人凝血因子Ⅷ cDNA 体外真核表达的初步研究

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。

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

OBJECTIVE: To evaluate the expression efficiency of a cDNA sequence of human clotting factor VIII (4.7 kb, 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/EBVVIII produced relatively high levels of FVIII activity (up to 0.7 units/ml and 2.0 units/ml from 24 h to 48 h, 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, psi-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.