摘要
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。
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 .
出处
《第四军医大学学报》
1991年第3期200-202,共3页
Journal of the Fourth Military Medical University
关键词
表皮生长因子
重组
DNA
Epidermal growth factor, dcoxyribonucletc acid reconbinant
gene synthesis
