表达狂犬病毒糖蛋白和核蛋白的重组腺病毒的生物学特性及免疫研究

对狂犬病毒G+N双基因复制缺陷型腺病毒穿梭载体进行转染,以获得融合表过狂犬病毒糖蛋白和核蛋白重组腺病毒,并对其进行生物学特性和免疫学研究.结果表明:将含有狂犬病毒G+N的重组穿梭质粒pAdTrack-CMV/G+N与腺病毒骨架载体pAdEasy-1在大肠杆菌细胞内同源重组,可获得带有目的基因的重组腺病毒载体pAd/G+N,经PacI酶切后转染HEK-293细胞,可获得带有目的基因的重组腺病毒.重组腺病毒在HEK-293细胞中连续传代15代次后,在细胞内的病变时间缩短为20 h以内.经测定,重组腺病毒对HEK-293细胞的TCID50为10-8.0.mL-1,用RT-PCR法可检测到狂犬病毒糖蛋白和核蛋白基因片段;重组腺病毒经口服免疫小白鼠,对狂犬病毒CVS毒株的免疫保护率可达80%.

The characteristics of biology and immunization of recombinant adenovirus expressing the rabies virus glycoprotein and nucleoprotein

The recombinant adenovirus vector containing the rabies virus(RV) glycoprotein(G) and nucleoprotein(N) was obtained by homologous recombination between the recombinant shuttle plasmid pAdTrack-CMV/G+N and pAdEasy vector in E.coli cells,which was designated as pAd/G+N.A recombinant adenovirus containing G and N genes were obtained by transfecting the Pac Ⅰdigested products of pAd/G+N to HEK-293 cells.The glycoprotein and nucleoprotein genes fragments of rabies virus could be detected in the recombinant adenovirus by RT-PCR.The infection time of the recombinant adenovirus was reduced to less than 20 hours after the virus was passaged in HEK-293 cells for 15 generations.Meanwhile,the TCID50 of recombinant adenovirus to HEK-293 cells reached about 10-8· mL-1.30 mice were randomly allocated into different groups and orally vaccinated with the recombinant adenovirus.All mice were challenged with rabies virus(CVS strains) in brain at 21 days post vaccination.The protection rate reached 80 % at 10 days post challenge.