【作者】 田华；

【导师】 孟继鸿； 王立新；

【作者基本信息】 东南大学 ， 免疫学， 2006， 硕士

【摘要】 戊型肝炎(Hepatitis E, HE)是一种经粪口途径传播的急性传染病,在世界各地尤其是发展中国家广泛流行,在发达国家也有散发。HE病死率约为0.4%,远较其他各型肝炎高,患者多为青壮年,孕期妇女病死率高达20%[1-2],给社会带来了很大危害。近年来发现在猪等动物中也有广泛的戊型肝炎病毒(Hepatitis E Virus,HEV)感染,使该病成为一种人畜共患病[18]。目前HE尚无特异的治疗方法,也无主动和被动免疫制剂可供预防。国内外学者均在致力于HEV疫苗的研究。而衡量疫苗是否有预防疾病作用的标准,就是看疫苗能否诱导机体产生中和抗体。中和抗体不同于一般的抗体,它能与病毒结构蛋白上的中和抗原表位结合,阻断病毒与敏感细胞的结合,从而阻止该病毒入侵敏感细胞,使病毒失去感染性,对机体有保护作用,对疾病的诊断、预防和预后起着重要作用。检测中和抗体的常用方法有动物体内试验和细胞培养体外试验两种,但目前对HEV敏感的动物只有非人灵长类动物[6],使用非人灵长类动物建立动物模型,价格昂贵,方法繁琐,试验周期长,动物饲养要求高,耗费大量的人力物力,难以在基层广泛使用。另外,由于HEV缺乏有效的细胞培养体系,传统的体外中和试验鉴定难以进行,1997年Meng [10]等发明了基于PCR和细胞培养的体外中和试验并用于HEV中和抗体的检测,但该体外中和试验需要细胞培养及RT-PCR技术,方法繁琐,也难以普遍推广应用。针对HEV中和抗体检测存在的问题,本研究的目的是建立一种简便易行的检测HEV中和抗体的方法,并进行初步评价。本研究利用我们先前获得的能够稳定分泌HEV中和性单克隆抗体(McAb)的杂交瘤细胞株,大量制备和纯化HEV中和性McAb,并进行酶标记,使待测样本中的HEV中和抗体与酶标记HEV中和性McAb竞争结合包被抗原上的HEV中和抗原表位,最后根据酶作用底物显色反应减弱的程度,实现对生物学样本中的HEV中和抗体的竞争酶联免疫检测。本研究分为三个部分,具体研究结果如下:一.HEV中和性单克隆抗体的大量制备、鉴定和纯化本实验室通过B淋巴细胞杂交瘤技术,以p166Chn和p166Bur免疫小鼠后获得3株杂交瘤细胞株5G5、1G10和3G1。这3株细胞所分泌的McAb均不与单独包被的GST发生结合,是针对HEV的特异性抗体。它们分泌的McAb能和7种不同基因型来源的p166均发生阳性反应,为p166共同型McAb。用这3株杂交瘤细胞制备出大量的McAb,并对McAb进行了HEV中和活性的鉴定,证实它们具有中和HEV的作用,是HEV中和性McAb。分别用正辛酸法,DEAE 52阴离子交换层析法,Protein-G亲和层析法3种不同的方法对McAb进行纯化,并对这3种纯化方法的结果进行比较。我们将纯化得到的产物系列稀释后进行间接ELISA检测抗体滴度,显示出用正辛酸法和Protein-G亲和层析的方法纯化的McAb滴度比DEAE 52阴离子交换层析法高,分别可达1:106(5G5),1:105(1G10)1:105(3G1)。经SDS-PAGE,纯化后的McAb仅在重、轻链位置出现明显条带,表明纯化后的McAb纯度较高。最终选用较经济、更多还原

【Abstract】 Hepatitis E virus (HEV) causes acute viral hepatitis worldwide; the highest incidence occurs in developing countries of Asia and Africa. The HEV is the causative agent of Hepatitis E (HE). HE is transmitted mainly by the fecal-oral route, usually through contaminated water, and occurs both in epidemic and in sporadic forms. The highest mortality occurs in infected pregnant women with case-fatality rates approaching 20% reported in many epidemics. HEV is a single-stranded positive-sense RNA virus overlapping open reading frames (ORFs). At present, HEV can be grouped into at least four major genotypes and several subtypes. This include genotypes I (several countries from Asia and Africa), II (Mexico, Nigeria), III (US, Argentina and Europe) and IV (China, Taiwan). Swine HEV in the US changed the epidemiology of HE. It raises the possibility of zoonotic spread of HEV. The study of distribution and transmission of HEV will be helpful to the control, diagnosis and treatment of HEV infection.Hepatitis E (HE) is endemic in China. However, an adequate level of information on the epidemiology of hepatitis E virus (HEV) has not been available so far because of the diagnostic methods. Now the diagnosis of HE mainly depends on immunodiagnostics. Problems remain both with the specificity of some assays, particularly when applied to seroepidemiological studies, and with the sensitivity of other assays for divergent strains of HEV and for the detection of past infection with HEV. The development of an enzyme immunoassay (EIA or ELISA) is now described using the ORF2 antigen expressed in E. coli, which provides a sensitive and specific assay for the detection and quantization of IgG anti-HEV in acute-and convalescent-phase sera. Fusion proteins of HEV ORF2 452~617aa (p166) of seven HEV strains clustered into different genotypes and subtypes were expressed in E. coli, and purified by the glutathione sepharose-4B affinity column, then identify the proteins by polyacrylamide gel electrophoresis (PAGE). At last we got seven pure recombinant proteins of the different genotypes and subtypes of HEV.Produced and purified monoclonal antibodies of 5G5,1G10,3G1.These three McAbs could react to all of the seven p166 recombinant proteins and they were all neutralizing antibody. Labeled antibody with HRP. Use the seven pure recombinant proteins and HRP 5G5 to establishment a competitive ELISA for detection of neutralizing antibody agaist HEV . Positive judging standard is> 50% of inhibition rate .To determine whether the ELISA could be used for detecting neutralizing antibodies,更多还原