摘要
采用点突变技术构建了带 6×His、Tat和Flag多个标记的pET HTF的质粒载体 ,利用基因重组技术构建pET HTF EGFP融合蛋白载体 .酶切和DNA测序证明 ,所构建的pET HTF和pET HTF EGFP载体正确 .BL2 1(DE3)表达融合蛋白 ,用Ni2 + 分离柱纯化His Tat Flag EGFP蛋白 ,并加入培养的NIH3T3细胞 .荧光显微镜观察显示 ,His Tat Flag EGFP融合蛋白进入细胞 .带His、Tat和Flag标记的质粒载体pET 14b HTF表达的融合蛋白能够进入细胞 。
pET HTF with 6×His, Tat and Flag was constructed by site directed mutagenesis method, fusion vector of pET HTF EGFP, pET 14b EGFP were constructed using gene recombination method. DNA sequencing analysis and enzyme digestion showed that the constructed vectors of pET HTF and pET HTF EGFP were correct. Tat EGFP was expressed in BL21(DE3) and purified using Qiagen Ni 2+ NTA agarose. Fusion protein was added to cultured NIH3T3 cells and fluorescence was observed using fluorescence microscope. Purified fusion protein could enter cultured NIH3T3 cells efficiently. The constructed vector pET HTF with 6×His, Tat and Flag which could transduct its fusion protein into cells provide a important tool in study of protein function and gene therapy.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2003年第3期354-358,共5页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金 (No. 3 0 0 3 0 0 60 )
国家杰出青年科学基金 (No .3 992 5 0 14 )
海外青年学者合作研究基金 (No.3 0 12 80 0 9)
国家高技术研究发展计划 ( 863 )课题 (No .2 0 0 1AA2 3 40 61)
广东省科技计划项目(No .A10 90 2 0 2 )资助~~
