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目的:应用非复制腺病毒表达系统构建表达人轮状病毒非结构蛋白4(NSP4)的重组腺病毒,初步评价其免疫保护效果。方法:构建含野生轮状病毒NSP4基因的穿梭质粒pshuttle-NSP4,与腺病毒骨架质粒pAdeasy经同源重组后在Ad-293细胞中包装获得pAd-NSP4重组腺病毒颗粒。电镜、RT-PCR、免疫荧光等方法鉴定病毒特征及在体外细胞中的表达。肌肉注射及滴鼻方式免疫小鼠,检测小鼠血清抗体效价及其中和保护效果。结果:获得了滴度为108.25CCID50/ml的重组腺病毒pAd-NSP4,免疫荧光检测到特异性目的蛋白的表达。二次免疫后肌肉注射和滴鼻小鼠的ELISA血清平均效价分别为1∶320和1∶1436.8;中和抗体效价1∶45.3和1∶71.8。结论:表达轮状病毒NSP4蛋白的非复制型重组腺病毒颗粒具有良好的免疫原性。滴鼻途径比肌肉注射可更加有效地诱导小鼠的免疫应答。
Objective: To construct a recombinant adenovirus expressing non-structural protein 4 of human rotavirus (NSP4) using non-replicating adenovirus expression system and evaluate its immunoprotective effect. METHODS: The shuttle plasmid pshuttle-NSP4 containing the wild-type rotavirus NSP4 gene was constructed and pAd-293 was packaged with adenovirus backbone plasmid pAdeasy to obtain recombinant adenovirus pAd-NSP4. Electron microscopy, RT-PCR, immunofluorescence and other methods to identify the virus characteristics and in vitro cell expression. Intramuscular and intranasal immunization mice to detect the serum antibody titer and its protective effect of neutralization. Results: The recombinant adenovirus pAd-NSP4 with a titer of 108.25 CCID50 / ml was obtained, and the expression of specific target protein was detected by immunofluorescence. The average titer of ELISA sera of mice injected intramuscularly and intranasally after secondary immunization was 1: 320 and 1:1436.8, respectively; neutralizing antibody titers 1:45.3 and 1:71.8. Conclusion: The non-replicating recombinant adenovirus expressing rotavirus NSP4 protein has good immunogenicity. Instillation of the nasal route induces an immune response in mice more effectively than intramuscular injection.