人GnRH及其转运肽基因合成及鉴定

Synthesis and Identification of Gene Encoding Human GnRH and Its Transporter

目的人工合成人GnRH及转运肽(TRS)基因。方法根据已发表的人GnRH基因mRNA序列以及转运肽(9个左旋精氨酸)基因核苷酸序列,设计一对核苷酸,再用含7mol/L尿素的变性聚丙烯酰胺凝胶电泳纯化,然后以这对核苷酸3′末端短互补序列退火、补齐,合成长达90bp的目的序列GnRH/TRS。将其亚克隆到pMD18T载体上,构建重组质粒pYC1。酶切鉴定筛选出阳性克隆pYC1,并测序。结果GnRH/TRS序列由90个核苷酸组成,扩增产物与原设计序列同源性达100%。结论已成功合成人GnRH/TRS基因,为进一步研究和应用奠定基础。

Objective To synthesize the gene encoding human GnRH and its transporter（TRS）. Methods Design a pair of nueleotide sequences according to the reported gene encoding human GnRH and its transporter and purify by PAGE with denaturing polyaerylamide gel containing 7 mol/L urea. Synthesize a 90 bp target sequence GnRH/TRS by filling-in of a short complementary sequence at 3＇-terminus of this pair of nucleotide sequences. Subclone the synthetic sequence to pMD18-T vector to construct recombinant plasmid pYC1. Identify the positive clones by restriction analysis and sequencing. Results The synthetic GnRH/TRS sequence consisted 90 nueleotides and showed a homology of 100% to that designed. Coneluslon Human GnRH/TRS gene was successfully synthesized. It laid a foundation of further study and application of GnRH/TRS.