Screening for CHO cell line stably expressing inducible costimulator protein and its biological activity / 第二军医大学学报

ABSTRACT

Objective:To construct a recombinant retroviral vector carrying human inducible costimulator (ICOS) gene,screen for CHO cell line stably expressing ICOS protein and to study its biological activity.Methods: ICOS cDNA was obtained from human peripheral blood mononuclear cells (PBMC) through RT-PCR and was cloned into retroviral vector to construct retroviral recombinant pMSCV-ICOS; the latter was then packed and the high-titer virus producing cells were screened.Then CHO cell was infected by this high-titer virus and the stable cell line was screened.CHO-ICOS cells were co-cultured with PBMC (the ratio of CHO-ICOS to PBMC being 11,12,15, and 110) in presence of substimulating dose of anti-human CD3 antibody.The proliferation of PBMC and the CD25 expression on T cells were examined by 3H-TdR incorporation method and flow cytometry,respectively.CHO-pMSCV cells co-cultured with PBMC (11) served as the negative control and PBMC served as blank control.Results: We successfully constructed the retroviral recombinant pMSCV-ICOS and obtained CHO cell line stably expressing ICOS protein.3H-TdR incorporation method and flow cytometry showed that,compared with the negative control group and the blank control group,co-culture with CHO-ICOS cells significantly inhibited the anti-CD3 antibody-induced activation and proliferation of PBMC(P