[PD-1 overexpression in HEK293T cells transfected with pMH3-PD1].

Objective To optimize the transfection conditions of HEK293T cells and compare the expression levels of human programmed death 1 (hPD-1) in different eukaryotic vectors to obtain the target protein efficiently. Methods L9(33) orthogonal test was designed to optimize the conditions of cell transfection.The DNA sequence of hPD-1 extracellular domain gene was amplified by PCR and then cloned into different vectors:pcDNA3.1, pCMV3 and pMH3, PD-1 recombinant protein was expressed in HEK293T cells transiently and the expression levels was evaluated by ELISA and Western blot analysis. Results The gene of hPD-1 extracellular domain was successfully cloned into pcDNA3.1 and pMH3 eukaryotic expression vectors, and the target protein was successfully expressed. Under the optimal transfection conditions, the expression level of hPD-1 recombinant protein in pMH3 vector was the highest, followed by that of pCMV3 and pcDNA3.1 vectors. Conclusion The extracellular domain of hPD-1 is successfully expressed and the expression level of pMH3-PD1 was the highest among the three vectors tested.