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《Sichuan Journal of Physiological Sciences》 2007-01
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Construction and identification of yeast bait plasmids carrying α-defensisns HNP-1 mature peptide cDNA fragment

Deng Lu-xia1,Peng Yuan-yuan1,Luo Lin2,Feng Yun1,Wu Qi1,Wang Bo-yao1,Huang Ning1(1.Research Unit of Infection and Immunity,West China School of Preclinical and Forensic Medicine,Sichuan University;2.Department of Anesthesia,The First Affiliated Hospital of Huaxi,Chengdu 610041)  
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 plasmid pGBKT7-HNP-1,then it was identified by DNA sequencing.The toxic action and self-activating effect of pGBKT7-HNP-1 was tested by aminoacid 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.
【Fund】: CMB基金(98-681);; 自然科学基金(30470763 30671963)资助
【CateGory Index】: R392
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