Ad5-knob蛋白在大肠杆菌中可溶性表达及其活性检测

采用PCR技术, 从AdEasy 1质粒DNA中获得knob全长序列, 克隆入pGEM T vector中。DNA测序鉴定后, 构建pQE 30/knob重组表达载体, 转化大肠杆菌M15 pREP4),IPTG诱导表达腺病毒5 型纤维蛋白的knob功能域(Ad5 knob), SDS PAGE分析,表达产物主要以可溶性蛋白的形式存在于细菌裂解液上清之中。经Ni2 NTA亲和层析一步分离纯化后,洗脱产物中Ad5 knob蛋白纯度超过95%。N 端氨基酸测序证实了纯化产物为Ad5 knob蛋白,细胞受体结合实验检测到所得蛋白能够与Hela细胞上的相应受体特异性结合。上述结果表明在大肠杆菌中已经高效表达了可溶性Ad5 knob蛋白,一步即纯化该蛋白,并具有良好的生物活性,为其下阶段的深入研究提供了重要的实验材料。

Soluble Expression, Purification and Characterization of Ad5-knob Protein

Ad5-knob gene from AdEasy plasmind DNA was amplified by polymerase chain reaction (PCR) and cloned into an E. coli expression vector pQE30 digested with BamH I and Hind III. After induction with IPTG, the transformed E. coli strain M15 expressed Ad5-knob gene efficiently. Moreover, the recombinant Ad5-knob protein mainly existed as a soluble protein. After one step purification with Ni~(2 )-NTA affinity chromatography, the protein was purified to nearly homogeneity. N-terminal amino acid sequencing indicated N-terminus of purified Ad5-knob. When FITC-labeled Ad5-knob transfers Hela cells, it can specially bind to the receptor to the surface of Hela cells and enter the cytosol. It suggests that the recombinant Ad5-knob protein identified and purified is to be suitable for nonviral gene carriers as a target protein in future studies.