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为了观察非病毒载体 pRC/RSV介导的人凝血因子Ⅷ基因在小鼠 32D细胞系中的表达 ,将B结构域缺失(△ 76 0aa - 16 39aa)的人FⅧcDNA(hFⅧBDcDNA)亚克隆至质粒载体pRC/RSV ,构建重组质粒载体 pRC/RSV hFⅧBDcDNA。经SuperFectTransfectionReagent转染小鼠 32D细胞系 ,分别采用一期法、ELISA法和RT PCR检测细胞培养上清液中人FⅧ的促凝活性 (hFⅧ∶C)和抗原含量 (hFⅧ∶Ag)以及细胞中hFⅧBDcDNA的转录。结果表明 :小鼠 32D细胞系培养上清液中的人FⅧ∶Ag最高达到了 4 5 0 .0 8毫微克 /(10 6细胞·2 4小时 ) ,hFⅧ∶C最高达到了 2 .0 1单位 /(10 6细胞·2 4小时 ) ,RT PCR可检测到 32D细胞中hFⅧBDcDNA转录的mRNA。结论 :非病毒质粒载体 pRC/RSV介导的人凝血因子Ⅷ基因能够在小鼠 32D细胞系中表达人FⅧ蛋白 ,所表达的FⅧ与正常人血浆中的野生型FⅧ具有相似的凝血活性。
To observe the expression of non-viral vector pRC / RSV-mediated human factor Ⅷ gene in mouse 32D cell line, human FⅧ cDNA (hFⅧBD cDNA) lacking the B domain (Δ76aa-1639aa) was subcloned into a plasmid vector pRC / RSV to construct recombinant plasmid vector pRC / RSV hFVIIIBD cDNA. The 32D cell lines were transfected with SuperFectTransfectionReagent. The procoagulant activity (hFVIII: C) and antigen content (hFVIII: Ag) of human FⅧ in the cell culture supernatant were detected by ELISA and RT-PCR. Transcription of hFⅧBD cDNA. The results showed that the highest level of human F Ⅷ: Ag in mouse 32D cell culture supernatant reached 405.58 ng / (10 6 cells · 24 h) and the highest hFⅧ: C reached 2.01 Unit / (10 6 cells · 24 hours), RT PCR detects mRNA transcribed by hFⅧBDcDNA in 32D cells. CONCLUSION: Human non-viral plasmid vector pRC / RSV-mediated human factor Ⅷ gene can express human FⅧ protein in mouse 32D cell line, and expressed FⅧ has similar coagulation activity to wild-type FⅧ in normal human plasma.