本文選題：鹿布氏桿菌病 + 原核表達(dá)��； 參考：《吉林農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：近年來(lái)我國(guó)鹿養(yǎng)殖業(yè)得到了迅速的發(fā)展,給我國(guó)來(lái)了巨大的經(jīng)濟(jì)收入。但目前我國(guó)梅花鹿嚴(yán)重的疫病流行影響著鹿養(yǎng)殖業(yè)的發(fā)展。布氏桿菌病(Brucellosis)就是其中重要的傳染疫病之一,作為常見(jiàn)的人畜共患病,該病廣泛流行于我國(guó)畜間,不僅帶來(lái)了極大的經(jīng)濟(jì)損失,還不斷威脅著人類和社會(huì)的安全。本研究旨在建立一種檢測(cè)布氏桿菌病的間接酶聯(lián)免疫吸附試驗(yàn)(iELISA)。并用建立的ELISA方法對(duì)吉林省梅花鹿主要養(yǎng)殖區(qū)鹿群進(jìn)行檢測(cè),為鹿布氏桿菌病防治工作提出有效建議。主要包括以下三個(gè)內(nèi)容:(1)布氏桿菌Bp26、Omp19、L7/L12基因的克隆與序列分析以實(shí)驗(yàn)室保存的布氏桿菌S2株為模板,根據(jù)Bp26、Omp19、L7/L12核苷酸序列設(shè)計(jì)三對(duì)特異性引物,經(jīng)PCR擴(kuò)增得到的大小分別為734bp、543bp、375bp目的片段,分別與pMD18-T Simple Vector連接,經(jīng)PCR、酶切鑒定,并測(cè)序。結(jié)果顯示,Bp26、Omp19、L7/L12基因均成功克隆到pMD18-T載體中。(2)布氏桿菌Bp26、Omp19、L7/L12基因的原核表達(dá)及反應(yīng)原性分析將三個(gè)陽(yáng)性重組克隆質(zhì)粒酶切后與原核表達(dá)載體pGEX-4T-1連接,構(gòu)建三段基因的重組表達(dá)質(zhì)粒將其轉(zhuǎn)化入到大腸桿菌感受態(tài)BL21(DE3)中,經(jīng)IPTG誘導(dǎo),摸索出目的基因最適表達(dá)條件,運(yùn)用透析法對(duì)目的蛋白進(jìn)行純化,對(duì)表達(dá)產(chǎn)物及純化產(chǎn)物進(jìn)行SDS-PAGE分析,得到分子量為54 ku、47 ku、41 ku的蛋白條帶。經(jīng)Western blot分析,結(jié)果表明目的蛋白均能與鹿布氏桿菌病的陽(yáng)性血清發(fā)生特異性結(jié)合,說(shuō)明Bp26、Omp19、L7/L12蛋白均具有良好的反應(yīng)原性。(3)間接ELISA方法的建立和吉林省主要養(yǎng)殖地區(qū)鹿布氏桿菌病血清學(xué)調(diào)查分別以純化的三段目的蛋白為包被抗原,通過(guò)摸索各項(xiàng)參數(shù),優(yōu)化反應(yīng)條件,初步建立三種抗原的間接ELISA檢測(cè)方法。經(jīng)綜合比較最終選定特異性強(qiáng),敏感性高,穩(wěn)定性較強(qiáng)的Bp26-ELISA方法對(duì)吉林省部分地區(qū)采集的254份樣品進(jìn)行檢測(cè),RBT陽(yáng)性檢出率為8.6%,ELISA陽(yáng)性檢率為10.2%。并為該地區(qū)布病的防治提出有效意見(jiàn)。
[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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.25

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 陳瑤;李明;;布魯氏菌bp26和OMP10基因的原核表達(dá)和鑒定[J];中國(guó)人獸共患病學(xué)報(bào);2006年04期

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本文編號(hào)：1860857