人凝血因子Ⅷ cDNA体外真核表达的初步研究

A preliminary study on the in vitro eukaryotic expression of human Factor Ⅷ cDNA

目的：建立人凝血因子ⅧｃＤＮＡ体外真核高效表达的体系。方法：将人ＦⅧＢ区大部分缺失（Δａ７６０１６３９）的ＦⅧｃＤＮＡ插入ｐＣＩ真核表达载体、ｐＧＲＥ５．２／ＥＢＶ载体和逆转录病毒载体ｐＭＳＣＶ，构建了５种表达框架，在体外转染和感染了多种靶细胞。结果：ｐＣＩⅧ在ＮＩＨ３Ｔ３细胞产生了低水平表达，而ｐＧＲＥ５．２／ＥＢＶⅧ和ｐＭＳＣＶⅧ系列分别在Ｈｅｌａ细胞和Ｂｏｓｃ２３逆转录病毒包装细胞中呈较高水平的表达。Ｂｏｓｃ２３细胞培养上清感染的ＮＩＨ３Ｔ３和３２ＤＣ不能有效地产生ＦⅧ。结论：在ＦⅧ体外表达及基因治疗的方案设计中，靶细胞的选择是一重要因素。

Objective:To evaluate the expression efficiency of a cDNA sequence of human clotting factor Ⅷ (4.7kb, B domain deleted)in in vitro systems. Methods: After insertion of the cDNA into several mammalian expression vectors, such as retroviral vector pMSCV, EB virus based vector pGRE5.2/EBV and eukaryotic expression plasmid pCI, the expression of these constructs were tested in a variety of cells. Results: All the three kinds of constructs pCI Ⅷ,pGRE5.2/EBV Ⅷ and pMSCV Ⅷ were able to direct FⅧ synthesis in NIH3T3, Hela and Bosc23 cells, respectively, while the pMSCV Ⅷ and pGRE5.2/EBV Ⅷ produced relatively high levels of FⅧ activity (up to 0.7 U/ml and 2.0 U/ml from 24h to 48h, respectively, after transfection with lipofectamine). The three forms of pMSCV Ⅷ vector worked in a similar efficacy in Bosc 23 cells, but this function was not detected in NIH3T3, ψ Crip and GP+E86 as well as 32DC13 cells in a transient transfection assay. Moreover, the NIH3T3 and 32DC13 cells infected with culture supernatant from pMSCV Ⅷ transfected Bosc23 cells (as packaging cells) unexpectedly did not produce detectable FⅧ activity. Conclusion:Apart from the design and construction of vectors, target cell selection may play a crucial role in the efficient expression of the FⅧ cDNA.