重叠片段法克隆全长人肝细胞生长因子cDNA

Cloning full-length cDNA of human hepatocyte growth factor by overlapping-fragments

通过重叠片段的ＲＴ－ＰＣＲ方法，从人胎盘组织克隆了人全长肝细胞生长因子ｃＤＮＡ片段（约２２００ｂｐ），经酶切证实和测序分析都表明该片段确为人ＨＧＦｃＤＮＡ。本文所用方法解决了扩增较长目的基因片段时，由于ＲＮＡ酶及反转录酶的ＲＮＡ酶Ｈ活性和ｍＲＮＡ二级结构等多种因素的影响。

To produce specific functional proteins,the molecular cloning and expression technology has been widely used.However,the first step of obtaining the target gene fragment is crucial and difficult,especially when the gene fragment is relatively long.Due to the influence of RNases,the secondary structure of mRNA and the RNases H activity of reverse transcriptase,it's rather difficult to generate long cDNA by RT PCR.Through synthesizing cDNA of hHGF by overlapping fragments,we have overcome such problems and cloned full length cDNA of hHGF successfully.