Expression and bioactivity of the fusion gene between extracellular portion of human ICOS and Fc portion of mouse IgG 2a
- VernacularTitle:可诱导共刺激分子和小鼠免疫球蛋白融合基因的分泌表达及生物学活性
- Author:
Jian WANG
;
Jun ZHANG
;
Qian SHEN
- Publication Type:Journal Article
- Keywords:
Inducible costimulator;
pSecTag2/Hygro A;
Fusion protein;
Eukaryotic expression
- From:
Chinese Journal of Immunology
1985;0(05):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To produce a fusion protein(ICOS-Ig)between extracellular portion of human ICOS and Fc portion of mouse IgG 2a and study on its biological activity.Methods:cDNA encoding the extracellular domain of human ICOS was prepared by RT-PCR from the RNA of the stimulated human peripherial blood mononuclear cells. The Fc portion of mouse IgG2a was cloned by PCR from the vector that contains the sequence-encoding Fc portion of mouse IgG2a.Two resulting amplified PCR products were ligated into a secrection mammalian expression vector, pSecTag2/Hygro A. The recombined vector was transfected into CHO cells by lipofectamine2000.Results:RT-PCR demonstrated the integration and mRNA synthesis of fusion gene. ELISA and Western blot analysis showed the expression of ICOS-Ig and its molecular weight was between 43-66 kD, its concentration was 5-25 ?g/ml. FACS analysis assured that ICOS-Ig had ligand specific binding activity. Addition of ICOS-Ig to MLR resulted in inhibition of T-cell proliferation and IL-2,IFN-? secretion.Conclusion:The fused gene ICOS-Ig was constructed and expressed successfully. It had the bioactivity of inhibition of T cell proliferation and cytokine secretion.
