本文選題：結核分枝桿菌 + Rv3480c��； 參考：《遵義醫(yī)學院》2017年碩士論文

【摘要】：目的:利用蛋白芯片技術篩選活動性結核病的特異性生物標志物,并將目的蛋白進一步克隆表達和純化。方法:用正常對照組、結核分枝桿菌潛伏感染組、活動性結核組病人血清與固定在芯片上的結核分枝桿菌菌體蛋白結合,再用抗人Ig M熒光標記二抗(cy5標記,呈現(xiàn)紅色)和抗人Ig G熒光二抗(cy3標記,呈現(xiàn)綠色)檢測,通過熒光掃描儀讀取信號,根據(jù)信號的強弱篩選特異性蛋白質。用primer 5.0軟件設計合成Rv3480c引物,PCR擴增Rv3480c全基因,構建Rv3480c-p ET28a融合基因,提取質粒DNA,雙酶切及測序分析驗證質粒DNA,將重組質粒轉化入表達宿主E.coli BL21(DE3)菌體內(nèi),不同濃度IPTG誘導蛋白質表達,考馬斯亮藍染色及Western blot驗證Rv3480c的表達,并通過反復低溫凍融、不同濃度尿素復性的方法進行包涵體中蛋白質的純化。結果:通過蛋白芯片技術最終篩選出15個與活動結核病診斷相關的特異性結核分枝桿菌蛋白,包括Rv3480c Ig M、Rv1860 Ig G、Rv2352c Ig M、Rv1597 Ig M、Rv2688c Ig M、Rv0049 Ig M、MT1560 Ig G、Rv2511 Ig G、Rv0350 Ig M、Rv0350 Ig G、Rv0270 Ig M、Rv1876 Ig M、Rv0494 Ig M、Rv2031c Ig G、Rv2450c Ig M(已申請國家發(fā)明專利,申請?zhí)?201610089179.1)。將熒光信號最強所對應的蛋白按照不同的組合,制成芯片,用正常對照組、結核分枝桿菌潛伏感染組、活動性結核組病人血清檢測該組合芯片,結果顯示其診斷活動性結核病的特異性為90.3%,靈敏度為85.4%。篩選出的15個蛋白中,結核分枝桿菌蛋白Rv3480c在抗原抗體結合反應中反應較強,可作為活動性結核病的特異性生物標志物之一。進一步成功構建Rv3480c-p ET28a融合質粒。0.2 m M IPTG在16℃條件下誘導蛋白質的表達,Western blot結果證明成功表達結核分枝桿菌Rv3480c蛋白質。考馬斯亮染色示Rv3840c蛋白表達于包涵體中,經(jīng)4M尿素溶解包涵體,分別用2M、1M尿素及1x PBS透析后,成功純化活動性結核病生物標志物Rv3480c蛋白質。結論:本研究使用蛋白芯片技術通過兩次芯片與血清反應,成功篩選出15個與活動結核病診斷相關的特異性結核分枝桿菌蛋白,這些蛋白組合對于區(qū)分活動結核病、潛伏感染、正常人群有較好的特異性(90.3%)和敏感性(85.4%)。我們通過基因克隆技術成功獲得Rv3480c重組蛋白,為其結構和功能的研究、新型抗結核藥物的研制以及結核病的血清學診斷奠定了基礎。在構建大腸桿菌重組表達載體體系及蛋白的表達、包涵體的純化過程中,我們對部分反應條件進行優(yōu)化,為更好的進行包涵體蛋白純化提供了思路。
[Abstract]:Objective: to screen the specific biomarkers of active tuberculosis by protein chip technique and to clone and express the target protein. Methods: normal control group, mycobacterium tuberculosis latent infection group and active tuberculosis group were used to bind to mycobacterium tuberculosis bacterial protein fixed on microarray and then labeled with anti-human IgM fluorescence labeled second antibody cy5. Red) and anti-human IgG fluorescent second antibody cy3 (green) were detected. The signal was read by fluorescence scanner and the specific protein was screened according to the intensity of the signal. Rv3480c whole gene was amplified by primer 5.0 software. Rv3480c-pET28a fusion gene was constructed, plasmid DNA was extracted, double enzyme digestion and sequencing analysis were performed to verify the plasmid DNA, and the recombinant plasmid was transformed into E. coli BL21DE3). The protein expression was induced by IPTG at different concentrations. Coomassie brilliant blue staining and Western blot were used to verify the expression of Rv3480c. The protein was purified by repeated freezing and thawing at low temperature and renaturation with different concentrations of urea. Results: fifteen specific Mycobacterium tuberculosis proteins related to the diagnosis of active tuberculosis were screened by protein chip technique. Including Rv3480c Ig MU, Rv1860Ig GV, Rv1860Ig GG, Rv1860Ig GG, Rv1860Ig / Rv1872c Ig MU Rv1597Ig MU, Rv2688c Ig Mf0049Ig MMT1560Ig / Rv2511Ig / Rv0350Ig / Rv0350Ig / Rv0270270Ig / Rv1876Ig / Rv1876Ig / Rv2031c / Rv2031c / Ig / Rv2450c (national patent application no. 201610089179.1c) The protein corresponding to the strongest fluorescent signal was made into a chip according to different combinations. The serum samples of patients with latent infection of Mycobacterium tuberculosis and active tuberculosis were used to detect the microarray with normal control group, latent infection group of Mycobacterium tuberculosis and active tuberculosis group. The results showed that the specificity of the diagnosis of active tuberculosis was 90.3 and the sensitivity was 85.4. Among the 15 selected proteins, Mycobacterium tuberculosis protein Rv3480c reacted strongly in antigen-antibody binding reaction and could be used as one of the specific biomarkers of active tuberculosis. Furthermore, Rv3480c-pET28a fusion plasmid was successfully constructed. The protein expression induced by 0.2 m IPTG at 16 鈩，

本文編號：2024741