狂犬病病毒糖蛋白重组人腺病毒的构建及免疫效果的初步研究

构建表达狂犬病病毒弱毒SRV9糖蛋白(GP)的重组人5型腺病毒,检测其对小鼠的免疫效果。将狂犬病病毒SRV9株GP基因的完整开放阅读框克隆到腺病毒表达系统中的穿梭质粒多克隆位点,构建重组穿梭质粒pac-Ad5CMV-Gs9,以罗氏转染液介导线性化骨架质粒和重组穿梭质粒共转染293AD细胞,细胞病变后取培养物进行PCR鉴定并电镜观察,在293AD细胞上测定病毒滴度。以106 TCID50重组腺病毒腹腔接种昆明小鼠,免疫后不同时段采尾静脉血通过荧光抗体病毒中和试验(FAVN)检测小鼠血清狂犬病中和抗体效价。正确构建重组穿梭质粒pacAd5CMV-Gs9;获得表达狂犬病病毒SRV9株GP蛋白的缺陷型重组人5型腺病毒;病毒滴度达到106 CFU/mL以上;腹腔接种小鼠14d后均产生了抗狂犬病中和抗体,有效保护率达90%。成功获得了表达狂犬病病毒GP基因的重组腺病毒,该腺病毒免疫小鼠可产生保护性中和抗体,为进一步开发新型兽用狂犬病疫苗奠定了物质基础。

Generation and preliminary immunological efficacy of a recombinant human adenovirus-rabies virus glycoprotein

To construct a recombinant human adenovirus type 5 expressing glycoprotein (GP) of attenuated rabies virus SRV9 and testing immunological efficacy on the immunized mice. Open reading frame of rabies virus GP gene of SRV9 strain was cloned into the shuttle vector of adenovirus expression system in multiple cloning sites to construct the recombinant shuttle plasmid pacAd5 CMV-Gs9, cotransfection was performed into 293AD cells mediated by FuGENE Transfection Reagent with linearized backbone plasmid and recombinant shuttle plasmid, cell cultures were collected after CPE appearance and were identified by PCR and electronmicroscopy, virus titer was measured in 293AD cells. Kunming mice were intraperitoneally injected with 10(6) TCID50 adenovirus, blood for serum preparation was collected through caudal vein pre-immune and post-immune and tested for VNA appearance by fluorescent antibody virus neutralization test (FAVN) detection. Recombinant shuttle plasmid pacAd5 CMV-Gs9 was constructed correctly. A recombinant human adenovirus type 5 was obtained expressing GP protein of rabies virus SRV9. The virus titer reached 10(6) CFU/mL at the least. All mice developed a certain amount of the anti-rabies neutralizing antibody 14 days after intraperitoneal inoculation, while the effective protection rates were 90%. In conclusion, Recombinant adenovirus expressing the rabies virus GP was constructed successfully and a certain amount of neutralizing antibodies were induced in mice, which laid the material foundation for further development of new rabies vaccine.