LCMV-CL13慢性感染小鼠模型的建立及其BCR突变分析

Establishment of chronic infection mouse model by LCMV-CL13 virus and analysis of BCR mutation

目的建立淋巴细胞性脉络丛脑膜炎病毒LCMV-CL13慢性感染小鼠模型,分析其作为B细胞受体高频突变研究模型的可能性。方法C57BL/6N小鼠经尾静脉途径接种2×10^(6)PFU剂量LCMV-CL13病毒,感染后第10、20、30、40、50、60、70天采集样品,通过qPCR检测组织病毒载量,流式检测分析外周血CD4^(+)T、CD8^(+)T、CD19^(+)B细胞及脾生发中心B细胞比例,免疫组库测序分析BCR V区基因丰度及突变率。结果在LCMV-CL13病毒感染小鼠体内检测到1×10^(6)copies/μL水平的病毒复制;在感染平台期,小鼠外周血CD4^(+)T细胞比例逐渐升高(13.15%±0.72%),CD8^(+)T细胞先降低(2.17%±0.40%)后逐渐恢复(6.65%±0.52%),CD19^(+)B细胞以及生发中心B细胞比例分别增加至(40.32%±0.46%)和(10.03%±0.60%);测序结果证明BCR重链V基因使用频率下降、突变率显著性升高(P<0.05)。结论成功建立LCMV-CL13慢性感染小鼠模型;此模型可用于BCR突变研究,为研究慢性病毒感染导致的B细胞高频突变奠定了基础。

Objective To establish an LCMV-CL13 chronic infection mouse model and analyze its use for B cell somatic hypermutation research.Methods C57BL/6N mice were inoculated with 2×10^(6) plaque-forming units of LCMV-CL13 virus via tail vein injection.The tissue viral load was then detected by quantitative polymerase chain reaction on days 10,20,30,40,50,60,and 70 post-infection.The CD4^(+)and CD8^(+)T cell and CD19^(+)B cell ratios in peripheral blood and the germinal center B cell percentage of the spleen were determined by flow cytometry,and the gene abundance and BCR V region mutation rate were analyzed using immune repertoire technology.Results LCMV-CL13-infected mice maintained a tissue viral load of 1×10^(6) copies/μL virus.The percentage of CD4^(+)T cells in the peripheral blood gradually increased in the infection plateau phase to(13.15%±0.72%),while the percentage of CD8^(+)T cells first decreased to(2.17%±0.40%)and then gradually recovered to(6.65%±0.52%),and the percentages of CD19^(+)B cells and germinal center B cells increased to(40.32%±0.46%)and(10.03%±0.60%),respectively.Sequencing result demonstrated that the frequency of V gene usage decreased and the mutation rate of the heavy chain CDR3 V gene increased 1.7-fold with increasing infection time.Conclusions We successfully established a LCMV-CL13 chronic infection mouse model.This model can be used to study BCR mutations,and provides a research tool for investigating B cell somatic hypermutation caused by chronic viral infection.