發(fā)布時(shí)間：2018-11-01 14:40

【摘要】：【目的】 布魯氏菌�。˙rucellosis，簡(jiǎn)稱布�。┦且环N全世界廣泛分布的人獸共患慢性細(xì)菌傳染病，由布魯氏菌（Brucella）感染機(jī)體引起，人間布魯氏菌病在我國(guó)被列為法定乙類傳染病。傳統(tǒng)的布魯氏菌病實(shí)驗(yàn)室檢測(cè)主要依賴于病原體的分離，該方法耗時(shí)耗力，且需專業(yè)的實(shí)驗(yàn)室，并不實(shí)用；隨著技術(shù)的發(fā)展，發(fā)現(xiàn)酶聯(lián)免疫吸附實(shí)驗(yàn)（ELISA）方法與補(bǔ)體結(jié)合實(shí)驗(yàn)（CFT）、傳統(tǒng)的試管凝集實(shí)驗(yàn)（SAT）相比，有更高的敏感度和特異度。本研究試圖選用布魯氏菌中具有代表性的重組蛋白作為抗原進(jìn)行酶聯(lián)免疫吸附實(shí)驗(yàn)（ELISA），從而建立以重組蛋白做為抗原的ELISA方法，為研制快速診斷布魯氏菌病的ELISA試劑盒奠定基礎(chǔ)。 【方法】 通過參考大量關(guān)于布魯氏菌相關(guān)的國(guó)內(nèi)外文獻(xiàn)，綜合云南省地方病防治所中心實(shí)驗(yàn)室對(duì)布魯氏菌診斷靶點(diǎn)的研究情況，選取外膜蛋白o(hù)mp10，核蛋白L7/12為診斷靶點(diǎn)進(jìn)行初步研究。 1.布魯氏菌omp10、L7/L12的克隆表達(dá)、純化 根據(jù)已發(fā)表的布魯氏菌外膜蛋白o(hù)mp10、核蛋白L7/L12基因設(shè)計(jì)兩對(duì)引物進(jìn)行擴(kuò)增，用BamHⅠ和NotⅠ或EcoRⅠ和NotⅠ將目的基因片段雙酶切純化與同樣雙酶切純化的載體PGEX-4T-1或PET32a（㧏）連接，之后將重組表達(dá)載體轉(zhuǎn)化到大腸桿菌BL21中。重組蛋白經(jīng)菌落PCR鑒定、雙酶切鑒定、Western-blot鑒定。應(yīng)用AKTA purifier色譜系統(tǒng)對(duì)重組蛋白進(jìn)行純化分析。 2.ELISA檢測(cè)方法的建立 用純化后omp10和L7/L12兩種重組蛋白做抗原對(duì)人、牛血清進(jìn)行檢測(cè)，以驗(yàn)證兩種蛋白用ELISA方法檢測(cè)人、牛布魯氏菌病的應(yīng)用價(jià)值。通過對(duì)純化蛋白濃度的測(cè)定，，確定不同包被濃度抗原的包被量后，進(jìn)行方陣滴定法確定ELISA最佳包被濃度。 【結(jié)果】 本實(shí)驗(yàn)成功構(gòu)建了含有布魯氏菌外膜蛋白o(hù)mp10和核蛋白L7/L12序列的原核表達(dá)載體PET32a/Omp10、PGEX-4T-1/Omp10和PGEX-4T-1/L7/L12，并經(jīng)過PCR、雙酶切鑒定，均證明確實(shí)插入了表達(dá)載體。利用IPTG誘導(dǎo)，PGEX-4T-1/Omp10和PGEX-4T-1/L7/L12在其宿主菌大腸桿菌BL21中均有蛋白表達(dá)，經(jīng)純化后以重組蛋白PGEX-4T-1/L7/L12和PGEX-4T-1/omp10做為抗原進(jìn)行的ELISA實(shí)驗(yàn)結(jié)果表明這兩種重組蛋白對(duì)人布魯氏菌病的檢測(cè)沒有實(shí)際的應(yīng)用價(jià)值，但對(duì)檢測(cè)動(dòng)物布魯氏菌病有一定潛力。
[Abstract]:[objective] brucellosis (Brucellosis,) is a chronic bacterial infection caused by brucella (Brucella) infection, which is widely distributed in the world. Human brucellosis is classified as a class B infectious disease in China. The traditional laboratory detection of brucellosis mainly depends on the isolation of pathogens. This method is time-consuming and labor-intensive and requires a professional laboratory, which is not practical. With the development of the technology, it was found that the Elisa (ELISA) method had higher sensitivity and specificity than the conventional (CFT), agglutination test (SAT). In this study, the representative recombinant protein of Brucella was selected as antigen to carry out the enzyme linked immunosorbent assay (ELISA),) to establish a ELISA method with recombinant protein as antigen. It lays a foundation for the development of ELISA kit for rapid diagnosis of brucellosis. [methods] by referring to a large number of domestic and foreign literatures on brucellosis, and synthesizing the research on the diagnostic targets of brucella in the central laboratory of Yunnan Provincial Institute of endemic Disease Prevention and Control, the outer membrane protein omp10, was selected. Nuclear protein L 7 / 12 was used as a diagnostic target. 1. Cloning and expression of Brucella omp10,L7/L12, purification and amplification of two pairs of primers based on the published L7/L12 gene of omp10, nucleoprotein of Brucella spp. The vector PGEX-4T-1 or PET32a (?) was digested by BamH 鈪

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