人IL-18结合蛋白A在CHO细胞中表达的研究

Study on expression of hIL-18BPa in CHO cells

目的 克隆人IL 18结合蛋白A (hIL 18BPa)的cDNA ,构建真核表达载体及其在CHO细胞中的表达。方法采用RT PCR ,自人白血病血细胞株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。以脂质体介导法 ,将其转染CHO细胞 ,用G4 18筛选抗性克隆 ,ELISA法测定其生物学活性。结果 获得的IL 18BPa的cDNA序列与GeneBank登录的cDNA序列一致。将hIL 18BPa插入载体pcDNA3.1后 ,成功地构建稳定表达的载体。转染CHO细胞后 ,获得可稳定表达hIL 18BPa的基因工程细胞株。经纯化后 ,证明具有良好的生物学活性。结论 成功地克隆hIL 18BPa基因 ,并实现了在CHO细胞中的稳定表达 。

Aim To clone hIL 18BPa cDNA, construct eukaryotic expression vector of hIL 18BPa,and then express it in CHO cells.Methods hIL 18BPa cDNA was obtained from Jurkat cells by using RT PCR and was cloned into PGEM T Easy vector. After the sequence was confirmed, the cDNA was inserted into vector pcDNA3.1 CHO cells were transfected with the recombinant vector via liposome. The CHO cells secreting hIL 18BPa protein in the culture superntant were obtained after selection by G418. Biological activity of positive clones was detec ted by ELISA. Results The sequence of hIL 18BPa cDNA obtained was identical with that published on GenBank. Mammalian recombinant expression vector was constructed. hIL 18BPa protein with biological activity was detected in the culture supernatant of the transfected cells. Conclusion hIL 18BPa with biological activity is expressed in CHO cells. The above results lay the foundation for further studying biological activity of hIL 18BPa protein.