人Delta-like4^(ext)-93-217原核表达、纯化及蛋白活性检测

Prokaryotic expression,purification and protein activity detection of human Delta-like4^(ext)-93-217

目的:原核表达、纯化融合蛋白TRX/hDll4ext-93-217,并对其活性进行检测.方法:采用PCR方法扩增hDll4ext-93-217多核苷酸序列,克隆入pMD18T载体.测序正确后,将其亚克隆入pET32a(+)原核表达载体,获得pET32a(+)-hDll4ext-93-217载体.以该载体转化E.coli菌株BL21,IPTG诱导其表达.以镍离子螯合柱纯化目的蛋白,采用SDS-PAGE,Western Blot方法鉴定目的蛋白的表达;采用萤光素酶报告基因系统检测目的蛋白活性.结果:通过PCR扩增获得了目的片段hDll4ext-93-217,成功构建了该蛋白的原核表达载体pET32a(+)-hDll4ext-93-217.采用SDS-PAGE,Western Blot等方法均可检测到目的可溶性融合蛋白TRX/hDll4ext-93-217的表达.融合蛋白TRX/hDll4ext-93-217在25℃,IPTG终浓度0.5mmol/L条件下诱导表达8h的蛋白表达量最高.以镍离子螯合柱纯化,获得纯化融合蛋白TRX/hDll4ext-93-217浓度为1.5g/L,其荧光素酶报告基因的刺激活性与空白对照组及TRX组相比较差异具有显著性(P=0.0154,P=0.0227,P<0.05).结论:通过原核表达获得的可溶性融合蛋白TRX/hDll4ext-93-217可活化Notch信号途径.

AIM： To express and purify the fusion protein TRX/ hDⅡ^ext -93-217 and to detect its protein activity. METHODS： The cDNA sequence of hDⅡ^ext-93-217 was amplified by PCR and cloned into the vector pMD18T. After sequencing, the correct fragment was cloned into prokaryotic expression plasmid pET32a （ ＋ ）. The target protein was expressed in E. coli BL21 induced by IPTG and the expression was detected by SDS-PAGE and Western blotting. The activity of purified target protein with NI-NTA was detected by luciferase report assay. RESULTS： The prokaryotic expression vector pET32a （ ＋ ）-hDⅡ^ext-93-217 was successfully constructed. The expression of the target protein, as a soluble fusion protein, was detected by SDS-PAGE and Western blotting, and the fusion purified protein of 1.5 g/L was obtained. The activity of the fusion protein TRX/hDⅡ^ext-93-217 to stimulate the reporter gene was significantly higher compared with control （P = 0. 0154 ） or TRX （P = 0. 0227） （ P 〈 0.05 ）. CONCLUSION ： A soluble fusion protein TRX/hDⅡ^ext-93-217 is successfully obtained, which can effectively activate the notch signaling pathway.