小鼠canstatin C端片段基因的克隆及其在大肠杆菌中的表达

为了构建小鼠canstatinC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canstatinC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canstatinC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canstatinC端片段氨基酸的同源性为61％。IPTG诱导mCan-C在大肠杆菌E．coliBL21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canstatinC端片段的cDNA,IPTG诱导mCan-C在大肠杆菌E．coliBL21中高效表达。小鼠canstatinC端片段的cDNA序列已收入GenBank,接受号为:AY502947。

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

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.