摘要
利用已成功高表达era基因的质粒pCE31翻译起始码上游的序列,去构建大肠杆菌新的外源基因表达载体。先合成特定序列的单链脱氧寡核苷酸,以改进的实验程序插入pJL6,其后再加上限制酶多克隆位点。所构建的pSM43和pSM53分别适合於不带翻译起始码(ATG)和带起始码的基因插入、表达非融合目的蛋白质之用。并已成功用於人肿瘤坏死因子、人骨形成蛋白、HIV蛋白酶、Duchenne肌营养不良等cDNA基因的高表达。
The upstream sequence of the translational initiation codon of era in pCE31, which successfully highly expressed era gene, has been used to construct plasmid for expression of exogenous genes in E.coli. Single strand oligodeoxynucleotide was synthesized and inserted into plasmid pLJ6 with improved protocol followed adding in with multiple restriction enzyme site. The resulting plasmids, pSM43 and pSM53 are suitable for expression of unfused protein encoded by gene with or without translational initiation codon repectively, and have been successfully used in high expression of human tumor necrosis factor, human bone morphogenetic factor, HIV protease, Duchenne muscule dystrophy and other cDNA genes.
关键词
表达载体
构建
翻译起始序列
质粒
Construction of expression vector
pSM43
pSM53
Translational initiation sequence