用改进的寡核苷酸诱导突变法改造人胰岛素前体基因

Construction of Human Proinsulin Gene Mutant by Improved Oligonucleotide Directed Mutagenesis

我们用改进了的寡聚核苷酸诱导突变法,将两个单一限制性内切酶位点引入人胰岛素前体B链与C链连接处附近,及C链与A铁连接处附近。用含U模板法将B链第30个残基密码子ACC改成ACG,从而引入MluⅠ位点。用修改了的缺口双链DNA突变法,将A链第4个残基密码于GAG改成CTG,引入了一个PstⅠ位点。突变效率的为17％-36％。这个突变体在人胰岛素前体结构及蛋白质折叠的研究中,将有利于更换不同的C-肽。

Two restriction endonuclease cleavage sites near the junction of B chain and C chain and the junction of C chain and A chain on the human proinsulin gene were created by improved oligonucleotide-directed mutagenesis. With the uracil template Oligonucleotide directed mutagenesis, we converted the codon of the 30th residue in the B chain from ACC into ACG to create a Mlu I site. By the modified gapped duplex DNA mutagenesis, we changed the condon of the 4th. residue in the A chain from GAG to CTG to create a Pst I site. The efficiency of mutant production was in the range of 17%-36%. This mutant would benefit the changing of C-peptide in the studies of proinsulin structure and protein foldings .