| 摘要: |
| 目的:将结核分枝杆菌Ag85A抗原基因克隆并在大肠杆菌中表达。方法:用PCR方法从结核分枝杆菌基因组中扩增Ag85A抗原基因,然后将其插入到原核表达载体pGEX5T中,构建成pGEX5TAg85A重组质粒。经IPTG诱导使该基因在E.coli k802菌中表达,亲和层析法纯化蛋白,Western印迹法验证表达蛋白的抗原性。结果:扩增出了约0.9 kb的单一条带,并成功地构建了pGEX5TAg85A重组子；经IPTG诱导后,Ag85A蛋白在k802菌中获得了表达,表达的蛋白条带大小约58 000,与预期结果相符;纯化后获得了较单一的蛋白条带。蛋白纯化前后均可被结核病患者血清特异地识别。结论:成功地克隆了Ag85A抗原基因,并将其在大肠杆菌中进行了表达、纯化,为将其应用于结核病诊断和防治的研究奠定了基础。 |
| 关键词: 结核分枝杆菌 Ag85A抗原 分子克隆 基因表达 |
| DOI：10.3724/SP.J.1008.2008.00121 |
| 投稿时间：2007-10-19修订日期：2007-12-14 |
| 基金项目:国家自然科学基金重点项目（30530660）. |
|
| Clone of Mycobacterium tuberculosis Ag85A gene and its prokaryotic expression |
| WANG Qing-min1,2,SUN Shu-han1* |
| (1.Department of Medical Genetics, College of Basic Medical Sciences, Second Military Medical University, Shanghai 200433, China) |
| Abstract: |
| Objective：To clone the Ag85A antigen gene of Mycobacterium tuberculosis and express it in E.coli k802. Methods: The Ag85A gene was amplified from the genome of Mycobacterium tuberculosis by PCR and was inserted into prokaryotic expressing vector pGEX5T to construct pGEX5TAg85A recombinant plasmid. The expression of Ag85A protein in E.coli k802 was induced by IPTG. The expressed protein was purified by affinity chromatography and the antigenicity of the expressed protein was confirmed by Western blotting assay. Results: A band about 0.9 kb in length was obtained by PCR and the recombinant plasmid pGEX5TAg85A was successfully constructed. A new band about 58 000 in length was observed after IPTG induction in E.coli. The relative molecular weight of the expressed protein was consistent with that expected. A single protein band of 58 000 in length was obtained after purification. The expressed protein could be specifically recognized by the sera of patients with tuberculosis patients. Conclusion: The Ag85A gene of Mycobacterium tuberculosis has been successfully cloned and expressed in E.coli k802, which paves a way for further studies on diagnosis and therapy of tuberculosis. |
| Key words: Mycobacterium tuberculosis Ag85A antigen molecular cloning gene expression |
