戊型肝炎病毒中和抗体竞争酶联免疫检测方法的建立及评价

发布时间：2018-12-12 21:16

【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、
[Abstract]:Hepatitis E (HE) is an acute infectious disease that is spread by the way of manure, and is widely popular in all parts of the world, especially in developing countries, and has also been distributed in developed countries. The mortality of HE is about 0.4%, which is much higher than that of other types of hepatitis. The mortality of pregnant women is 20%[1-2], which brings great harm to society. In recent years, it has been found that a wide range of hepatitis E virus (HEV) infection in pigs and other animals has caused the disease to become a human-animal disease[18]. At present, HE has no specific treatment method, nor is it active and is The dynamic immune preparation can be used for prevention, and both domestic and foreign scholars are committed to The study of the HEV vaccine, which measures whether the vaccine has a standard to prevent disease, is to see if the vaccine can be induced The neutralizing antibody is different from the general antibody, and can be combined with the neutralizing antigen epitope in the viral structural protein to block the binding of the virus and the sensitive cell, thereby preventing the virus from invading the sensitive cells, and protecting the body the effect, the diagnosis and prevention of the disease, and an animal model is established by using the non-human primate animal, the price is high, the cost is high, the method is complicated, the test period is long, the animal feeding requirement is high, a large amount of manpower and material resources are consumed, It is difficult to use extensively at the grass-roots level. In addition, due to the lack of an effective cell culture system in the HEV, the traditional in vitro neutralization test identification is difficult to carry out, and in 1997, Meng[10] et al. developed in vitro neutralization test based on the PCR and cell culture and used in the detection of the neutralizing antibody in the HEV, but the in vitro neutralization test requires cell culture and the method is complex, It is also difficult to popularize the application in general. The purpose of this study is to establish a simple and convenient method for detecting the neutralizing antibody in the HEV, aiming at the problems existing in the detection of the antibody and the antibody in the HEV. The present study makes use of the previously obtained hybridoma cell lines capable of stably secreting the neutralizing monoclonal antibody (McAb) of the HEV, a large number of preparation and purification of the HEV, and the sexual Mc and carrying out enzyme labeling so that the HEV in the sample to be tested and the antibody in the sample to be tested compete with the antibody and the sex McAb in the HEV to bind to the HEV in the antigen and the epitope of the antigen, and finally, the antibody and the antibody in the biological sample are realized according to the degree of the color reaction of the enzyme action substrate to decrease, competitive enzyme-linked immunosorbent assay. the present invention The study was divided into three parts, and the specific results were as follows: A. A large number of preparation, identification and purification of the neutralizing monoclonal antibody in the HEV were carried out by B-lymphocyte hybridoma technique, p166Chn and p166B. Three hybridoma cell lines 5G5, 1G10, and 3G1 were obtained after the mice were immunized. b is not bound to a separately coated GST, is a specific antibody against an HEV. The McAb secreted by them can be combined with 7 different groups A lot of McAb was prepared by using the three hybridoma cells, and the activity of the McAbs in the HEV was investigated. They were identified by positive caprylic acid method, DEAE 52 anion exchange chromatography and Protein-G affinity chromatography. The results of the three purification methods were compared, and the results of the three purification methods were compared. After the product family was diluted, the titer of the antibody was detected by indirect ELISA, and the concentration of the McAb of the purified McAb was higher than that of the DEAE 52 anion exchange chromatography with the method of n-octanoic acid and Protein-G affinity chromatography, respectively. 06 (5G5), 1: 105 (1G10) 1: 105 (3G1). in heavy and light chain position only
【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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