Previous Articles Next Articles
ZHANG Wei,CHEN Pinlin,LEI Ming,CHENG Ying,CHEN Huan,CAO Xianying,DU Li,ZHANG Donglin,LIU Tao,XU Shiying,FU Yunnan,QI Chao,WANG Fengyang
Online:
Published:
张巍, 陈品林, 雷明, 成鹰, 陈欢, 曹献英, 杜丽, 张冬琳, 刘涛, 许世英, 符运南, 祁超, 王凤阳
Abstract: 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.
Key words: CDC42, Cloning, Expression, Hainan Eld’s Deer (Cervus eldi hainanus), Purification
摘要: 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。
关键词: 海南坡鹿, CDC42, 克隆, 原核表达, 纯化
ZHANG Wei,CHEN Pinlin,LEI Ming,CHENG Ying,CHEN Huan,CAO Xianying,DU Li,ZHANG Donglin,LIU Tao,XU Shiying,FU Yunnan,QI Chao,WANG Fengyang. Cloning and prokaryotic expression and purification of CDC42 cDNA from Hainan Eld’s Deer[J]. .
张巍, 陈品林, 雷明, 成鹰, 陈欢, 曹献英, 杜丽, 张冬琳, 刘涛, 许世英, 符运南, 祁超, 王凤阳. 海南坡鹿CDC42 cDNA 的克隆、原核表达及纯化[J]. .
0 / / Recommend
Add to citation manager EndNote|Ris|BibTeX
URL: http://www.mammal.cn/EN/
http://www.mammal.cn/EN/Y2011/V31/I1/97