重组人凝血因子Ⅶ稳定转染细胞株的构建及鉴定OA

Objective To construct a stable monoclonal human embryonic kidney 293(HEK293)cell line ex-pressing recombinant human coagulation factor Ⅶ(rhFⅦ)and evaluate the expression level and procoagulant bioac-tivity of rhFⅦ.Methods The plasmid pCDNA3.1-EGFP-FⅦ was transfected into HEK293 cells to verify the effec-tiveness of the transfection system.The plasmid pCDNA3.1-FⅦ was transfected into HEK293 cells,and monoclo-nal stable transfected cell lines were selected using geneticin(G418).The transcription of the FⅦ gene was identi-fied by reverse transcription polymerase chain reaction(RT-PCR).The expression level of rhFⅦ in the superna-tant of the monoclonal stable transfected cell line was detected by sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis and Western blot.The concentration of rhFⅦ was determined by enzyme-linked immunosorbent assay(ELISA),and the procoagulant activity of rhFⅦ was measured by human coagulation factor Ⅶ potency assay.Re-sults HEK293 cells transfected with pcDNA3.1-EGFP-FⅦ showed green fluorescence,indicating that rhFⅦ was successfully expressed in the supernatant of HEK293 cells after transient transfection with pcDNA3.1-FⅦ.The monoclonal stable transfected cell line was obtained by G418 screening.RT-PCR identified that the FⅦ gene was integrated into the genome of the monoclonal stable transfected cell line.The cell viability was good as detected by Cell Counting Kit-8,and a single band of rhFⅦ was obtained by purification of the cell supernatant.The highest rhFⅦ expression was(1.27±0.09)mg/L,and the highest procoagulant activity was(380.29±13.80)%.Conclu-sion The monoclonal HEK293 cell lines which can express rhFⅦ protein efficiently and stably with excellent pro-coagulant bioactivity is successfully screened.