛋白酶,RNA解链酶以及RNA聚合酶。ORF2起于核苷酸第5107位,延伸1980bp,终止于Poly(A)上游63个核苷酸处,长约2kb,编码病毒衣壳蛋白,为主要的结构基因编码区,其编码的氨基酸第5-22位信号肽序列。ORF3含有369个核苷酸,与ORF1轻度重叠(1bp),与ORF2广泛重叠(328bp),编码产物为磷蛋白,与细胞的支架及HEV特异性免疫活性有关。然而急性戊型肝炎病例不仅存在于发展中国家,而且在发达国家也有很多受感染的病人。发达国家的病人感染途径至今仍然是一个谜,但是许多人都认为动物很有可能是个重要的传播者。最近在美国猪源HEV的发现证明了猪很有可能传播HEV。为了了解我国国内HEV的流行情况和我国HEV的基因型,我们实验对此进行了详细的调查与研究。本研究采用两步夹心ELISA方法对我国部分省区猪的戊型肝炎病毒(Hepatitis E virus, HEV)感染情况进行了调查,实验共采集全国18个省、市、自治区的48个养猪场不同月龄的1706头猪,其中2月龄以下猪有521只,2-6月龄的猪有611只,6月龄以上的猪有574只,结<WP=9>果显示其中1126份为HEV抗体阳性,平均阳性率为66.00%,在所有检测的48个猪场中,仅有一个猪场没有检测到HEV抗体,有6个猪场抗体阳性率在20%以下;有13个猪场抗体阳性率为20%-60%,猪群抗体阳性率达到60%-90%有16个,阳性率高于90%的猪场有12个,同时HEV的抗体阳性率随着月龄变化而有所不同,其中6月龄以上的猪的抗体阳性率最高为90.94%,其次为2-6月龄的猪的抗体阳性率为70.05%,最低的是2月龄以下的仔猪抗体阳性率为33.78%,这些结果表明猪群的抗体阳性率有可能随着月龄的增长而有所升高,说明我国大部分集约化养殖场均存在HEV感染。同时本实验还在抗体阳性率较高的某个猪场采集了血清和粪便样品,用巢式PCR对其进行克隆和扩增,结果在猪粪中成功扩增并克隆了HEV ORF2 N端的主要抗原决定区的部分基因片断,同时还对这一片断进行了原核表达载体的构建;但是在血液中没有扩增成功。该段目的基因片段与已经发表的HEV基因型I中的印度株,HEV基因型II中的墨西哥株,HEV基因型III中的美国HEV us-1株,中国1株人的HEV,日本1株HEV以及列入基因型IV的台湾株基因序列等10个地方的基因序列进行比较, 序列分析表明核苷酸序列的同源性最高为86.4%,最低的也为73.0%;氨基酸的同源率最高为99.1%,最低也为94%。 同时,把目的基因片断已经成功克隆到PET-30a(+),为下一步原核表达做好准备。更多还原

【Abstract】 Hepatitis E , the principal cause of enterrically transmitted, was once considered non-A non-B hepatitis. It is a very important public health concern in many developing countries where epidemics of hepatitis E are common. The disease generally affects young adults.Although the overall mortality rate associated with HEV infection is low, it is reportedly as high as 20% in infected pregnant women. The causative agent of hepatitis E , hepatitis E virus(HEV), is a single-stranded positive-sense RNA virus without an envelope. HEV is generally transmitted by the fecal-oral route. The genomic RNA of HEV is about 7.5kb and contains three open reading frames(ORFs). ORF1 is predicted to encode viral nonstructural proteins, ORF2encodes the putative capsid protein, and ORF3 encodes a cytoskeleton-associated phosphoprotein.However Sporadic cases of clinical hepatitis E not only occur in these countries but also occur uncommonly in patients with no know epidemiological exposure to HEV in industrialized countries. The source of infection in industrialized countries.is unknow but it has been hypothesized that animals might serve as a reservoir for HEV in both settings. The recent identification of swine HEV in U.S pigs and the demonstration of its ability to infect across species have lent credence to this hypothesis.. In order to learn the situation in Chinese, we start this experiment on HEV.This research adopt ELISA method to invest the situation in Chinese some provinces, of pigs if it is infected hepatitis E. We have gathered 1706 swine serum from the 48 pig farms of 18 nation-wide province city autonomous regions, These swine have different month age which have 521 pigs below 2 mouths age and have 611 pigs between 2-6 months age and <WP=11>have 574 pigs about 6 month age . The result show that in which, 1126 copies of serum is HEV antibody positive. Average positive rate is as high as 66% . all 48 pig farms of was detected, and the result show that only one pig farm have no HEV antibody, there is 6 pig farms which is antibody positive rates below 20% there is 13 pig farms which is antibody positive rates between 20%-60%; there is 16 pig farms which is antibody positive rates between 60%-90%; there is 12 pig farms which is antibody positive rates above 90%. At the same time the antibody positive rate of HEV make change along with different month . The swine of 6 mouth age of antibody positive rate is the highest and it is 90.47%, secondly the swine of 2-6 mouth age of antibody positive rate is high and it is 70.05% . The swine of under 2 mouth age of antibody positive rate is low and it is only 33.78%. These results show the antibody positive rate of pig crowd go up possibility along with month make increase. At the same time, this experiment have gathered serum and fecal from the pig farm which antibody positive rate is higher certain. We handle after them and, draw RNA, from them.The result of transcribe used in nested PCR to increase and clone. As a result we have cloned HEV ORF2 N ends fight decision mainly the partial gene segments of district which is 348 bp. This section gene compare with have published Indian in HEV gene type , I Mexican in HEV gene type II and HEV gene type III in American HEV us-1 , one Chinese persons HEV and one Japanese HEV as well as place the Taiwan of gene type IV gene sequence et al . The result of sequence analysis show this section nucleotide acid sequenc is smililarity with the Taiwan of gene type IV which is 86.4% and is divergence from Mexican in gene type II which is 73.0%; this section amino sequence is <WP=12>Smililarity with one Chinese person highest, which is 99.1%; and Smililarity with with Indian of gene typeIminimum which is also 94%. We can know from the result that the change level of nucleotide acid is great , but amino acid is less. This can explain this section gene change greater, however amino acid relatively conservative. At the same time, this section gene have cloned PET-30 a(+) successfully for 348 bp purpose gene segments, and i更多还原