人血小板生成素基因的克隆及CHO稳定细胞系的建立

Cloning of Human Thrombopoietin Gene and Establishment of CHO Cell Lines Stably Expressing TPO Gene

以人肝cDNA为模板克隆了人血小板生成素(Human Thrombopoietin,hTPO)基因,利用基因重组技术构建了带有新霉素抗性基因(neo)筛选标记的pcDNA3.1(+)-hTPO真核表达载体,将重组质粒瞬时转染293T细胞,用鼠抗人TPO单抗Western blot检测TPO蛋白的瞬时表达;再将重组质粒转染中国仓鼠卵巢细胞(Chinese Hamster Ovary,CHO),应用400μg/mL的G418筛选克隆,经PCR及Western blot验证,获得了3株hTPO蛋白表达水平不同的CHO细胞系,为获得大量蛋白并进行活性功能试验及临床应用奠定基础。

Human thrombopoietin （Human Thrombopoietin, hTPO） gene was cloned by PCR using the human liver cDNA as template, then fragment of hTPO gene was cloned into pcDNA3. 1 （＋） eukaryotic expression vector which has the neomycin resistance gene （neo） selection marker, named as pcI）NA3. 1 （＋）-hTPO. First, the recombinant plasmid was transiently transfected into 293T cells. The transient expression of TPO in eukaryotic cells was determined by Western blot using mouse anti-human TPO mon oclonal antibody; then Chinese hamster ovary cells （Chinese hamster ovary, CHO） were transfected with pcDNA3.1 （＋）-hTPO recombinant plasmid, using G418 of 400 /,g/ml to select CHO stable cell lines, and verified by PCR and Western blot. The above results showed that three CHO cell lines stably express- ing TPO gene were established. The cell lines may get a large number of proteins applied to actual medical and activity functional experiments and even laid a certain foundation for the next stage.