Construction of cDNA library from the induced human peripheral blood lymphocytes(Hu-pBL)

Abstract

Lymphocytes were isolated from human periperal blood by Ficollpaque and immune interferon was induced in isolated lymphocytes in culture by combined treatment with a phorbol ester (12-o-tetracanoylphorbol 13-acetate, TPA) and Concanavalin A. Total RNA was isolated from mononuclear cells induced with TPA and Con A, and passed through oligo-(dT)-cellulose column to obtain mRNA. Poly(A)-containing RNA was transcribed into cDNA by AMV reverse transcriptase under appropriate conditions which increase the yield of full length cDNA. After alkaline hydrolysis of mRNA, the second cDNA strands were synthesized with hairpin loop as a primer. The loop of the ds-cDNA was specifically cleaved under appropriate conditions. cDNAs larger than 700 bp were purified on a preparative agarose gel. After a tailing step with dCTP, cDNAs were annealed to plasmid DNA tailed with dGTP. Recombinant plasmid DNAs were transformed in $\underline{E}. \underline{coil}$ LE392.