结核杆菌抗原HSP65基因在大肠杆菌中的表达及纯化

目的 :为研制预防结核病疫苗 ,选取结核杆菌HSP65蛋白为免疫抗原 ,将结核分枝杆菌HSP65抗原基因在大肠杆菌中表达、纯化。方法 :将HSP65的全长cDNA插入到原核表达载体pGEX5T中 ,构建成pGEX5T HSP65重组质粒。将质粒转化到E .coli.K80 2细菌 ,用IPTG诱导HSP65表达 ,然后用亲和层析的方法进行纯化 ,最后用Western blot方法确认表达蛋白的特异性。结果 :获得了pGEX5T HSP65重组子 ,HSP65蛋白在k80 2菌中获得了表达 ,表达的蛋白条带大小约 86kDa ,与预期的结果相符。表达产物经亲和层析后获得了较单一的蛋白条带 ;表达及纯化的蛋白在纯化前后均可被结核病患者血清特异地识别。为进一步研究其在结核病诊断和防治中的应用打下了基础。

The Expression and Purification of HSP65 Antigen from Mycobacterium tuberculosis in E.Coli

Objective:To develop a vaccine against tuberculosis,HSP65 antigen was chosed.The HSP65 antigen was expressed and purifed in E.coli k802. Methods:The full-length cDNA encoding HSP65 protein was inserted into prokaryotic expressing vector-pGEX5T.HSP65 protein was purified by affinity chromatography after expression induced by IPTG.Finally,the protein was confirmed by Western-blot,using the serum from patients infected with tuberculosis.Results: The recombinant plasmid pGEX5T-HSP65 was successfully constructed.The relative molecular weight of the expressed protein was about 86kDa which was consistent with the respected.The expressed protein was purified successfully by using affinity chromatography and could be specially recognized by the sera from tuberculosis patients.Conclusion: The HSP65 protein of Mycobacterium tuberculosis was expressed in E.coli.k802 and was purified successfully.It will be helpful in further studies on diagnosis and therapy of tuberculosis.;