结核分枝杆菌CFP10-ESAT6-PPE68融合蛋白的表达及其在结核病诊断中的初步应用

发布时间：2018-04-21 03:14

本文选题：结核分枝杆菌 + CFP10-ESAT6-PPE68　；参考：《重庆医科大学》2013年硕士论文

【摘要】：目的：构建结核分枝杆菌CFP10-ESAT6-PPE68融合基因原核表达质粒，并在大肠杆菌中诱导表达融合蛋白。通过亲和层析法分离、纯化所表达的目的蛋白，将其作为一种特异性抗原，间接ELISA法检测临床已确诊为结核病（包括肺结核和肺外结核）病人的血清，统计分析检测的特异性、准确性和敏感性，为其在临床上的进一步应用打下基础。方法： 1.以结核分枝杆菌H37Rv菌株的DNA作为模板，PCR法扩增ESAT6和PPE68基因，利用基因拼接技术中的Gene-SOEing法扩增ESAT6-PPE68融合基因，克隆至原核表达质粒pET-32a(+)中，构建重组表达质粒pET-32a(+)-ESAT6-PPE68。再分别以H37Rv株DNA和重组质粒pET-32a(+)-ESAT6-PPE68为模板扩增CFP10和ESAT6-PPE68基因，Gene-SOEing法获取目的基因CFP10-ESAT6-PPE68，克隆至pET-32a(+)载体中，构建重组表达质粒pET-32a(+)-CFP10-ESAT6-PPE68，并用酶切和测序鉴定。 2.将构建成功的质粒pET-32a(+)-CFP10-ESAT6-PPE68转化入大肠杆菌BL21(DE3)中，IPTG诱导其大量表达，用Western blot鉴定重组蛋白。用亲和层析法分离、纯化所表达的融合蛋白。 3.将纯化的融合蛋白作为一种特异性抗原，间接ELISA法检测已临床确诊为结核病（包括250例肺结核和66例肺外结核）病人血清，，58例正常人血清，统计分析检测的特异性、准确性和敏感性。结果： 1.重组表达质粒pET-32a(+)-CFP10-ESAT6-PPE68经内切酶作用后在1782bp处可见CFP10-ESAT6-PPE68目的基因片段，片段大小与预期一致。基因测序结果显示重组质粒的碱基序列与理论值相符。 2.成功诱导表达CFP10-ESAT6-PPE68融合蛋白，SDS-PAGE分析诱导产物的相对分子量约为77KDa，包括相对分子量约20KDa的Trx条带；相对分子量约为11KDa的CFP10；相对分子量约9KDa的ESAT6；相对分子量约为37KDa的PPE68。Western blot分析诱导表达蛋白可与结核分枝杆菌感染BALB/c小鼠的血清发生特异性反应，在相对分子量约77KDa处可见明显条带。融合蛋白用亲和层析法分离、纯化后，Bandscan软件分析目的蛋白的纯度约为87.3%。 3.将纯化后CFP10-ESAT6-PPE68融合蛋白作为特异性诊断抗原，以临床确诊结核病人作为金标准，58例正常人血清作为阴性对照，统计分析结果表明融合蛋白检测肺结核病人和肺外结核病人的特异性分别为94.8％和98.3%；准确性分别为63.6%和93.5%；敏感性分别为56.4%和89.3%。结论： 1.将CFP10、ESAT6和PPE68三种特异性基因成功融合并插入原核表达质粒pET-32a(+)中，并在大肠杆菌BL21(DE3)中诱导表达及鉴定。 2.CFP10-ESAT6-PPE68融合蛋白作为一种特异性抗原在诊断结核分枝杆菌感染方面有一定的参考价值，特别是针对肺外结核病的诊断。
[Abstract]:Objective: The prokaryotic expression plasmid of CFP10-ESAT6-PPE68 fusion gene of Mycobacterium tuberculosis was constructed and the fusion protein was induced and expressed in Escherichia coli. The expressed target protein was purified by affinity chromatography and used as a specific antigen. Indirect ELISA assay was used to detect the serum of patients with tuberculosis (including tuberculosis and extrapulmonary tuberculosis). Accuracy and sensitivity lay the foundation for further clinical application. Methods: 1. The ESAT6 and PPE68 genes were amplified by DNA from H37Rv strain of Mycobacterium tuberculosis. The fusion gene of ESAT6-PPE68 was amplified by Gene-SOEing in gene splicing technique. The ESAT6-PPE68 fusion gene was cloned into the prokaryotic expression plasmid pET-32a() and the recombinant expression plasmid pET-32a (-ESAT6-PPE68) was constructed. Then the target gene CFP10-ESAT6-PPE68 was amplified from H37Rv strain DNA and recombinant plasmid pET-32a (-ESAT6-PPE68). The target gene CFP10-ESAT6-PPE68 was cloned into pET-32a() vector. The recombinant expression plasmid pET-32a (-CFP10-ESAT6-PPE68) was constructed and identified by enzyme digestion and sequencing. 2. The constructed plasmid pET-32a (-CFP10-ESAT6-PPE68) was transformed into Escherichia coli BL21DE3, and its expression was induced by IPTG. The recombinant protein was identified by Western blot. The fusion protein was purified by affinity chromatography. 3. The purified fusion protein was used as a specific antigen. Indirect ELISA assay was used to detect the serum of 58 healthy people with clinically diagnosed tuberculosis (including 250 cases of pulmonary tuberculosis and 66 cases of extrapulmonary tuberculosis). Accuracy and sensitivity. Results: 1. The recombinant expression plasmid pET-32a (-CFP10-ESAT6-PPE68) was treated with endonuclease and the target gene fragment of CFP10-ESAT6-PPE68 was found in 1782bp, and the size of the fragment was the same as expected. The results of gene sequencing showed that the base sequence of the recombinant plasmid was consistent with the theoretical value. 2. SDS-PAGE analysis showed that the relative molecular weight of the product was about 77kDa, including the Trx band of 20KDa. CFP10 with a relative molecular weight of 11KDa, ESAT6 with a relative molecular weight of 9KDa and PPE68.Western blot with a relative molecular weight of 37KDa could induce a specific reaction with the serum of BALB/c mice infected with Mycobacterium tuberculosis. Obvious bands were observed at the relative molecular weight of about 77KDa. The fusion protein was separated by affinity chromatography and purified by Bandscan software. The purity of the target protein was about 87.3. 3. The purified CFP10-ESAT6-PPE68 fusion protein was used as the specific diagnostic antigen, and the serum of 58 normal persons was used as the negative control. The results of statistical analysis showed that the specificity of fusion protein in detecting pulmonary tuberculosis and extrapulmonary tuberculosis was 94.8% and 98.3%, the accuracy was 63.6% and 93.55.The sensitivity was 56.4% and 89.3%, respectively. Conclusion: 1. The three specific genes of CFP10 ESAT6 and PPE68 were successfully fused and inserted into the prokaryotic expression plasmid pET-32a (), and were induced and identified in E. coli BL21DE3. As a specific antigen, 2.CFP10-ESAT6-PPE68 fusion protein has a certain reference value in the diagnosis of Mycobacterium tuberculosis infection, especially for the diagnosis of extrapulmonary tuberculosis.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R378.911

【参考文献】