用于酵母双杂交的α-防御素HNP-1成熟肽重组诱饵载体的构建和鉴定

Construction and identification of yeast bait plasmids carrying α-defensisns HNP-1 mature peptide cDNA fragment

目的:构建和转化人α-防御素HNP-1成熟肽的酵母诱饵重组质粒,为通过酵母双杂交研究其相互作用蛋白分子打下基础。方法:用RT-PCR扩增HNP-1成熟肽基因,将该基因片断与腺病毒载体pBluescript-SK-II定向重组;用酶切和测序鉴定重组质粒;回收目的片断,将其连接入酵母表达载体pGBKT7,构建重组诱饵表达质粒pGBKT7-HNP-1,测序鉴定。缺陷培养方法检测重组诱饵载体有无毒性和自激活作用。结果序列分析表明HNP-1成熟肽重组诱饵表达质粒构建成功,重组诱饵表达质粒对酵母细胞无毒性作用,无自激活效应发生。结论构建用于酵母双杂交的α-防御素HNP-1成熟肽重组诱饵载体,为找出与人α-防御素HNP-1成熟肽相互作用蛋白分子打下了基础。

Objective： To construct and transform yeast bait plasmid carrying α-defensisns HNP-1 mature peptide cDNA fragment and provide a basis for researching its interactive proteins molecules. Methods.. The cDNA sequence of the HNP-1 mature peptide gene was amplified by RT-PCR. The amplification product was directly ligated to vector pBluescript-SK-Ⅱ. Insert-contained plasmid Was confirmed by restriction endonuclease analysis and DNA sequencing. Then HNP-1 mature peptide gene wa Reclaimed and cloned into bait expression vector pGBKT7 and constructed the recombinant bait ptasmid pGBKT7-HNP-1, then it was identified by DNA sequencing The toxic action and self-activating effect of pGI3KT7-HNP-1 was tested by aminoacld dropout nutrient culture analysis. Results：The DNA sequencing analysis showed that the HNP-1 mature peptide gene was cloned into bait plasmid pGBKT7 correctly. It did not show toxicity and autonomous activation to yeast AH109 cells. Conclusions the construction of the recombined plasmid Was capable to be used as the fusion bait plasmid in yeast two-hybrid system Ⅲ, it might be a base to find the interactive proteins of HNP- 1 mature peptide.