耐药结核病血清标志物的筛选及Rv0350的克

发布时间：2018-01-30 05:56

本文关键词： 结核分枝杆菌耐药标志物蛋白芯片 Rv0350　出处：《遵义医学院》2017年硕士论文　论文类型：学位论文

【摘要】：目的:筛选耐药结核分枝杆菌血清学特异分子标志物并体外克隆、表达及纯化Rv0350目的蛋白。方法:收集2011年到2015年就诊遵义医学院附属医院结核病患者及健康体检者血清标本,与结核蛋白芯片杂交筛选特异性蛋白;结合前期试验筛选出的特异性蛋白,组合制作成蛋白小芯片,再收集血清标本与小芯片杂交,进一步筛选出耐药结核病候选标志物。设计目的基因引物,行PCR扩增目的基因,用pET28a与目的基因构建重组质粒,然后转染至BL21(DE3)感受态细胞,IPTG体外诱导目的蛋白,利用镍亲和层析获取纯化的重组蛋白。结果:初次筛选出100个特异性蛋白,第二次杂交筛选出了8个与耐药相关的抗原,分别为Rv0350、Rv0363c、Rv2396、Rv1100、Rv3509c、Rv3653、Rv0865和Rv2031c,其组合诊断耐药结核病的特异度、灵敏度分别为80.4%、80%。我们已经申请一项发明专利(申请号CN201610089177.2)。体外成功克隆、表达和纯化Rv0350蛋白。结论:成功筛选出8个与耐药相关的结核分枝杆菌特异抗原,其中Rv0350诊断特异性较好。利用分子生物学技术可成功获得纯化的结核分枝杆菌Rv0350抗原,为耐药结核病检测试剂盒的研发奠定了基础。
[Abstract]:Objective: to screen the serological specific molecular markers of drug-resistant Mycobacterium tuberculosis and clone them in vitro. Methods: from 2011 to 2015, the serum samples of tuberculosis patients and healthy people in affiliated hospital of Zunyi Medical College were collected. The specific protein was screened by hybridization with tuberculosis protein chip. Combined with the specific protein selected from the previous experiment, the protein microarray was made, and then the serum samples were collected and hybridized with the microchip to further screen the candidate markers of drug-resistant tuberculosis. The target gene primers were designed. The target gene was amplified by PCR and the recombinant plasmid was constructed with pET28a and target gene. The purified recombinant protein was obtained by nickel affinity chromatography. Results: 100 specific proteins were screened for the first time and 8 antigens related to drug resistance were screened by the second hybridization, respectively, which were Rv0350. Rv0363c1, Rv2396, Rv1100, Rv3509cFV, Rv3653, Rv0865 and Rv2031c. the specificity of Rv2396, Rv1100, Rv3653, Rv0865 and Rv2031cin the diagnosis of drug-resistant tuberculosis. The sensitivity is 80.4 and 800.We have applied for an invention patent (application number CN201610089177.2) and cloned successfully in vitro. Conclusion: eight Mycobacterium tuberculosis specific antigens associated with drug resistance were successfully screened. The purified Rv0350 antigen of Mycobacterium tuberculosis can be successfully obtained by molecular biological technique, which lays a foundation for the development of drug-resistant tuberculosis detection kit.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R52

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