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人VEGF基因昆虫杆状病毒表达载体的构建
Construction of Insect Baculovirus Expression Vector of Human VEGF Gene
【摘要】 为了在昆虫杆状病毒表达系统表达人血管内皮细胞生长因子 (VEGF)基因 ,用HindⅢ对 pcDNA3 .1 /VEGF进行酶切 ,回收 5 75bp的VEGF片段 ,与 pFastBacI载体连接 ,得到含VEGF基因的转座载体 pFast/VEGF ,NcoI酶切鉴定方向 ;将其转化到含有穿梭载体杆粒 (Bacmid)的DH1 0Bac感受态细胞 ,得到重组杆状病毒穿梭载体Bacmid/VEGF ,采用琼脂糖凝胶电泳、PCR扩增对重组转座载体、穿梭载体进行鉴定。将Bacmid/VEGF转染sf 9细胞 ,进行病毒重组及噬斑筛选。结果 :琼脂糖电泳得到杆粒 (Bacmid)DNA条带 ,PCR扩增得到 5 75bp的VEGF片段 ,病毒液在 1 0 - 7稀释度时出现噬斑 ,约 5 0个 /孔。说明已成功构建了重组杆状病毒穿梭载体并重组出杆状病毒。
【Abstract】 Using Hind Ⅲ enzyme to digest pcDNA3.1/VEGF, the VEGF fragment (575 bp) was obtained. The recombinant transposition vector pFast/VEGF including VEGF gene was constructed and identified with NcoI enzyme reaction. It was transformed into DH10Bac competent cell to recombine baculovirus shuttle vector Bacmid/VEGF. Agrose electrophoresis and PCR amplication were used to determine recombinant vector. The Bacmid/VEGF was transfected into sf-9 cell to screen virus plaque. As a result, agrose electrophoresis observed bacmid DNA band, PCR amplication obtained 575 bp VEGF fragment, virus solution appeared plaque in diluting to 10 -7 concentration(about 50 plaques/well).
【Key words】 human VEGF； recombinant baculovirus vector； PCR； plaque screening；
- 【文献出处】 首都医科大学学报 ,Journal of Capital University of Medical Sciences , 编辑部邮箱 ,2004年02期
- 【分类号】Q78
- 【被引频次】1
- 【下载频次】106