TAT-EGFP融合蛋白的表达纯化及穿膜活性的研究

Expression and purification of TAT-EGFP fusion protein and study of its penetrating activity

目的在大肠埃希菌中高效表达TAT-EGFP融合蛋白并鉴定纯化,然后进行穿膜活性的研究。方法以本室前期构建的重组质粒pQE-EGFP为模板,采用PCR方法特异性扩增TAT-EGFP基因序列,随后克隆于原核表达载体pQE-31,所构建的重组质粒经测序鉴定后转化大肠埃希菌JM109,IPTG诱导表达;SDS-PAGE和Western blotting鉴定表达蛋白质,经Ni2+-金属螯合亲和层析纯化,将纯化蛋白加入体外培养的小鼠黑色素瘤B16细胞中,荧光显微镜下观察TAT蛋白的穿膜活性。结果成功构建了pQE-TAT-EGFP重组质粒的,转化大肠埃希菌JM109,经IPTG诱导,目的蛋白表达率为25%,SDS-PAGE、Western blotting初步测定目的蛋白的相对分子质量(Mr)约为30160,纯化得到了目的融合蛋白TAT-EGFP。并在体外培养的B16细胞中证实TAT-EGFP融合蛋白穿透生物膜的能力。结论通过对TAT-EGFP融合蛋白穿膜活性的分析,证实了TAT具有穿膜活性,为TAT-PEⅢ融合蛋白抑瘤活性的研究提供了理论依据,也为生物大分子药物进入组织细胞内发挥治疗作用奠定了基础。

Objective To purify the TAT-EGFP fusion protein and investigate its transduction efficiency. Methods By using pQE- EGFP plasmid as template, a pair of primers were designed for amplification of TAT- EGFP gene by PCR. In this primer pair, the sequence encoding the 11-amino-acid HIV TAT domain and flanking glycine residues were added to the 5＇terminal of the forward primer. The TAT-EGFP PCR products were purified, and then cloned into pQE-31 vector. The recombinant pQE-TAT-EGFP was transformed into E. coli JM109 and induced with IPTG for protein expression. The expression of TAT-EGFP was identified by SDS-PAGE and Western blotting, and then purified by Ni^2＋ -metal chelate affinity chromatography. The purified fusion protein was added into cultured B16 ceils in vitro and the transduction efficiency was detected using fluorescence assay. Results The recombinant plasmid pQE-TAT-EGFP was constructed successfully. The results of SDS- PAGE and Western blotting showed that the molecular weight of the expressed TAT-EGFP fusion protein was 30 160. After purification, the TAT-EGFP fusion protein could cross the cytomembrane of B16 cells in vitro. Conclusion This experiment demonstrates the possibility of TAT to directly deliver protein into cells, which provides a basis for the research on the antitumor activity of TAT-PE Ⅲ fusion protein.