小鼠canstatin C端片段基因的克隆及其在大肠杆菌中的表达  

Cloning and Expression of Mouse Canstatin C-fragment cDNA in E. coli

作　　者：侯卫红[1] 田芳[1] 王建民[1] 王天云[1] 柴玉荣[1] 陈华艳[1] 薛乐勋[1] 

机构地区：[1]郑州大学医学院细胞生物学研究室,郑州450052

出　　处：《中国生物工程杂志》2005年第6期51-55,共5页China Biotechnology

基　　金：国家自然科学基金资助项目(30270031);河南省重大科技攻关资助项目(0122032500);河南省杰出人才创新基金资助项目(0221001900)

摘　　要：为了构建小鼠canstatin C端片段的原核表达载体并在大肠杆菌中表达。以小鼠肝脏组织总RNA为模板,通过RT-PCR扩增小鼠canstatin C端片段(mCan-C)基因,克隆到pMD18-T载体中并进行序列分析。将mCan-C基因定向克隆于原核表达载体pET30a(+)中,构建表达载体pET/ mCan-C,转化大肠杆菌BL21(DE3),IPTG诱导表达。结果表明,小鼠canstatin C端片段的cDNA 长度为399bp,含有1个终止密码,编码132个氨基酸,与已知的人canstatin C端片段氨基酸的同源性为61%。IPTG诱导mCan-C在大肠杆菌E.coli BL21中表达,表达量约占菌体总蛋白量的28%,重组蛋白主要以包涵体形式存在。首次克隆了小鼠canstatin C端片段的cDNA,IPTG诱导mCan-C在大肠杆菌E.coli BL21中高效表达。小鼠canstatin C端片段的cDNA序列已收入GenBank,接受号为:AY502947。To construct prokaryotic expression vector for C-fragment of mouse canstatin(mCan-C) and to express recombinant mCan-C in E . coli BL21, total RNA was extracted from mouse liver tissue by Trizol Reagent, and mouse canstatin C-fragment cDNA was amplified by RT- PCR, then cloned into vector pMD18-T for sequencing. PET/mCanC recombinant plasmid was constructed and expressed in E. coli BL21 with induction of IPTG. mCan-C cDNA was 396bp encoding 132 amino acids. The sequences of amino acid share 61 % homology with human canstatin C-fragment. mCan-C was expressed in E. coli BL21 with amount of 28 % of the total bacterial proteins after being induced by IPTG for 4h. The expressed products existed mainly as inclusion bodies. It is concluded that mouse canstatin C-fragment cDNA has been cloned and mCan-C is highly expressed in E. coli BL21.

关 键 词：CANSTATIN C端片段 基因克隆 大肠杆菌 原核表达 内源性血管生成抑制因子 

分 类 号：R331.3[医药卫生—人体生理学] Q78[医药卫生—基础医学]