TAT-GFP-ER融合蛋白的原核表达与鉴定

The Prokaryotic Expression and Bio-activity Examination of TAT-GFP-ERβ Protein

从前列腺中提取总RNA,RT-PCR获得ERβ基因,将其联入pEGFP载体,得到重组质粒pEGFP-ERβ,再将上述质粒的GFP-ERβ片段亚克隆于pTAT-2.1质粒,形成pTAT-GFP-ERβ重组质粒.在BL21工程菌中IPTG诱导表达TAT-GFP-ERβ融合蛋白.用Western Blot鉴定纯化的融合蛋白的免疫原性.用纯化的蛋白转导事先转染ERE-Luc报道质粒的Hela细胞,用Luciferase Assay鉴定转导蛋白的生物学活性.证明TAT-GFP-ERβ蛋白具有完整的生物活性.

Use RT-PCR to get ERβ fragment from prostate tissue and ioin it to pEGFP vector. Use restriction enzyme to cut GFP-ERβ fragment from pEGFP-ERβ plasmid, join the fragment to pTAT2.1 plasmid, then express the combinational plasmid in BL21 E. coli bacteria with IPTG inducement, Use the western blot to prove the antigenicity of TAT-GFP-ERβ protein, transduce purified TAT-GFP-ERβ protein into Hela cells with ERE-Luc reporter plasmid, use luciferase Assay to exam transduce protein bio-activity. The result show the TAT- GFP-ERβ protein has completely bioactivity.