Construction and screening of cDNA library of alveolar macrophages / 解放军医学杂志

ABSTRACT

Objective To construct cDNA library of alveolar macrophages after lipopolysaccharide (LPS) and dexamethasone (Dex) treatment for exploring the protein molecule interactive with glucocorticoid receptor (GR) in condition of inflammation. Methods After the cultured AMs were treated with LPS (10mg/ml) and Dex (10 -5 mol/L) for 4h, total RNA was extracted from AMs, then the cDNA was synthesized from total RNA of AMs and amplified using primers SMARTⅢ TM and CDSⅢoligo (dT) as the base of recombination. The purified PCR products as well as the linearized plasmid pGADT7-Rec were co-transformed into the competent yeast AH109. They were recombined by yeast homologous recombinase in the yeast cells and became the active cyclic plasmid. The transformed yeasts grew in the SD/-Leu plates. All the growing clones were harvested and then constituted the cDNA library. Furthermore, the bait pGBKT7-rGR transformed the yeast AH109 of library was constructed, and the protein molecule interactive with GR was screened. Results cDNA library of AMs was constructed with high multiplication and good capacity. 1.07?106 recombinants were obtained from the cDNA library. The amplified PCR fragments were between 0.3-1.5kb in size. One true positive clone, obtained by screening the cDNA library, was confirmed to be BAG-1 by sequencing and BLAST. Conclusion The yeast two-hybrid cDNA library of AMs was successfully constructed by Clontech SMART method; GR can interact with BAG-1 in yeast cells.