结核分支杆菌ESAT-6真核表达质粒的构建及蛋白表达的鉴定

目的 用6kDa早期分泌性抗原靶蛋白(ESAT-6)基因构建真核表达重组质粒pcDNA3．1( )-ESAT-6，并鉴定其在真核细胞(COS-7)中的蛋白表达。方法 以结核杆菌H37Rv株基因组DNA为模板，用PCR对ESAT-6基因进行扩增，将扩增的产物连接于测序载体pUCm—T上，经测序反应确定无误后，再将PCR反应产物克隆于真核表达载体pcDNA3．1( )上。并用脂质体介导真核表达重组质粒pcDNA3．1( )-ESAT-6转染真核细胞COS-7，72h后，通过SDS—PAGE和免疫印迹鉴定ESAT-6基因表达的蛋白。结果 用结核杆菌基因ESAT-6构建重组真核表达质粒pcDNA3．1( )-ESAT-成功，通过SDS—PAGE证明重组质粒转化的细胞内有一分子量6kDa的特异蛋白，免疫印迹证明该6kDa蛋白能与抗ESAT-6单克隆抗体反应。结论 结核杆菌早期分泌性蛋白ESAT-6真核表达重组质粒成功构建，该质粒转染的细胞能够产生、分泌结核杆菌早期分泌性蛋白ESAT-6。

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

Aim To construct eukaryotic expression vector of ESAT-6 and investigate transient expression of protein in eukaryotic cells.Method The gene encoding for protein ESAT-6 was amplified from M.tuberculosis H37Rv genomic DNA by using PCR.PCR product was cloned into sequencing vector pUCm-T.Sequence of ESAT-6 gene was proved by sequencing reaction.ESAT-6 gene was ligated to eukaryotic expression vector pcDNA3.1(+).Eukaryotic expression vector pcDNA3.1(+)-ESAT-6 were transfected into eukaryotic cells(COS-7) by means of LipofectamineTM2000,and investigate transient expression of protein in eukaryotic cells with SDS-PAGE and Western Blotting.Results ESAT-6 gene was successfully inserted into the eukaryotic expression vector pcDNA3.1(+).Transient expression of protein of ESAT-6 gene in eukaryotic cells(COS-7) was confirmed by SD-PAGE and Western Blotting.Conclusion We have obtained recombinant pcDNA3.1(+)-ESAT-6 and confirmed its protein expression in eukaryotic cells(COS-7).