结核分枝杆菌免疫优势抗原ESAT-6真核表达载体的构建及蛋白表达的鉴定

目的　克隆并表达结核分枝杆菌早期分泌性抗原靶ESAT 6的编码基因 ,为研究其免疫功能奠定基础。方法　以结核分枝杆菌H37Rv株基因组为模板 ,用PCR对ESAT 6基因进行扩增 ,扩增产物克隆到真核表达载体pcDNA3 1/Zeo( )中。对重组表达载体pcDNA ESAT 6进行酶切、PCR及测序鉴定。经鉴定无误的重组质粒用DEAE 葡聚糖法转染COS 7细胞 4 8h后 ,用Trizol法提取转染细胞总RNA进行RT PCR鉴定。结果　重组质粒pcDNA ESAT 6构建成功 ,并能在COS 7细胞中有效表达。结论　结核分枝杆菌早期分泌性抗原靶ESAT 6真核重组表达质粒pcDNA ESAT 6构建成功 ,为进一步研究ESAT 6和CFP 10的免疫原性、免疫保护性及其二者之间的相互作用奠定了基础

Construction and expression of eukaryotic expression recombinant plasmid of Mycobacterium tuberculosis ESAT-6

Objective To construct eukaryotic expression vector of ESAT-6 and investigate transient expression of its protein in COS-7 cells.Methods ESAT-6 coding sequence esxA was amplified from Mycobacterium tuberculosis H37Rv genomic DNA by using PCR.PCR product was cloned into eukaryotic expression vector pcDNA3.1/Zeo ( ).Eukaryotic expression vector pcDNA-ESAT-6 were transfected into eukaryotic cells COS-7 by means of DEAE-dextran,and transient expression of its protein was investigated in eukaryotic cells with RT-PCR.Results ESAT-6 coding sequence was successfully inserted into the eukaryotic expression vector pcDNA3.1/Zeo ( ) and efficiently expressed in eukaryotic cells (COS-7).Conclusion The recombinant pcDNA-ESAT-6 could be successfully constructed and expressed efficiently in COS-7.