结核抗原ESAT-6基因的克隆及在大肠埃希菌中的表达

目的：将结核分支杆菌ESAT抗原编码区基因克隆，并使其在大肠埃希菌中表达。方法：培养并抽提结核分支杆菌基因组，采用聚合酶链反应（PCR）扩增编码ESAT的全长cDNA，并插入到原核表达载体pGEX5T中，构建pGEX5T-ESAT重组质粒，IPTG诱导使该基因在k802菌中表达，并免疫印迹（Western blot)方法确证表达蛋白的特异性。结果：PCR扩增获得了单一的条带，大小约0.3kb；全自动测序与Genbank中登录的序列完全相同。获得了pGEX5T-ESAT重组子并在k802菌中表达，该蛋白可被结核病患者血清特异地识别。结论：扩增和克隆结核杆菌ESAT抗原的全长cDNA并在大肠埃希菌中获得表达，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。

Cloning and expression of ESAT cDNA coding region of mycobacterium tuberculosis in E.Coli

Objective To clone and express the gene encoding ESAT antigen of Mycobacterium tuberculosis (chinese stain). Methods Genome of mycobacterium tuberculosis was extracted. Then, the full length cDNA encoding ESAT protein was amplified by PCR and cloned into prokaryotic expressing vector pGEX5T and sequenced. pGEX5T ESAT was expressed in k802 Ecoli, and the expressed protein was determined by western blot using the sera from ten patients with tuberculosis. Results One specific band of 0.3kb or so was obtained and sequencing result was identical to that reported from Genbank. The expressed protein could be specifically recognized by the sera from tuberculosis patients. Conclusions The full length cDNA of Mycobacterium tuberculosis (Chinese strain) ESAT protein was cloned and expressed successfully, which will be helpful in further studies on diagnosis and treatment of tuberculosis.