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This study aimed to construct a stable cell line of human embryonic kidney epithelial cells 293 (HEK293) expressing recombinant human coagulation factor Ⅸ (rhFⅨ) and to identify the procoagulant biological activity of the expressed product. The recombinant plasmid pcDNA3.1-FⅨ was transfected into HEK293 cells, and a monoclonal stable transfection cell line was obtained through geneticin (G418) pressure screening. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine whether the FⅨ gene was transcribed and expressed in the monoclonal stable transfection cell line. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot were used to identify the expression of rhFⅨ; the concentration of rhFⅨ was determined by bicinchoninic acid assay (BCA), and the procoagulant activity of rhFⅨ was determined by activated partial thromboplastin time (APTT). The results showed that after G418 (200 µg·mL-1) pressure screening, 5 monoclonal stable cell lines could be normally transcribed, and the protein expression could be achieved through the transcription-translation process; the protein in the cell supernatant was purified and showed a single band of rhFⅨ; the expression level of rhFⅨ reached (1.58 ± 0.10) mg·L-1, and the maximum procoagulant activity was (66.8 ± 0.5)%. The established HEK293 monoclonal stable transfection cell line can efficiently and stably express rhFⅨ and has good procoagulant activity, providing a cell source for promoting the pharmacological evaluation and preclinical research of rhFⅨ.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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