摘要
目的构建泛醌肩动子（PUB）驱动的携带犬凝血因子Ⅸ（cFⅨ）基因的慢病毒三质粒表达载体，将其包装成病毒后，观察是否能在体外获得有效的cFⅨ表达。方法采用改良的磷酸钙沉淀法将包装质粒ANRF、包膜蛋白质粒VSV—G和构建的重组转移质粒PTK164共转染293T包装细胞产生慢病毒颗粒，病毒以MOI3：1比例感染培养的第三代骨髓基质细胞和293T细胞，用一期法测定细胞培养上清中cFⅨ活性。结果体外成功培养出骨髓基质细胞，经病毒感染后的骨髓基质细胞和293T细胞均能表达FIX因子，活性分别为（26．30±2．10）％和（19．70±1．53）％，与对照组[（1．00±0．05）％]比较，差异有统计学意义。结论成功构建PUB启动子驱动的cFⅨ基因的三质粒慢病毒表达载体，包装成病毒颗粒后成功转染至培养的骨髓基质细胞和293T细胞。
Objective To construct a three pLasmids LentiviraL vector containing canine coagulation factor Ⅸ （cFⅨ） gene with ubiquinone promoter（PUB） and observe the expression of cFⅨ gene. Methods Lentivirus was generated by transient three-plasmid transfection, nameLy, the VSV-G envelope expression cassette, the ANRF packaging plasmid and the PTK 164 plasmid. Viral particles were used to infect the target cell, third passage mesenchymal stem cells （MSCs） and 293T cell respectively at MOI 3： 1. The cFIⅨ activity was detected in cultured cells with one-stage clotting assay. Results The MSCs were obtained in vitro. The lentivirus infected MSCs and 293T cells all expressed the active factor IX with the activity of （26.30 ± 2.10）% and （ 19.70 ± 1.53）%, respectively, which are significantly higher than that of control （ 1.00 ± 0. 05） %. Conclusions The Lentiviral vector of three plasmids with ubiquinone promoter（PUB） was constructed and can transfect the MSCs and 293T cells.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2008年第9期583-586,共4页
Chinese Journal of Hematology
基金
国家自然科学基金（30370606）
