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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1800bp的特异性片段;ELISA表明,其中6个克隆rhIL-12表达水平较高(312.69～719.10ng/L),最高表达量达到719ng/L(5×104个细胞,48h);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(2ng/LBlasticidin)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。
OBJECTIVE: To transfect CHO cells with rhIL-12 eukaryotic expression plasmid (pcDNA6 / v5-his-p70) and screen the clones efficiently and stably. The biological activity of the expressed product was analyzed. Methods: The pcDNA6 / v5-his-p70 was transfected into CHO cells by polyethylenimine (PEI). The positive clones were screened by Blasticidin and cloned by monoclonal antibody. The expression was confirmed by RT-PCR The expression of rhIL-12 in each clone was detected by ELISA. The biological activity of rhIL-12 was analyzed by lymphocyte proliferation assay and intracellular cytokine staining. At the same time, the positive clones were screened for expression stability. Results: The RT-PCR results showed that all the 20 positive clones amplified a 1800 bp specific fragment. ELISA showed that rhIL-12 was highly expressed in 6 of the 6 clones (312.69 ~ 719.10 ng / L), with the highest expression (5 × 104 cells, 48h). The biological activity analysis showed that the expression of rhIL-12 could significantly increase the CD4 / IFN-γ and CD8 / IFN- γ double labeling positive cell clones frequency; and, can significantly improve blood donors peripheral blood PBMC PHA / HCMV stimulated proliferative response. The positive clones were passaged for 6 months under the pressure of 2ng / LBlasticidin and the expression level did not change significantly. Conclusion: rhIL-12 eukaryotic expression vector (pcDNA6 / v5-his-p70) can be efficiently and stably expressed in CHO cells, and its expressed product has good biological activity.