稳定表达可诱导共刺激分子CHO细胞株的筛选及其生物学活性鉴定

Screening for CHO cell line stably expressing inducible costimulator protein and its biological activity

目的:构建携带人可诱导共刺激分子(inducible costimulator molecule,ICOS)基因的重组逆转录病毒载体,筛选稳定表达ICOS蛋白的CHO细胞株,并对其生物学活性进行鉴定。方法:RT-PCR法从人PBMC中获取ICOScDNA,定向克隆到逆转录病毒载体上,制备逆转录病毒重组体pMSCV-ICOS;经病毒包装细胞包装后抗性筛选出产高滴度病毒的细胞株,用高滴度病毒上清感染CHO细胞,抗性筛选出稳定表达细胞株。将CHO-ICOS细胞和PBMC以不同比例(1:1、1:2、1:5、1:10)共培养,加入亚刺激剂量(200ng/ml)的抗人CD3抗体,采用3H-TdR掺入法检测PBMC的增殖,流式细胞仪检测T细胞表面活化分子CD25表达情况,并以CHO-pMSCV细胞和PBMC以1:1比例共培养作为阴性对照以及未处理的PBMC作为空白对照。结果:成功构建逆转录病毒重组体pMSCV-ICOS,筛选出稳定表达ICOS蛋白的CHO细胞株。3H-TdR掺入法和流式细胞仪检测结果表明,与阴性对照及空白对照相比,CHO-ICOS细胞与PBMC共培养能抑制CD3抗体诱导的PBMC增殖及活化(P<0.05或P<0.01),细胞比例为1:1时抑制率最大,分别为(68±5.9)%、(44.08±3.26)%。结论:成功建立具有生物学活性的膜稳定表达人ICOS蛋白的CHO细胞株,为今后研究奠定了基础。

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 anti- body. The proliferation of PBMC and the CD25 expression on T cells were examined by 3 H-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. 3 H-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 activa- tion 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 1 ： 1 ,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.