TAT-NEP1-40融合蛋白的表达、纯化及蛋白转导功能的鉴定

为大量获得高纯度具有蛋白转导功能并可以穿过血脑屏障的融合蛋白TAT—NEP1-40，将构建好的表达载体pTAT—NEP1—40转化大肠杆菌BL21（DE3）进行表达后。将融合蛋白进行纯化和复性，鉴定蛋白转导功能。带有重组质粒pTAT—NEP40的大肠杆菌经IPTG诱导后，主要以包涵体形式表达，融合蛋白的分子量大小为14kD，其表达量占菌体总蛋白量的31．59％，纯化后目的蛋白纯度达95．6％，通过免疫组化染色显示融合蛋白可以穿过血脑屏障进入脑组织。上述结果表明利用pTAT—NEP1-40重组质粒，可以成功地表达TAT—NEP1—40；纯化后该蛋白具有穿过血脑屏障的功能，为NEP1-40功能的进一步研究提供了基础。

Expression, Purification and Transduction Activity Identification of TAT-NEP1-40

To get sufficient quantity of pure biologically TAT-NEP1-40 and verify its penetrability across the blood braid barrier(BBB), the pTAT-NEP1-40 plasmid was transformed and expressed in the host cells E. coli BL21(DE3), the recombinant protein was identified by SDS-PAGE and purified by Ni-NTA . TAT-NEP1-40 was observed in the brain with immunofluorescence at 6 h after injection i.p. into rats. SDS-PAGE analysis suggested the bacteria contained a 14 kD protein expressed in inclusion body when induced by IPTG. The protein could be purified up to 95.0% and conld cross BBB into brain parenchyma. This research provided groundwork for the further studies of NEP1-40 function.