Fusion protein expression of extracellular portion of human inducible costimulator and Fc portion of mouse IgG2a and its bioactivity in vitro / 第二军医大学学报

ABSTRACT

Objective:To study the bioactivity of inducible costimulator Ig(ICOS-Ig) as an inhibitor of ICOS-B7RP-1 costimulatory pathway in vitro. Methods: cDNA encoding the extracellular domain of human ICOS was prepared by RT-PCR from the RNA of the stimulated human peripheral blood mononuclear cells. The Fc portion of mouse IgG2a was cloned by PCR from the vector containing the sequence-encoding Fc portion of mouse IgG2a.The above 2 PCR products were ligated into a clone vector pGL-3-Basic. The fusion gene was then cloned and ligated into a mammalian expression vector pcDNA4/HisMAX A. The recombined vector was transfected into CHO cells by Lipofectamine2000 and the expression of the fusion protein was identified by Western blot. The mixture lymphoproliferation reaction(MLR) of the lymphocytes derived from BALB/c and C57BL mice was used to detect the fusion protein function in vitro. Results: Western blot analysis showed the expression of fusion protein, with the molecular weight being 43 000-66 000. FACS analysis assured that expression products had ligand specific binding activity. MLR was inhibited by the fusion protein. Conclusion: The constructed recombinant fusion protein has ligand specific binding activity and can inhibit the lymphoproliferation.