坦布苏病毒NS5蛋白单克隆抗体的制备与鉴定及快速检测胶体金试纸条方法的建立
发布时间：2018-11-25 11:12
【摘要】：2010年春夏之交,我国江浙和山东等地区均报道了以一种引起鸭产蛋率大幅下降、生长发育迟缓、卵巢出血为主要症状的急性传染病,之后广泛传播到我国的大部分地区,并给蛋鸭的养殖业造成了很大的经济损失。感染鸭群主要表现为采食量和产蛋量的急速下降,依照发病的日龄不同,死亡率为5%~10%之间。经过国内多个试验室的诊断和病毒的分离,可以确定该病是由黄病毒引起的,隶属黄病毒属(Flavivirus)、恩塔亚病毒群(Ntaya virus group)的鸭坦布苏病毒(duck Tembusu virus,DTMUV)。研究坦布苏病毒蛋白的结构功能及建立快速检测方法,是控制该病的有效措施。本研究建立了快速检测坦布苏病毒的胶体金检测方法,成功地制备了抗坦布苏病毒NS5蛋白的单克隆抗体,为防治该病提供了技术支持。本研究内容分为以下两个部分:1.坦布苏病毒NS5蛋白单克隆抗体的制备本研究利用本试验室构建的PET-28a+NS5原核表达载体,成功转化入大肠杆菌BL21细胞,并纯化产生的坦布苏病毒NS5蛋白。利用纯化的NS5蛋白免疫6-8周龄的BALB/c小鼠,经过细胞融合、间接ELISA筛选和有限稀释亚克隆法获得了3株稳定分泌针对坦布苏病毒NS5蛋白的单克隆抗体A4D1、B4B8和C12D2,并对A4D1和B4B8进行相关特性鉴定。经ELISA效价测定,两株单抗的细胞上清效价均为1:100,诱导产生腹水的效价分别为1:128000和1:64000。间接ELISA结果表明,两株单抗只与DTMUV反应,无交叉反应性。Western blot和IFA结果表明,两株单抗均可与体外纯化的NS5蛋白以及坦布苏病毒发生特异性反应。抗坦布苏病毒NS5蛋白单克隆抗体的成功研制,为坦布苏病毒的免疫学诊断及致病机理的研究奠定基础。2.坦布苏病毒胶体金检测试纸条方法的建立利用试验室保存的抗坦布苏病毒E蛋白单克隆抗体A12D3为金标抗体,制备的鼠抗E蛋白多克隆抗体为包被抗体,建立了双抗体夹心胶体金试纸条检测坦布苏病毒的方法。用该试纸条可特异性检测坦布苏病毒,在10~15 min即可观察检测结果。利用建立的胶体金试纸条的方法和RT-PCR对50份临床样品进行检测比较,结果显示两者符合率为93.8%。该试纸条重复性好,特异性高,可用于坦布苏病毒的临床快速诊断。
[Abstract]:At the turn of spring and summer in 2010, Jiangsu, Zhejiang, Shandong and other regions in China reported an acute infectious disease characterized by a sharp drop in duck laying rate, stunted growth and ovarian hemorrhage, and then spread widely to most areas of China. And to the egg duck breeding industry caused great economic losses. The main characteristics of infected ducks were the rapid decrease of feed intake and egg production. According to the age of the infected ducks, the mortality rate was between 510% and 10%. It was confirmed that the disease was caused by yellow virus, which belonged to (Ntaya virus group) of (Flavivirus), Entavirus group (duck Tembusu virus,DTMUV). To study the structure and function of Tambusuvirus protein and to establish a rapid detection method are effective measures to control the disease. In this study, a colloidal gold assay for rapid detection of TBV was established, and monoclonal antibodies against TBV NS5 protein were successfully prepared, which provided technical support for the prevention and treatment of TBV. The content of this study is divided into the following two parts: 1. Preparation of Monoclonal Antibody against Tambusuvirus NS5 protein in this study the PET-28a NS5 prokaryotic expression vector constructed in our laboratory was successfully transformed into Escherichia coli BL21 cells and the resulting NS5 protein was purified. The purified NS5 protein was used to immunize 6-8 week-old BALB/c mice. Through cell fusion, indirect ELISA screening and limited dilution subcloning, three strains of monoclonal antibodies A4D1B4B8 and C12D2 secreted against NS5 protein of TBV were obtained. The related characteristics of A4D1 and B4B8 were identified. The titer of supernatant of the two McAbs was 1: 100, and the titer of induced ascites was 1: 128000 and 1: 64000, respectively. Indirect ELISA results showed that the two McAbs reacted only with DTMUV, while the non-cross-reactive. Western blot and IFA showed that the two McAbs could react specifically with the purified NS5 protein and Tambusuvirus in vitro. The successful development of monoclonal antibody against NS5 protein of TBV lays a foundation for the immunological diagnosis and pathogenesis of TBV. 2. The Establishment of TBV Colloidal Gold Test Strip the monoclonal antibody (A12D3) against tambusuvirus E protein was used as gold labeled antibody and the polyclonal antibody of mouse anti-E protein was coated antibody. A double antibody sandwich colloidal gold test strip for detection of tambusuvirus was established. The test strip can be used to detect tambusuvirus specifically, and the results can be observed at 10 ~ 15 min. 50 clinical samples were detected and compared by using the method of colloidal gold test strip and RT-PCR. The results showed that the coincidence rate of the two methods was 93.884%. The test strip has good reproducibility and specificity and can be used for rapid clinical diagnosis of TBV.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3
本文编号：2355898
[Abstract]:At the turn of spring and summer in 2010, Jiangsu, Zhejiang, Shandong and other regions in China reported an acute infectious disease characterized by a sharp drop in duck laying rate, stunted growth and ovarian hemorrhage, and then spread widely to most areas of China. And to the egg duck breeding industry caused great economic losses. The main characteristics of infected ducks were the rapid decrease of feed intake and egg production. According to the age of the infected ducks, the mortality rate was between 510% and 10%. It was confirmed that the disease was caused by yellow virus, which belonged to (Ntaya virus group) of (Flavivirus), Entavirus group (duck Tembusu virus,DTMUV). To study the structure and function of Tambusuvirus protein and to establish a rapid detection method are effective measures to control the disease. In this study, a colloidal gold assay for rapid detection of TBV was established, and monoclonal antibodies against TBV NS5 protein were successfully prepared, which provided technical support for the prevention and treatment of TBV. The content of this study is divided into the following two parts: 1. Preparation of Monoclonal Antibody against Tambusuvirus NS5 protein in this study the PET-28a NS5 prokaryotic expression vector constructed in our laboratory was successfully transformed into Escherichia coli BL21 cells and the resulting NS5 protein was purified. The purified NS5 protein was used to immunize 6-8 week-old BALB/c mice. Through cell fusion, indirect ELISA screening and limited dilution subcloning, three strains of monoclonal antibodies A4D1B4B8 and C12D2 secreted against NS5 protein of TBV were obtained. The related characteristics of A4D1 and B4B8 were identified. The titer of supernatant of the two McAbs was 1: 100, and the titer of induced ascites was 1: 128000 and 1: 64000, respectively. Indirect ELISA results showed that the two McAbs reacted only with DTMUV, while the non-cross-reactive. Western blot and IFA showed that the two McAbs could react specifically with the purified NS5 protein and Tambusuvirus in vitro. The successful development of monoclonal antibody against NS5 protein of TBV lays a foundation for the immunological diagnosis and pathogenesis of TBV. 2. The Establishment of TBV Colloidal Gold Test Strip the monoclonal antibody (A12D3) against tambusuvirus E protein was used as gold labeled antibody and the polyclonal antibody of mouse anti-E protein was coated antibody. A double antibody sandwich colloidal gold test strip for detection of tambusuvirus was established. The test strip can be used to detect tambusuvirus specifically, and the results can be observed at 10 ~ 15 min. 50 clinical samples were detected and compared by using the method of colloidal gold test strip and RT-PCR. The results showed that the coincidence rate of the two methods was 93.884%. The test strip has good reproducibility and specificity and can be used for rapid clinical diagnosis of TBV.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3
【引证文献】
相关会议论文 前1条
1 张琳;胡北侠;张秀美;;4株鸭坦布苏病毒包膜蛋白基因的分子进化分析及其表达[A];中国畜牧兽医学会兽医公共卫生学分会第三次学术研讨会论文集[C];2012年
，本文编号：2355898
本文链接：https://www.wllwen.com/yixuelunwen/dongwuyixue/2355898.html
