人表皮生长因子基因化学及酶促合成与克隆

作者利用DNA自动合成仪及酶促反应合成了全长175bp的人表皮生长因子基因,由计算机辅助进行基因序列、化学合成片段和酶切位点的设计,片段退火预测以及重复顺序和发夹结构的搜寻,所设计的基因具有多种克隆及表达途径。全基因先由DNA合成仪合成8个片段,经5端磷酸化及退火,再由DNA聚合酶合成4个缺口片段,用连接酶连接而形成,将基因插入pUC12质粒,转化JM103大肠杆菌,20ng的连接质粒转化后,在含X-gal及氨苄青霉索的营养琼脂平板上获得约200个白色克隆。随机选择7个克隆提取质粒作限制性内切酶鉴定,4个符合实验设计,取1个克隆作插入基因的DNA序列分析,结果与设计完全相符。

Chemical and enzymatic synthesis and cloning of human epidermal growth factor gene

Human cpidermal growth factor gene was synthcsicd by DNA synthesizer and cnzymes. Computer assisted to design the gene sequence , and restrictases , and to detect the repeated and self-complementary sequence .The designed gene sequence has a virablc cloning and expressiveways . The whole gene was formed by annealing the chemical synthesizing fragments, filling the 4 spaces by DNA polymerase and ligating the nicksby ligase , and was cloned into plasmid pUC12 .The recombinant was transformed into E. coli JM103 and screened by X-gal and ampicillin, hence 200 while clones was obtained . The possive clone was isolated and characterized by restrictases and DNA scquencing .