Construct And Identify The Yeast Two-Hybrid System Of α-defensin HNP-1 Mature Peptide

Objective To construct and identify the yeast two-hybrid systeme of a-defensin HNP-1 mature peptide, in an attempt to find the interaction molecule of HNP-1 whith lung epthlieal cells.Method The cDNA fragment encoding HNP-1 mature peptide was amplified by RT-PCR from the extracted total RNA in cultured HL-60 cells. Firstly, The fragment was cloned into pBluescript-SK-II vector, then subcloned into the " bait plasmid" pGBKT7(named as pGBKT7-HNP-1). After identificated with restricted endonu- clease digestion and sequencing, the bait plasmid was transformed into the yeast cell AH109 by LiAc method. The automatic transcriptional activation and toxicity effect of the transformants were tested in dropout nutrient culture medium SD/Trp^-, SD/Ade^-/Trp^-, SD/His^-/Trp^-, SD/Leu^-/Trp^- or SD/Trp^- respectively. The mRNA was isolated from total RNA of A549 cell by magnetic bead, and then the A549 cell cDNA library was constructed by SMART technique. Through LD-PCR to obtain ds cDNA,then it was transformed into Y187 to inform A549 cell library.The library capcity was identified through assaying coefficient of performance. The both pGBKT7—HNP-1,A549 ds cDNA and library plasmid pGART7/Rec¹ were introduced into yeast AH109 cells in turn. The positive clones was screened byβ-galactosidase activity and amino acid dropout nutrient culture analysis, and further identified by colony PCR. Reasult the DNA secquence analysis showed the insert in the recombinant plasmid pBluescript-SK-II—HNP-1 and the recombinant bait plasmid pGBKT7—HNP-1 were correct.The yeast AH109 transformed recombinant bait plasmid could grow well on SD/Trp^- plate and none could survive on SD/Ade^-/Trp^-, SD/His^-/Trp^-, SD/Leu^-/Trp^-plate. After being cultered in SD/Trp^- liquid medium for 16 hours,the OD₆₀₀ could live up to requisition in yeast system.The dropout nutritient culture confirmed that recombinant bait plasmid had no automatic transcriptional activation and toxicity effects to yeast AH109. The capacity of the A549 cDNA library was 1.7×10⁶ transfomants/3μg pGADT7/Rec^-. About four hundred candidate positive clones were obtained by double hybrid screening. The fragments of some positive clones were between 100 to 1000bp.Conclusion The recombinant bait plasmid pGBKT7—HNP-1 and A549 cDNA library was constructed succefully. It laid a basis on going yeast screen to obtain positive clone and further study of the mature peptide HNP-1 protein.