狂犬病病毒核蛋白和糖蛋白基因的克隆及其腺病毒穿梭载体的构建

Cloning of nucleoprotein and glycoprotein genes and construction of recombinant adenovirus shuttle vector with N and G genes

用狂犬病病毒CVS毒株经小鼠脑腔攻击后从发病鼠脑组织中提取总RNA,用带接头的特异性引物分别扩增出了狂犬病病毒核蛋白(N)基因和糖蛋白(G)基因。将N基因和G基因分别克隆入质粒载体pMD18T后,对筛选的含有N基因和G基因的重组质粒进行了全基因序列测定和分析。随后将狂犬病病毒N基因和G基因分别从其各自的克隆载体上双酶切消化,同时亚克隆入腺病毒穿梭载体pAdTrackCMV。经卡那霉素抗性和酶切筛选,最终成功构建了含有N基因和G基因的重组穿梭载体pAdTrackCMVNG。

The total rabies viral RNA wasisolated from the brain tissues of 4-weeks-old Kunming mice which had been attacted by rabies virus CVS strain. The nucleoprotein(N) gene and glycoprotein(G) gene of rabies virus were amplified by RT-PCR and then cloned into pMD18-T plasmid,respectively. The sequencing and analyzing with DNAStar showed that the N and G genes were derived from rabies virus CVS strain with high neuropathogenicity.Toconstruct recombinant adenovirus, the N and G genes were respectively cleaved from the pMD18-T plasmids by double restriction endonuclease,and then cloned into shuttle vector pAdTrack-CMV coinstantaneously. The recombinant shuttle plasmid of pAdTrack-CMV-N-G was obtained after being selected by kanamycin resistance and being screened by restriction endonuclease.