IL-33基因重组和表达不影响重组狂犬病病毒体外表型特征

IL-33 gene recombination and expression does not affect the phenotypic characteristics of rabies virus in vitro

目的 构建白细胞介素33(IL-33)基因重组的狂犬病病毒,明确表达IL-33对重组病毒体外表型特征的影响。方法从狂犬病强毒株感染的小鼠脑内扩增获得的IL-33基因,通过反向遗传操作技术插入亲本病毒LBNSE基因组的G、 L基因之间,并拯救过表达IL-33的重组病毒;将重组狂犬病毒rLBNSE-IL33和亲本毒株LBNSE感染BSR仓鼠肾细胞或NA小鼠神经瘤细胞;在感染复数=0.01的情况下,测序和荧光抗体病毒中和试验检测重组病毒在传代过程中的稳定性;检测病毒滴度病灶形成单位(FFU)绘制多步生长曲线(感染复数=0.01);细胞毒性检测试剂盒检测细胞活性;ELISA检测不同感染复数感染细胞上清中IL-33的含量。结果 拯救获得过表达IL-33的rLBNSE-IL33,rLBNSE-IL33能够稳定连续传代至少10代,且毒价约为108FFU/mL;rLBNSE-IL33能够以剂量依赖的形式高水平表达IL-33,但检测被LBNSE感染的细胞上清,没有检测到IL-33的高表达;检查rLBNSE-IL33和亲本毒株LBNSE在BSR和NA细胞中5 d内的滴度,显示无明显差别,具有相似的生长动力学特性;过表达IL-33对感染细胞增殖及活性无显著影响。结论 IL-33基因重组和表达对狂犬病病毒体外表型特征无显著影响。

Objective To create a recombinant rabies virus overexpressing IL-33 and to clarify the effect of IL-33 overexpression on the phenotypic characteristics of recombinant virus in vitro.Methods The IL-33 gene was obtained and amplified from the brain of a highly virulent strain of rabies infected mouse.It was then inserted between the G and L genes of the parental virus LBNSE genome by reversing genetic manipulation and rescuing a recombinant virus overexpressing IL-33.BSR cells or mouse NA cells were infected with recombinant rabies virus(rLBNSE-IL33)and the parental strain LBNSE.Sequencing and fluorescent antibody virus neutralization assay was employed to detect the stability of recombinant virus at multiplicity of infection=0.01.Viral titres focal forming units(FFU)were detected to plot multi-step growth curves(multiplicity of infection=0.01).Cytotoxicity assay kit was used to detect cellular activity.ELISA was adopted to identify the IL-33 in the supernatant of infected cells of different multiplicity of infection.Results Rescued rLBNSE-IL33 overexpressing IL-33 remained stable for at least 10 consecutive generations and had virus titers of approximately 10°FFU/mL.rLBNSE-IL33 was able to express IL-33 at high levels in a dose-dependent manner,but no high expression of IL-33 was detected in the supernatant of cells infected by LBNSE.Examination of the titers of rLBNSE-IL33 and the parental strain LBNSE in BSR and NA cells over 5 days showed no significant differences and similar kinetic properties in growth.Overexpression of IL-33 had no significant effect on the proliferation and activity of infected cells.Conclusion Overexpression of IL-33 does not significantly affect the phenotypic characteristics of recombinant rabies virus in vitro.