重组人IL12在CHO细胞中高效表达克隆筛选及产物生物学活性.doc

重组人IL12在CHO细胞中高效表达克隆筛选及产物生物学活性 　 作者：吕锐, 张文卿*, 于红, 李丹 【摘要】 　　目的: 重组人白细胞介素12(rhIL12)真核表达质粒(pcDNA6/v5hisp70)转染CHO细胞, 筛选高效稳定表达克隆; 并对表达产物进行生物学活性分析。方法: 采用聚乙烯亚胺(PEI)将pcDNA6/v5hisp70转入CHO细胞; 用杀稻瘟菌素(Blasticidin)筛选阳性表达克隆, 并进行单克隆扩增; RTPCR进行表达鉴定; ELISA检测各克隆rhIL12的表达量; 应用淋巴细胞增生实验及细胞内细胞因子染色方法分析rhIL12的生物学活性。同时, 对筛选出的阳性克隆进行表达稳定性观察。结果: RTPCR结果显示, 选取的20个阳性克隆均可扩增出1 800 bp的特异性片段; ELISA 表明, 其中6个克隆rhIL12表达水平较高(312.69～719.10 ng/L), 最高表达量达到719 ng/L (5×104个细胞, 48 h); 生物学活性分析表明, 表达的rhIL12可明显提高献血员外周血PBMC对 PHA/HCMV反应的CD4/IFNγ及CD8/ IFNγ双标记阳性细胞克隆的频数; 并且, 可明显提高献血员外周血PBMC对PHA/HCMV刺激的增生反应效能。阳性克隆在维持量的筛选药物(2 ng/L Blasticidin)压力下传代6个月, 其表达水平无明显变化。 结论: rhIL12真核表达载体(pcDNA6/v5hisp70)能在CHO细胞中高效稳定表达; 其表达产物具有良好的生物学活性。 【关键词】 白细胞介素 12 中国仓鼠卵巢细胞; 基因表达 　　［Abstract］ AIM: To transfect the constructed recombinant human IL12 (rhIL12) eukaryotic expression plasmid (pcDNA6/v5hisp70) into CHO cells and screen the efficiently and stably expressed clones; and to identify the biological activities of rhIL12. METHODS: pcDNA6/v5hisp70 was transfected into CHO cells using polyethyleneimine (PEI) and positive clones were screened with Blasticidin as well as single clone amplification. Then the expression clone was identified by RTPCR and the rhIL12 expression of each clone was measured using ELISA. Finally, biological activities of the expressed rhIL12 were analyzed with proliferation of lymphocytes in vitro and intracellular cytokine staining(ICS) . The stability of the selected clones was observed as well. RESULTS: The RTPCR results showed that a specific fragment of 1 800 bp could be amplified in all positive clones, among which six clones had rather high expression of 312.69 ng/L-719.10 ng/L with the highest expression of 719 ng/L (5×104 per cells, 48 h) by ELISA test. Biological activities analysis indicated that the expressed rhIL12 could significantly increase the percentage of CD4+IFNγ+ and CD8+ IFNγ+ cells in normal PBMC as well as the proliferation of PBMC to PHA/HCMV stimu