| 摘要: |
| 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ICOS cDNA，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(11、12、15、110)共培养，加入亚刺激剂量(200 ng/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 11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11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。 |
| 关键词: 可诱导共刺激分子 重组逆转录病毒载体 转染 |
| DOI：10.3724/SP.J.1008.2008.00254 |
| 投稿时间：2007-10-09修订日期：2008-01-22 |
| 基金项目:国家高技术研究发展计划(“863”计划) (2002AA214091). |
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| Screening for CHO cell line stably expressing inducible costimulator protein and its biological activity |
| DING Qing-li,LIU Meng-lei,LONG Xian-lian,SHEN Qian* |
| (Department of Laboratory Medicine，Changhai Hospital，Second Military Medical University，Shanghai 200433,China) |
| 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. |
| Key words: inducible costimulator recombinant retroviral vector transfection |
