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对人胰岛素基因组基因真核表达载体 ( p CMA/m INS)进行 Xho 酶切 ,回收的含人胰岛素基因组基因的 1 60 8bp片段与线性化的杆状病毒载体 p Fast Bac 进行连接 ,获得重组载体 p Fast/m INS。将该重组载体转化 DH1 0 Bac感受态细菌 ,在体内进行重组 ,并经 2次抗性与蓝白斑筛选 ,得到杆状病毒重组载体 Bacmid/m INS。将该载体转染 Sf9细胞 ,获得了重组杆状病毒 ,经 Tricine-SDS-PAGE和 Western-blotting检测 ,重组杆状病毒在 Sf9细胞中的表达产物是胰岛素原 ,而细胞培养上清中未检测到胰岛素和胰岛素原
Xho digestion of the human insulin gene eukaryotic expression vector (pCMA / m INS) was performed to ligate the 1608 bp fragment containing the human insulin gene and the linearized baculovirus vector p Fast Bac to obtain a recombinant vector p Fast / m INS. The recombinant vector was transformed into DH1 0 Bac competent cells, and then recombined in vivo. The recombinant baculovirus recombinant Bacmid / m INS was obtained after twice resistance and blue-white screening. The recombinant baculovirus was transfected into Sf9 cells. Tricine-SDS-PAGE and Western-blotting results showed that the recombinant baculovirus expressed pro-progenitor cells in Sf9 cells, but not in cell culture supernatants To insulin and proinsulin