结核分枝杆菌培养滤液蛋白32的克隆表达及血清学诊断应用

发布时间：2018-09-19 14:54

【摘要】： 目的:构建结核分枝杆菌培养滤液蛋白32(culture filtrate protein 32,CFP32)的重组质粒,在大肠杆菌中表达并纯化重组蛋白CFP32,探讨其在结核病血清学诊断中的应用价值。方法:PCR扩增CFP32基因片段,将其克隆到pET-21a表达载体。优化表达条件,Ni-NTA亲和层析方法纯化重组CFP32蛋白。建立检测CFP32抗体的酶联免疫吸附试验(ELISA)方法,并用于检测160例临床血清标本中的CFP32抗体。结果:克隆了cfp32基因,构建了表达质粒pET21a-cfp32,IPTG诱导在大肠杆菌中得到高表达CFP32蛋白包涵体,SDS-PAGE电泳在30 KD处得到条带,纯化得到高纯度CFP32蛋白,PBS复性后测得蛋白含量为3.46 mg/mL。建立并优化了CFP32检测结核抗体的ELISA方法。97例临床确诊的肺结核患者中62例CFP32抗体阳性,敏感性为63.9%;25例非结核病患者和38例健康人血清CFP32抗体阳性2例,特异性为96.8%。结论:成功在大肠杆菌中获得高效表达CFP32蛋白,血清抗体检测表明重组的CFP32蛋白有希望成为结核病血清学诊断的有效候选蛋白之一。
[Abstract]:Objective: to construct the recombinant plasmid of mycobacterium tuberculosis culture filtrate protein 32 (culture filtrate protein 32 CFP32 and express and purify the recombinant protein CFP32, in Escherichia coli to explore its application value in tuberculosis serological diagnosis. Methods CFP32 gene fragment was amplified by PCR and cloned into pET-21a expression vector. Purification of recombinant CFP32 protein by Ni-NTA affinity chromatography. An enzyme linked immunosorbent assay (ELISA) was established to detect CFP32 antibodies in 160 clinical serum samples. Results: the cfp32 gene was cloned, and the expression plasmid pET21a-cfp32,IPTG was constructed to induce the inclusion body of high expression CFP32 protein in Escherichia coli. SDS-PAGE electrophoresis was carried out at 30 KD. After renaturation of CFP32 protein with high purity, the protein content was 3.46 mg/mL.. A ELISA method for detection of tuberculosis antibody by CFP32 was established and optimized. 62 cases were positive for CFP32 antibody. The sensitivity was 63.9% in 25 cases of non-tuberculosis and 38 cases in healthy persons. The specificity was 96.8%. Conclusion: CFP32 protein was successfully expressed in Escherichia coli. The detection of serum antibody showed that the recombinant CFP32 protein might be one of the effective candidate proteins for the serological diagnosis of tuberculosis.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R378;R446.6

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