融合蛋白TAT-EGFP的表达及其对膀胱癌细胞和组织的穿膜活性鉴定

Expression of TAT-EGFP Fusion Protein and Verification of Its Transmem-brane Activity Into Bladder Cancer Cells and Tissues

目的构建穿膜肽-增强型绿色荧光蛋白(TAT-EGFP)融合蛋白的原核表达系统,研究该融合蛋白在体外对人膀胱癌细胞(EJ细胞)和膀胱癌组织的跨膜作用。方法以pEGFP-1载体为模板,设计包含TAT序列的引物,用PCR技术扩增TAT-EGFP基因,扩增产物插入载体pET30a,构建成重组质粒pET30a-TAT-EGFP。将重组质粒转入大肠杆菌BL21中,IPTG诱导TAT-EGFP融合蛋白表达。表达产物用十二烷基硫钠-聚丙烯酰胺凝胶电泳鉴定,Ni-NTA Superflow Cartvidge亲和层析柱纯化融合蛋白。将融合蛋白TAT-EGFP加入培养的EJ细胞和膀胱癌组织,荧光显微镜观察TAT-EGFP融合蛋白进入EJ细胞和膀胱癌组织的情况。结果成功构建了高表达pET30a-TAT-EGFP重组子,纯化了分子质量约为31 kD的融合蛋白TAT-EGFP。不同浓度的TAT-EGFP融合蛋白对膀胱癌细胞均无明显毒性。TAT-EGFP融合蛋白具有穿过膀胱癌细胞和膀胱癌组织的作用。结论通过对TAT-EGFP融合蛋白表达纯化及活性分析,证实TAT的蛋白转导作用,为肽类及生物大分子药物进入组织细胞内发挥治疗作用提供了理论基础。

Objeetlve To construct an prokaryotic expression system of cell-penptrating peptides-enhanced green fluorescent protein（TAT-EGFP） fusion protein,and investigate its transmem-brane effect in vitro on human bladder cancer cell（ EJ cells） and tissues. Methods With pEGFP-1 cartier as a template, a primer that contained TAT sequences was designed. Amplification of TAT-EGFP gene was done by PCR, and its product was inserted into pET30a vector to construct recombinant plasmid pET30a-TAT-EGFP. The recombinant vector was transformed into escherichia coli BL21 and TAT-EGFP fusion protein was induced with IPTG. The expressed fusion protein was purified by Ni-NTA Superflow Cartridge and tested by SDS-PAGE electrophoresis,which was added into cultured EJ cells and bladder cancer tissues in vitro. Observation on TAT-EGFP fusion protein transformed into EJ cells and bladder cancer tissues was performed by fluorescence microscopy. Results A high expression pET30a-TAT-EGFP recombinant was constructed successfully, purified molecular mass of TAT-EGFP fusion was about 31 kD. Different concentrations of TAT-EGFP fusion protein showed no significant toxicity on bladder cancer cells, but showed a transmem-brane effect on EJ cells and bladder cancer tissues. Conclusion The transduction effect of TAT protein is confirmed based on the purification and activity analysis of TAT-EGFP fusion protein expression,which provides a theoretical basis for applying peptide and biological macromolecule drugs to tissues and cells.