布氏杆菌病间接ELISA方法的建立及吉林省梅花鹿主要养殖地区鹿布病血清学调查

发布时间：2018-05-08 09:27

本文选题：鹿布氏杆菌病 + 原核表达　；参考：《吉林农业大学》2015年硕士论文

【摘要】：近年来我国鹿养殖业得到了迅速的发展,给我国来了巨大的经济收入。但目前我国梅花鹿严重的疫病流行影响着鹿养殖业的发展。布氏杆菌病(Brucellosis)就是其中重要的传染疫病之一,作为常见的人畜共患病,该病广泛流行于我国畜间,不仅带来了极大的经济损失,还不断威胁着人类和社会的安全。本研究旨在建立一种检测布氏杆菌病的间接酶联免疫吸附试验(iELISA)。并用建立的ELISA方法对吉林省梅花鹿主要养殖区鹿群进行检测,为鹿布氏杆菌病防治工作提出有效建议。主要包括以下三个内容:(1)布氏杆菌Bp26、Omp19、L7/L12基因的克隆与序列分析以实验室保存的布氏杆菌S2株为模板,根据Bp26、Omp19、L7/L12核苷酸序列设计三对特异性引物,经PCR扩增得到的大小分别为734bp、543bp、375bp目的片段,分别与pMD18-T Simple Vector连接,经PCR、酶切鉴定,并测序。结果显示,Bp26、Omp19、L7/L12基因均成功克隆到pMD18-T载体中。(2)布氏杆菌Bp26、Omp19、L7/L12基因的原核表达及反应原性分析将三个阳性重组克隆质粒酶切后与原核表达载体pGEX-4T-1连接,构建三段基因的重组表达质粒将其转化入到大肠杆菌感受态BL21(DE3)中,经IPTG诱导,摸索出目的基因最适表达条件,运用透析法对目的蛋白进行纯化,对表达产物及纯化产物进行SDS-PAGE分析,得到分子量为54 ku、47 ku、41 ku的蛋白条带。经Western blot分析,结果表明目的蛋白均能与鹿布氏杆菌病的阳性血清发生特异性结合,说明Bp26、Omp19、L7/L12蛋白均具有良好的反应原性。(3)间接ELISA方法的建立和吉林省主要养殖地区鹿布氏杆菌病血清学调查分别以纯化的三段目的蛋白为包被抗原,通过摸索各项参数,优化反应条件,初步建立三种抗原的间接ELISA检测方法。经综合比较最终选定特异性强,敏感性高,稳定性较强的Bp26-ELISA方法对吉林省部分地区采集的254份样品进行检测,RBT阳性检出率为8.6%,ELISA阳性检率为10.2%。并为该地区布病的防治提出有效意见。
[Abstract]:In recent years, deer farming in China has been developing rapidly, which has brought huge economic income to our country. But the serious epidemic of sika deer affects the development of deer breeding. Brucellosis is one of the most important infectious diseases. As a common zoonosis, Brucellosis is widely spread among livestock in China, which not only brings great economic losses, but also threatens the safety of human beings and society. The aim of this study was to establish an indirect enzyme-linked immunosorbent assay (Elisa) for detection of brucellosis. The established ELISA method was used to detect deer herd in main breeding areas of sika deer in Jilin Province, and effective suggestions were put forward for the prevention and control of brucellosis in deer. The cloning and sequence analysis of Bp26Omp19L7 / L12 gene of Brucella spp. Using S 2 strain of Brucella spp preserved in laboratory as template, three pairs of specific primers were designed according to the nucleotide sequence of Bp26Omp19L7 / L12. The size of the target fragment was 734 BP, 543 BP and 375 BP, respectively, which were ligated with pMD18-T Simple Vector, and identified by PCR, restriction endonuclease digestion, and sequenced. The results showed that Bp26Omp19L7 / L12 gene was successfully cloned into pMD18-T vector.) the prokaryotic expression of Bp26Omp19L7 / L12 gene and its reactivity were analyzed. The three positive recombinant plasmids were digested and ligated with prokaryotic expression vector pGEX-4T-1. The recombinant expression plasmid of the three-segment gene was constructed and transformed into the Escherichia coli (BL21DE3). After induction by IPTG, the optimal expression conditions of the target gene were found, and the target protein was purified by dialysis. The expressed product and purified product were analyzed by SDS-PAGE, and the protein band with a molecular weight of 54 Ku-47 Ku-41 ku was obtained. The results of Western blot analysis showed that the target proteins could bind specifically to the positive serum of brucellosis deer. The results showed that Bp26Omp19L7 / L12 protein had good reactivity. The establishment of indirect ELISA method and serological investigation of brucellosis in Jilin Province took the purified three-segment target protein as the coating antigen, and explored the parameters of Bp26Omp19L7 / L12 protein by exploring the parameters of Bp26Omp19L7 / L12 protein, and the serological investigation of brucellosis in Jilin Province. The indirect ELISA detection method for three antigens was established by optimizing the reaction conditions. After comprehensive comparison, the Bp26-ELISA method with strong specificity, high sensitivity and strong stability was selected to detect the positive rate of Bp26-ELISA in 254 samples collected from parts of Jilin Province. The positive rate of Elisa was 8. 6% and the positive rate of Elisa was 10. 2%. And put forward effective advice for the prevention and cure of brucellosis in this area.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.25

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