Construct And Identify The Yeast Two-Hybrid System Of α-defensin HNP-3 Mature Peptide

Objective To construct the yeast two-hybrid system of HNP-3 mature peptide for studying the interaction protein molecule of HNP-3 with lung tissue.Methods The cDNA fragments encoding HNP-3 mature peptide wereamplified by RT-PCR from the extracted total RNA in cultured HL-60 cells. The fragment was cloned into pBluescript-SK-II vector. After verified with restriction endonuclease digestion of EcoR I and Sal I, the vector was inserted into the "bait plasmid " pGBKT7. After confirmation with restricted endonuclease digestion and sequence analysis, the plasmid was transformed into the yeast cell AH109, and its toxicity and transcriptional activation was tested both by auxotrophic selective culture. The mRNA was isolated from total RNA of lung tissue by magnetic bead, and the cDNA library was construacted by SMART technique, and the library capacity was identified through assaying coefficient of performance. The pGBKT7-HNP-3, ds cDNA and library plasmid pGADT7/Rec" were inserted into the yeast cell AH109 in turn. The positive clones was screened byβ-galactosidase activity and auxotrophic selective culture method.Results DNA sequencing indicated that the inserted fragment in pBluescript-SK-II vector was HNP-3 mature peptide gene sequence, and the subcloned recombinant pGBKT7-HNP-3 was match. The recombinant bait plasmid did not have self-activating effect and did not show toxicity to yeast AH109 cell.The capacity of the cDNA library was consistent with the standard of the libraryconstruction. About one hundred and ninety candidate posistive clones wereobtained by double hybrid screening.Conclusion The recombinant bait plasmid of HNP-3 mature peptide and lungtissue cDNA library was constructed successfully. The candidated clone screenedby yeast two hybried was helpful for investigating the proteins molecule in lungtissue interacting with HNP-3 mature peptide.