人白细胞介素12在哺乳动物细胞中的稳定表达及其生物活性的研究

Expression of human IL-12 in mammalian cell and study on its biological activities

目的采用遗传基因工程方法获得具有生物活性的人白细胞介素12，探索其治疗肿瘤和慢性肝炎的可行性。方法采用已克隆的国人IL-12基因序列，利用内部核糖体切人位点（IRES）完成IL-12P35、P40双亚基共表达载体的构建，通过转染CHODHFR缺陷细胞，双抗夹心ELISA法筛选阳性克隆，MTX加压扩增，PCR检测其基因整合，T淋巴细胞增殖和诱生γ干扰素实验检测其生物活性。结果获得了稳定高效表达人IL-12的工程细胞系，表达产物为70×10^3左右的糖蛋白。结论本研究得到的基因重组人IL-12具有良好的生物活性，有强的诱导产生γ干扰素的能力和诱导活化T淋巴细胞增殖能力。

Objective To obtain recombinant human interleukin 12 by genetic engineering, and to explore possibility of its clinical application in treatment of tumor and chronic hepatitis. Methods Bicistronic expression vector P35-IRES-P40 was constructed for the simultaneous translation of IL-12 p35 and p40 cDNA subunit through internal ribosomal entry sites （IRES）. pCI-dhfr-P35-IRES-P40 vector was constructed for expression in CHO- DHFR- cells. Positively cloned cells were screened by means of ELISA. Pools of clones with increased expression of IL-12 could be generated by selection in methotrexate. To determine the biological activities of rhIL-12, PHA- activated lymphoblasts proliferation assay and IFN-γ induction assay were used in this study. Results Genetically engineered cells expressing hIl-12 were obtained and all the cell lines showed the stabile expression of rhIL-12 in high efiqciency and good growth properties. Conclusion rhlL-12 have good biological activities, it can stimulate activation and proliferation of T cells and induce production of IFN-γ.