【作者】 王宇；

【导师】 粟硕； 邢刚；

【作者基本信息】 南京农业大学 ， 兽医硕士（专业学位）， 2023， 硕士

【摘要】 盖他病毒（Getah Virus,GETV）是一种单股正链的RNA病毒,属于披膜病毒科甲病毒属成员。可通过蚊虫媒介传播感染多种哺乳动物,在包括中国在内的许多东亚国家的猪、马、牛等动物中暴发,影响了养殖行业的健康发展。母猪感染可导致生殖系统障碍,马匹感染可导致淋巴结肿大、发烧和后躯肿大等症状。GETV基因组有两个开放阅读框（ORF1和ORF2）,分别编码非结构蛋白（NSP1、NSP2、NSP3和NSP4）和结构蛋白（Capsid,E3、E2、6K和E1）。目前,RT-PCR和血清学检测方法是检测GETV最常用的手段,但前者存在操作复杂、容易交叉污染的情况,后者存在感染早期检测抗体空白导致结果不准确的情况,因此有必要建立一个快速、敏感、可靠且适用于感染动物早期诊断的检测方法。本试验首先制备出可直接靶向GETV病原的抗体,在此基础上建立了针对GETV双抗体夹心ELISA方法,具体研究内容如下:1.GETV的单、多克隆抗体的制备本试验以纯化后的GETV全病毒作为免疫原,每次以200μg剂量免疫三只BALB/c小鼠,三次免疫后,经间接ELISA法测定选择血清效价较高且达到10^-5的小鼠进行细胞融合,最终获得四株单克隆抗体并分别命名为:1E1、1E12、2B5和1A9。经Western Blot鉴定四株单克隆抗体均能够特异性与GETV发生反应;间接免疫荧光鉴定结果显示四株单克隆抗体均可特异性与GETV发生反应;在BHK-21细胞上验证中和试验结果发现这四株单克隆抗体不具有中和活性。本研究制备出的抗GETV单克隆抗体能够特异性识别单个表位,可以作为双抗体夹心ELISA检测的检测抗体,从而减少非特异性结合以避免假阳性结果的出现。将GETV上清进行纯化,以GETV全病毒作为免疫原,以600μg的剂量的全病毒免疫新西兰大白兔,三次免疫后从达到效价的新西兰大白兔心脏收集血液,进行蛋白A亲和层析的方法纯化血清,获得GETV兔源多克隆抗体。由于多克隆抗体与靶蛋白有多个表位结合位点,亲和性更高,拟将其作为双抗体夹心ELISA检测的捕获抗体,为后续GETV的检测提供试验材料。2.双抗体夹心ELISA检测方法的建立在本试验中,利用GETV兔源多克隆抗体作为捕获抗体,酶标单克隆抗体HRP-2B5作为检测抗体,通过对ELISA检测方法各条件的优化,摸索最优的双抗体夹心体系,从而建立了一种用于检测GETV全病毒的双抗体夹心ELISA方法。当P/N>2.0且OD_450nm≥0.2时,即为阳性。该方法最低检测量为10³TCID₅₀/m L。在检测GETV、PEDV、PDCo V和TGEV时,仅GETV为阳性,其余四种病毒检测为阴性,表明该方法有较好的特异性;该方法的变异系数小于10%;用该方法检测临床的20份阳性组织样品和10份阴性组织样品,发现阳性符合率为90%（18/20）,阴性符合率为90%（9/10）。以上结果表明,该方法的建立为GETV的鉴别诊断及流行病学调查提供了更简便、准确且直观的手段,具有潜在的经济效益。更多还原

【Abstract】 Getah Virus（GETV）is a single stranded positive stranded RNA virus that belongs to the genus A of the family PHimoviridae.Various mammals can be transmitted and infected through mosquito vectors,causing outbreaks in pigs,horses,cows and other animals in many East Asian countries,including China,posing a serious threat to the aquaculture industry.Infection in sows can lead to reproductive system disorders,while infection in horses can cause symptoms such as enlarged lymph nodes,fever,and enlarged hindquarters.The GETV genome has two open reading frames（ORF1 and ORF2）,encoding non-structural proteins（NSP1,NSP2,NSP3,and NSP4）and structural proteins（Capsid,E3,E2,6K,and E1）.At present,RT-PCR and serological detection methods are the most commonly used methods for detecting GETV.However,the former has complex operations and is prone to cross contamination,while the latter has inaccurate results due to blank antibodies in early infection detection.Therefore,it is necessary to establish a fast,sensitive,reliable and suitable detection method for early diagnosis of infected animals.This experiment first prepared antibodies that can directly target the pathogen of GETV,and based on this,established a sandwich ELISA method for double antibodies against GETV.The specific research content is as follows:1.Preparation of Monoclonal and Polyclonal Antibodies for GETVThis experiment uses purified GETV whole virus as the immunogen,three BALB/c mice were immunized with a dose of 200μg,and after three immunizations,mice with high serum titers and reaching 10^-5 were selected for cell fusion using indirect ELISA.Finally,four monoclonal antibodies were obtained and named 1E1,1E12,2B5,and 1A9,respectively.According to Western blot analysis,all four monoclonal antibodies were able to react specifically with GETV;The indirect immunofluorescence identification results showed that all four monoclonal antibodies could react specifically with GETV;The results of the neutralization test on BHK-21 cells showed that these four monoclonal antibodies did not exhibit neutralizing activity.The anti GETV monoclonal antibody prepared in this study can specifically recognize a single epitope and serve as a detection antibody for double antibody sandwich ELISA detection,thereby reducing non-specific binding and avoiding false positive results.Purify the supernatant of GETV and use the entire GETV virus as the immunogen,immunize New Zealand white rabbits with a dose of 600μg,collect blood from the heart of the rabbits that have reached the titer,and purify the serum using protein A affinity chromatography to obtain rabbit derived polyclonal antibodies against GETV.Due to the multiple epitope binding sites and higher affinity between polyclonal antibodies and target proteins,it is proposed to use them as capture antibodies for double antibody sandwich ELISA detection,providing experimental materials for subsequent detection of GETV.2.Establishment of a dual antibody sandwich ELISA detection methodIn this experiment,using rabbit derived polyclonal antibodies from GETV as capture antibodies and enzyme-linked monoclonal antibody HRP-2B5 as detection antibodies,the optimal double antibody sandwich system was explored by optimizing the conditions of the ELISA detection method,and a double antibody sandwich ELISA method for detecting the entire virus of GETV was established.When P/N>2.0 and OD450nm≥0.2,it is considered positive.The minimum detection amount of this method is 10³TCID₅₀/m L.When detecting GETV,PEDV,PDCo V,and TGEV,only GETV was positive,while the other four viruses were negative,indicating that this method has good specificity;The coefficient of variation of this method is less than10%;Using this method to detect 20 clinical positive tissue samples and 10 negative tissue samples,it was found that the positive coincidence rate was 90%（18/20）and the negative coincidence rate was 90%（9/10）.The above results indicate that the establishment of this method provides a more convenient,accurate,and intuitive means for the differential diagnosis and epidemiological investigation of GETV,and has potential economic benefits.更多还原