双抗体夹心ELISA检测猪戊型肝炎病毒抗原方法的建立及流行病学调查

发布时间：2018-06-02 14:33

本文选题：猪戊型肝炎病毒 + P1蛋白　；参考：《吉林大学》2017年硕士论文

【摘要】：戊型肝炎病毒(Hepatitis E,HEV)属于戊型肝炎病毒科戊型肝炎病毒属中的成员,是引起人类和动物戊型肝炎病毒感染的病原体。近年研究发现,戊型肝炎作为一种人畜共患病,在人-猪的流行与传播中扮演重要角色,严重威胁着人类的健康,目前认为人HE的流行与传播主要是由猪戊型肝炎病毒引起的。有研究发现从猪体内分离的HEV与当地人分离出的HEV核苷酸序列同源性较高,与猪接触的职业人群血清抗HEV抗体均高于非职业人群,表明HEV在人和猪种间传播可能进行跨种传播。目前,HEV分为四种基因型,其中基因1型和2型主要感染人;基因3型和4型主要感染猪等家畜,同时也可以感染人和非人灵长类,猪感染HEV以基因3型为主。人HEV主要通过消化道感染,以青壮年感染率最高,孕妇感染病死率高达20%。猪感染HEV一般不出现临床症状,常呈现隐性感染,但作为HEV的自然宿主,可能参与引起人类的戊型肝炎。近年来食源性戊型肝炎流行情况严重,已成为全球关注重要的公共卫生问题,因此,掌握猪群HEV的感染情况,已成为保障人身安全的重要措施,对防控人戊型肝炎具有重要的公共卫生学意义。目前有关本病的实验室诊断方法有很多,如RT-PCR、ELISA、免疫印记、血清中和实验、免疫荧光显微实验、免疫电镜实验和免疫胶体金等技术。最常用的诊断方法是RT-PCR和ELISA方法。RT-PCR方法具有操作简单、敏感性高、特异性强等优点,但该方法易出现假阳性、所需检测设备昂贵、仅限于实验室诊断、难以检测大量临床样品。ELISA方法多用于检测病毒感染产生的抗体,难以用于本病的早期、快速诊断。因此,迫切需要建立一种特异、敏感、快速与简便用于HEV临床诊断的实验室检测方法。本研究应用RT-PCR扩增出编码HEV病毒核衣壳P1蛋白的核苷酸序列,并将其克隆到原核表达载体p GEX-4T-1中,构建出p GEX-4T-1-P1重组表达质粒。应用表达、纯化的HEV P1蛋白,制备抗P1蛋白的单克隆抗体和多克隆抗体,并以此建立了检测HEV病毒抗原的双抗体夹心ELISA方法。试验结果显示,建立的检测HEV病毒抗原的双抗体夹心ELISA方法特异、敏感、快速与简便,可用于大规模HEV感染的流行病学调查。以建立的夹心ELISA方法对吉林与辽宁部分地区猪群感染HEV进行了调查,发现所调查地区均存在着不同程度的HEV感染。该结果将为快速检测HEV抗原提供有效方法,同时为揭示人类戊型肝炎发生与流行提供一定的理论依据。
[Abstract]:Hepatitis E virus (HEV) is a member of hepatitis E virus genus of hepatitis E virus family, and is the pathogen causing hepatitis E virus infection in humans and animals. In recent years, it has been found that hepatitis E, as a zoonotic disease, plays an important role in the epidemic and transmission of human and pig, and it is a serious threat to human health. At present, it is believed that the prevalence and transmission of human HE is mainly caused by porcine hepatitis E virus. It was found that the nucleotide sequence of HEV isolated from pigs was highly homologous to that of HEV isolated from local people, and the serum anti HEV antibodies of the occupational population in contact with pigs were higher than those of non-occupational population, indicating that the transmission of HEV between human and pig species might be interspecies transmission. At present, there are four genotypes of HEV, among which gene type 1 and type 2 mainly infect human, gene 3 and type 4 mainly infect domestic animals such as pigs, but also can infect human and non-human primates. The main type of HEV infection in pigs is gene 3. The infection rate of human HEV was the highest in young adults, and the mortality rate of pregnant women was as high as 20%. Porcine infection with HEV usually shows no clinical symptoms and often shows recessive infection, but as a natural host of HEV, it may be involved in the human hepatitis E. In recent years, the epidemic situation of foodborne hepatitis E has become an important public health issue of global concern. Therefore, mastering the situation of HEV infection in pigs has become an important measure to ensure personal safety. It has important public health significance for prevention and control of hepatitis E in human. At present, there are a lot of laboratory diagnostic methods for this disease, such as RT-PCRV ELISA, immune imprinting, serum neutralization, immunofluorescence microscopy, immunoelectron microscopy and immuno-colloidal gold. The most commonly used diagnostic methods are RT-PCR and ELISA methods. RT-PCR has the advantages of simple operation, high sensitivity and strong specificity. However, this method is prone to false positives, and the detection equipment required is expensive, which is limited to laboratory diagnosis. It is difficult to detect a large number of clinical samples. Elisa method is often used to detect antibodies produced by virus infection, and is difficult to be used in the early and rapid diagnosis of this disease. Therefore, there is an urgent need to establish a specific, sensitive, rapid and simple laboratory detection method for clinical diagnosis of HEV. In this study, the nucleotide sequence encoding the nucleocapsid P1 protein of HEV virus was amplified by RT-PCR and cloned into prokaryotic expression vector p GEX-4T-1 to construct the recombinant expression plasmid of p GEX-4T-1-P1. The monoclonal antibody and polyclonal antibody against P1 protein were prepared by using the expressed and purified HEV P1 protein, and a double antibody sandwich ELISA method for the detection of HEV virus antigen was established. The results showed that the double antibody sandwich ELISA method for detecting HEV virus antigen was specific, sensitive, rapid and simple, and could be used in the epidemiological investigation of large-scale HEV infection. A sandwich ELISA method was used to investigate the infection of HEV in swine populations in Jilin and Liaoning provinces. It was found that there were different degrees of HEV infection in all the areas investigated. The results will provide an effective method for rapid detection of HEV antigens and provide a theoretical basis for revealing the occurrence and prevalence of human hepatitis E.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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