海南坡鹿CDC42 cDNA的克隆、原核表达及纯化

Cloning and prokaryotic expression and purification of CDC42 cDNA from Hainan Eld's Deer

应用RT-PCR方法对海南坡鹿细胞分裂周期蛋白42(Cell division cycle 42,CDC42)编码区进行扩增,将扩增产物与PET-42a载体连接,重组质粒鉴定正确后,进行生物信息学分析;构建pET42a-hdCDC42表达载体,经IPTG诱导表达,将表达产物进行SDS-PAGE、可溶性分析、纯化及Western blot分析。结果表明,海南坡鹿CDC42含有1个576 bp的开放阅读框,编码191个氨基酸。经IPTG诱导表达后,得到一个带有His-tag和GST的约54 kD的融合蛋白,用抗His单克隆抗体进行Western blot,得到一条约54 kD的特异性抗体结合带,表明海南坡鹿CDC42原核表达载体成功构建并表达。

Cell division cycle 42（CDC42） cDNA of Hainan Eld＇s Deer（Cervus eldi hainanus）was amplified by RT-PCR,subcloned into a pET42a vector and transformed into E.coli host cells.Protein expression was induced by IPTG and analyzed by SDS-PAGE and Western blot.The results showed that the CDC42 cDNA from Hainan Eld＇s Deer（hdCDC42）contained a 576 bp ORF encoding 191 amino acids.A 54 kD fusion protein with a Histag was induced by IPTG and confirmed by Western blot using anti His-tag monoclonal antibody.The results indicate that prokaryotic expression vector of CDC42 from Hainan Eld＇s Deer was constructed and expressed successfully.