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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。
Recombinant human adenovirus type 5 expressing the attenuated rabies virus SRV9 glycoprotein (GP) was constructed and its immune effect on mice was tested. The complete open reading frame of the GP gene of rabies virus SRV9 was cloned into the shuttle plasmid multi-cloning site in the adenovirus expression system to construct the recombinant shuttle plasmid pac-Ad5CMV-Gs9. The shuttle plasmid was co-transfected into 293AD cells. After the cells were harvested, the cultures were identified by PCR and electron microscopy. The virus titer was determined on 293AD cells. Kunming mice were inoculated intraperitoneally with 106 TCID50 recombinant adenovirus, and the tail vein blood was collected at different time after immunization to detect the serum rabies neutralizing antibody titers by fluorescent antibody virus neutralization test (FAVN). Correct construction of recombinant shuttle plasmid pacAd5CMV-Gs9; Obtain defective recombinant adenovirus type 5 of GP of SRV9 strain of rabies virus; The titer of virus reached above 106 CFU / mL; Anti-rabies neutralization Antibody, effective protection rate of 90%. The recombinant adenovirus expressing GP gene of rabies virus was successfully obtained. The adenovirus immunized mice can produce protective neutralizing antibodies, which lays the material foundation for further development of novel veterinary rabies vaccine.