【摘要】 利用大肠杆菌内质粒间同源重组的方法,将狂犬病病毒G基因插入腺病毒基因组的E1基因区,构建了带有狂犬病病毒G基因的重组腺病毒质粒。重组质粒经Pme酶切,线性化后转染293细胞,成功获得了均一的复制缺陷型重组腺病毒。荧光显微镜下能观察到重组腺病毒GFP报告基因的表达。用PCR方法证实了重组腺病毒基因组中含有G基因,RT-PCR方法可检测到G基因的转录产物mRNA,Westernblotting方法能检测到重组腺病毒在29细胞中表达的G蛋白。此重组腺病毒在293细胞连续传代10次,PCR方法都能扩增出G基因目的条带。试验结果表明,狂犬病病毒G基因已成功重组到腺病毒基因组中,不但能稳定表达,而且能在重组腺病毒基因组中稳定存在。更多还原

【Abstract】 A recombinant plasmid comprising adenovirus genome with rabies virus glycoprotein gene in E1 region was constructed by homologous recombination.The plasmid was transfected into 293 cells after being digested with PacⅠ,then a homogeneous E1-deleted replication defective adenovirus recombinant expressing GP was packaged out successfully.Under fluorescence microscope,GFP expressed by the recombinant could be observed.G gene of the recombinant adenovirus and its transcript product could be detected by PCR or RT-PCR.At last,glycoprotein was also detected in 293 cells infected with the recombinant by Western blotting.G gene of the recombinant adenovirus always could be detected by PCR after 10 series generation in cells.The results indicated:G gene of rabies virus was recombined into adenovirus genome,and it could not only be expressed by the recombinant but also exist stably in the adenovirus genome.更多还原