人凝血因子Ⅶ在CHO-K1细胞中的表达与鉴定

Expression and identification of human coagulation factor Ⅶ in CHO-K1 cells

目的在CHO-K1细胞中表达人凝血因子Ⅶ(human coagulation factorⅦ,hFⅦ)。方法利用脂质体转染法,将表达质粒pcDNA3.1-FⅦ和空载体pcDNA3.1分别转染CHO-K1细胞,经900μg/ml G418筛选抗性细胞株。收集细胞培养上清,采用ELISA法检测细胞培养上清中rhFⅦ的水平;PT法检测细胞培养上清的凝血时间;Westernblot法检测细胞培养上清中rhFⅦ的表达。结果共获得3株抗性细胞,分别命名为CHO-FⅦ01～03,空载体转染的细胞命名为CHO-CK;CHO-FⅦ01～03细胞培养上清中均检测到了rhFⅦ,但含量均低于阳性对照;CHO-CK细胞培养上清的凝血时间分别为CHO-FⅦ01～03细胞培养上清的2.4、2.1和3.1倍;CHO-FⅦ01～03细胞培养上清中含目的蛋白rhFⅦ,相对分子质量约为50 000。结论成功在CHO-K1细胞中表达了hFⅦ,为下一步hFⅦ的深入研究及应用奠定了基础。

Objective To express recombinant human coagulation factor Ⅶ（rhFⅦ）in CHO-K1 cells.Methods CHOK1 cells were transfected with plasmid pcDNA3.1-FⅦ and empty vector pcDNA3.1 respectively,from which G418-resistant cell strains were screened with 900 μg/ml G418.The cell culture supernatant were harvested and determined for rhFⅦ level by ELISA,for coagulation time by prothrombintime（PT） method,and for expression of rhFⅦ by Western blot.Results Three G418-resistant cell strains were obtained and named as CHO-FⅦ01 ～ 03 respectively,while that transfected with empty vector as CHO-CK.The rhFⅦs were detected in all the culture supernatants of CHO-FⅦ01 ～ 03 cells,of which the contents were lower than that in positive control group.The coagulation times of culture supernatant of CHO-CK cells were 2.4,2.1 and 3.1 times of those of CHO-FⅦ01 ～ 03 cells respectively.The culture supernatants of CHO-FⅦ01 ～ 03 cells contained rhFⅦ with a relative molecular mass of about 50 000.Conclusion Recombinant hFⅦ was successfully expressed in CHO-K1 cells,which laid a foundation of further study on hFⅦ.