大肠杆菌表达质粒pSM43及pSM53的构建

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。

Construction of Plasmids pSM43 and pSM53 for Expression Genes in Escherichia coli

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.