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目的 克隆人IL-18结合蛋白A（hIL-18 BPa）的 cDNA,观察其在COS-7中的表达。方法 采用RT-PCR自人白血病细胞株jurkat中获得hIL-18BPa的cDNA,将其克隆至T-vector中,经测序确证后,将该基因定向插入真核表达载体pcDNA3.1（+）中。脂质体介导法将其转染COS-7细胞,72h后收集上清纯化,用BCA法测定hIL-18 BPa的含量,用ELISA法测定其活性。结果获得的IL-18BPa的cDNA序列与gene bank登录的cDNA序列一致。上清液中hIL-18 BPa含量为1.4mg/L,并具有良好的生物学活性。结论 成功克隆hIL-18 BPa基因,并实现了在COS-7细胞中的瞬时表达,为研究其活性奠定了基础。
Objective To clone the cDNA of human IL-18 binding protein A (hIL-18 BPa) and observe its expression in COS-7. Methods The cDNA of hIL-18BPa was obtained from human leukemia cell line jurkat by RT-PCR and cloned into T-vector. After sequencing, the gene was inserted into eukaryotic expression vector pcDNA3.1 (+). The cells were transfected into COS-7 cells by liposome-mediated method. After 72 hours, the supernatants were collected and purified for purification. The content of hIL-18 BPa was determined by BCA method and its activity was determined by ELISA. Results The cDNA sequence of IL-18BPa obtained was identical to that of gene bank. The content of hIL-18 BPa in supernatant was 1.4 mg / L and had good biological activity. Conclusion The hIL-18 BPa gene was successfully cloned and transiently expressed in COS-7 cells, which laid the foundation for the study of its activity.