Construction of eukaryotic expression vector of a novel recombinant protein B7-2-PE40KDEL having immunological suppression effect

Objective:To construct an eukaryotic expression vector containing fusion protein B7-2-PE40KDEL which can be expressed in vivo to selectively kill CD28 highly expressing cells and induce immune tolerance.Method:The cDNA of B7-2-PE40KDEL was amplified by PCR from the plasmid pRSETA-B7-2-PE40KDEL and the eukaryotic expression plasmid pcDNA3.1/Zeo(+)-B7-2-PE40KDEL was constructed by inserting the B7-2-PE40KDEL cDNA into the vector pcDNA3.1/Zeo(+).The recombination plasmid was transfected into RPE-CHO cells and the expression of the fusion protein was identified by RT-PCR,Western blot and ELISA.Its biological feature of cytotoxity was measured by MTT.Results:The relative molecular mass of the fusion protein was about 71×103.Over 0.23μg/L of B7-2-PE40KDEL fusion protein was produced per 106 transfected cells in 24h.The cell killing effect was significant on Jurkat cells which expressed high level CD28 receptor and only a small proportion of CD28-negative Raji cells was killed.Conclusion:In vivo production of B7-2-PE40KDEL by mammalian cells is feasible and has potential therapeutic application.