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【目的】研究狂犬病病毒Flury鸡胚低代毒株(Flury LEP)在基因组P-M位增加糖蛋白基因(G基因)的重组表达对病毒致病力的影响。【方法】利用反向遗传操作技术,构建了P、M基因之间额外插入G基因的重组狂犬病病毒Flury LEP株(rLEP-PGM),并对重组病毒的生物学特性及对小鼠的致病性进行了初步研究。【结果】亲本株和重组病毒具有相似的生长特性,LEP和rLEP-PGM在BHK-21细胞的生长滴度分别为4×106 FFU/mL和2.5×106 FFU/mL,在小鼠神经母细胞(NA)的生长滴度分别为4×107 FFU/mL和2.5×107 FFU/mL;嗜神经指数均为1;Western blot显示,rLEP-PGM在感染细胞的G蛋白表达量比LEP显著提高;小鼠感染试验显示,rLEP-PGM与LEP脑内注射小鼠的LD50分别为3 FFU和1 FFU,肌肉注射途径的LD50分别为4×104 FFU和3.2×105 FFU。【结论】P、M基因之间插入一个额外的G基因能够提高G蛋白的表达水平,同时增强重组病毒外周侵入中枢神经系统的能力。
【Objective】 To investigate the effect of Flury LEP of rabies virus on the virulence of the virus by increasing the expression of the glycoprotein gene (G gene) at the P-M site of the genome. 【Method】 Recombinant rabies virus Flury LEP strain (rLEP-PGM) with inserted G gene between P and M genes was constructed by using reverse genetic manipulation technique. The biological characteristics of the recombinant virus and pathogenicity in mice Sex conducted a preliminary study. 【Result】 The results showed that the parent strain and recombinant virus had similar growth characteristics. The growth titers of LEP and rLEP-PGM in BHK-21 cells were 4 × 106 FFU / mL and 2.5 × 106 FFU / mL, respectively. In mouse neuroblasts (NA) were 4 × 107 FFU / mL and 2.5 × 107 FFU / mL, respectively. The index of nephropathy was all 1. Western blot showed that the expression of G protein in rLEP-PGM was significantly higher than that in LEP. Mouse infection tests showed that the LD50 of mice injected with rLEP-PGM and LEP was 3 FFU and 1 FFU respectively, and the LD50 of intramuscular route was 4 × 10 4 FFU and 3.2 × 10 5 FFU, respectively. 【Conclusion】 The insertion of an additional G gene between P and M genes can increase the expression of G protein and enhance the ability of the virus to invade the central nervous system.