摘要
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。
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).
出处
《中国人兽共患病杂志》
CSCD
北大核心
2003年第5期65-68,47,共5页
Chinese Journal of Zoonoses
