Construction and identification of a recombinant expression vector encoding OX40-IgG1 fusion gene

Objective To construct a eukaryotic expression vector encoding human OX40-IgG1 fusion gene,express OX40Ig fusion protein with high biological activity,and make preparation for the gene therapy of induction of composite tissue allograft transplants immune tolerance.Methods The cDNA of extracellular domain of human OX40 gene and IgG1 Fc gene were synthesized and inserted into pUC57(+) vector,and then subcloned into eukaryotic expression vector pDC315(+) to construct the plasmid pDC315-OX40Ig.The constructed recombinant plasmid was identified by PCR,restriction enzymes and DNA sequence analysis.After these identifications,the recombinant plasmid was transfected into NIH/3T3 cells with Lipofectamine 2000.Expression and secretion of OX40Ig was confirmed by SDSPAGE and Western Blotting,and the inhibitory effect of OX40Ig on MLR in vitro was observed by MTT assay.Results A DNA fragment about 1320 bp was obtained.The OX40Ig gene sequence of pDC315-OX40Ig was consistent with GenBank.Restriction enzymes and PCR identification proved that the OX40Ig gene was correctly cloned into expression vector.SDS-PAGE and Western Blotting analysis showed that OX40Ig fusion protein was expressed in NIH/3T3 cells culture supernatant successfully.The relative molecular mass(Mr) of the expression protein was 48000,which wasaccorded with the predicted Mr value.It was confirmed that the OX40Ig protein can inhibit MLR in vitro.Conclusion The eukaryotic expression vector pDC315-OX40Ig was successfully constructed and OX40Ig fusion protein can inhibit lymphocytes proliferation.This study has laid the foundation for the intervention of composite tissue allograft rejective reaction.