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 11,12,15, and 110) 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 (11) 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<0.05);the maximal inhibitory rates of activation and proliferation were obtained when the ratio of CHO-ICOS to PBMC was 11, being (68±5.9)% and (44.08±3.26)%, respectively. Conclusion: CHO cell line stably expressing ICOS protein has been successfully established, which lays a foundation for future study.