活动性结核病特异性生物标志物的筛选及Rv3480c的克

发布时间：2018-06-16 02:11

  本文选题：结核分枝杆菌 + Rv3480c　； 参考：《遵义医学院》2017年硕士论文

【摘要】：目的:利用蛋白芯片技术筛选活动性结核病的特异性生物标志物,并将目的蛋白进一步克隆表达和纯化。方法:用正常对照组、结核分枝杆菌潜伏感染组、活动性结核组病人血清与固定在芯片上的结核分枝杆菌菌体蛋白结合,再用抗人Ig M荧光标记二抗(cy5标记,呈现红色)和抗人Ig G荧光二抗(cy3标记,呈现绿色)检测,通过荧光扫描仪读取信号,根据信号的强弱筛选特异性蛋白质。用primer 5.0软件设计合成Rv3480c引物,PCR扩增Rv3480c全基因,构建Rv3480c-p ET28a融合基因,提取质粒DNA,双酶切及测序分析验证质粒DNA,将重组质粒转化入表达宿主E.coli BL21(DE3)菌体内,不同浓度IPTG诱导蛋白质表达,考马斯亮蓝染色及Western blot验证Rv3480c的表达,并通过反复低温冻融、不同浓度尿素复性的方法进行包涵体中蛋白质的纯化。结果:通过蛋白芯片技术最终筛选出15个与活动结核病诊断相关的特异性结核分枝杆菌蛋白,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(已申请国家发明专利,申请号:201610089179.1)。将荧光信号最强所对应的蛋白按照不同的组合,制成芯片,用正常对照组、结核分枝杆菌潜伏感染组、活动性结核组病人血清检测该组合芯片,结果显示其诊断活动性结核病的特异性为90.3%,灵敏度为85.4%。筛选出的15个蛋白中,结核分枝杆菌蛋白Rv3480c在抗原抗体结合反应中反应较强,可作为活动性结核病的特异性生物标志物之一。进一步成功构建Rv3480c-p ET28a融合质粒。0.2 m M IPTG在16℃条件下诱导蛋白质的表达,Western blot结果证明成功表达结核分枝杆菌Rv3480c蛋白质。考马斯亮染色示Rv3840c蛋白表达于包涵体中,经4M尿素溶解包涵体,分别用2M、1M尿素及1x PBS透析后,成功纯化活动性结核病生物标志物Rv3480c蛋白质。结论:本研究使用蛋白芯片技术通过两次芯片与血清反应,成功筛选出15个与活动结核病诊断相关的特异性结核分枝杆菌蛋白,这些蛋白组合对于区分活动结核病、潜伏感染、正常人群有较好的特异性(90.3%)和敏感性(85.4%)。我们通过基因克隆技术成功获得Rv3480c重组蛋白,为其结构和功能的研究、新型抗结核药物的研制以及结核病的血清学诊断奠定了基础。在构建大肠杆菌重组表达载体体系及蛋白的表达、包涵体的纯化过程中,我们对部分反应条件进行优化,为更好的进行包涵体蛋白纯化提供了思路。
[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，

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