Cloning of human interleukin-10 cDNA from in vitro-cultured peripheral blood mononuclear cells

Objective To clone human interleukin-10 cDNA from in vitro cultured peripheral blood mononuclear cells.Methods Peripheral blood mononuclear cells were separated from human peripheral venous blood with lymphocyte separation fluid and in vitro cultured for 24 h with RPMI 1640 containing concanavalin A.RNA was extracted from PBMCs,human interleukin-10 cDNA was amplified by RT-PCR and cloned into pUCm-T vector,and recombinant was transformed into competent cells DH5α.Miniprep DNA was prepared after blue-white color screening.PCR amplifying,restriction mapping and recombinant sequencing were performed.Results The 560 bp fragment amplified by RT-PCR was the same as the anticipated one in size.Miniprep DNA was prepared from randomly isolated white colony,the fragment obtained from PCR amplification,restriction mapping was the same as the anticipated one in size and sequencing showed its complete homology to hIL-10cDNA published in Genebank.Conclusion The cloning of human interleukin-10 cDNA from PBMCs and recombination with pUCm-T vector is feasible and relatively simple,which provides dependable prerequisite for further subcloning and gene expression study.