Construction of HNP-3 Mature Peptide Bait Plasmid of Yeast Two-hybrid System and Detecting Its Self-activating and Toxic Effect / 航天医学与医学工程

ABSTRACT

Objective To construct the bait plasmid of HNP-3 mature peptide in yeast two-hybrid system and examine whether the recombinant bait plasmid has self-activating and toxicity effect.Methods Using RT-PCR technique,the cDNA fragments of HNP-3 mature peptide gene were amplified from the extracted RNA in cultured HL-60 cells.The fragment was firstly cloned into pBluescript-SK-II vector,confirmed by sequencing,then sub-cloned into the bait plasmid pGBKT7 and identified with PCR and sequence analysis techniques.The recombinant plasmid was introduced into the yeast cell AH109,and its self-activating and toxicity effect was tested by auxotrophic selective culture.Results DNA sequencing indicated that the inserted fragment in pBluescript-SK-II vector was HNP-3 mature peptide gene sequence,and the sub-cloned recombinant pGBKT7-HNP-3 was no mismatch.The recombinant bait plasmid didn't have self-activating effect and did not show toxicity to yeast AH109 cell.Conclusion The bait plasmid of HNP-3 mature peptide was constructed successfully.This was helpful for investigating the proteins interacting with HNP-3 mature peptide by yeast two-hybrid technique.