密码子优化的牛精蛋白基因在大肠杆菌中的表达

以PCR的方法得到牛精蛋白基因的基因,去其内含子,得到牛精蛋白cDNA,克隆入原核表达载体pGEX-2T中,组装成表达载体pGEX-2T-PE,利用大肠杆菌偏好的编码精氨酸的密码子CGT将野生型牛精蛋白基因中编码精氨酸的稀有密码子（AGA或AGG）替换掉,通过基因合成得到密码子优化的牛精蛋白的基因,将其克隆入原核表达载体pGEX-2T中,组装成表达载体pGEX-2T-PS。将这两个表达载体分别转化入大肠杆菌表达菌株BL21中,经IPTG诱导,同样条件下,野生型牛精蛋白基因无法得到表达,密码子优化的牛精蛋白基因能够得到良好的表达,表达产物约占细菌总蛋白的18％,将表达蛋白纯化,进行DNA－蛋白结合实验,发现其能与DNA发生非特异性的结合。

Expression of Optimized-code Bull Protamine in E.coli.

Bull protamine genomic gene was cloned by PCR amplification. After deletion of its intron, the cDNA was cloned into pGEX-2T to construct expression vector pGEX-2T-PE. Optimized-code bull protamine gene was synthesized, the code (CGT) which E.coli. is partial to was substituted for the rare codes(AGA or AGG), the gene was cloned into pGEX-2T to construct expression vector pGEX-2T-PS. The two expression plasmids were transfered into E.coli BL21, induced by IPTG, wild bull protamine gene can not be expressed while optimized bull protamine can be highly expressed, the production was approximatly 18% of the total germ protein. Biochemical assay demonstrated that the purified protamine is capable of binding double stranded DNA.