人生长激素释放因子基因的合成和克隆

SYNTHESIS AND CLONING OF THE HUMAN GROWTH HORMONE RELEASING FACTOR GENE

本文报道人生长激素释放因子(Leu^(27),Met^(44))hGRF(1-44)基因的合成和克隆。选用大肠杆菌高效表达所偏爱的简并密码子,用计算机辅助设计合成基因的顺序,用固相亚磷酸酰胺法合成了hGRF的6个寡聚核苷酸片段,长度分别为39至51个核苷酸,总共141个碱基对。通过酶促连接反应构建完整的hGRF基因,并直接克隆到pUC-19质粒中,克隆的宿主菌为E.coli JM83。通过抗性筛选、阳性标记筛选、限制酶分析和分子杂交确定阳性克隆株。用M13双脱氧末端终止法对克隆基因序列分析,证实合成和克隆的hGRF基因序列完全正确。

The human growth hormone releasing factor(hGRF) gene has been synthesized and cloned. Computer programming was applied to the design of optimized segments for the synthesis of the hGRF gene using preferred codons for expression in E.coli. Six segments with length ranging from 39 to 51 nucleotides were synthesized by solid-phase phosphoramidite method. The entire gene consisting of 141 base pairs was constructed by enzymatic ligation of all synthetic segments and then cloned into plasmid vector pUC-19. The positive colonies were confirmed by screening of ampicillin resistance, inactive β-galactosidase, analysis by restriction enzymes and dot-blot hybridization. The DNA sequence of the cloned synthetic hGRF gene was proved correct by M13 dideoxynucleotide chain termination method.