汉滩病毒诱导乳鼠组织热休克蛋白表达及其与病毒抗原蛋白的相关性

Hantaan virus induces heat-shock protein 70 expression in brain of suckling mice and its correlation with viral proteins

目的 探讨汉滩病毒 (HV)感染诱导乳鼠组织热休克蛋白 (HSPs)表达及热休克蛋白与病毒蛋白的相互关系。方法 出生后 2～ 3d的昆明乳鼠 ,每只脑内接种 10 0个半数致死量的HV0 .0 3ml,8d后乳鼠发病死亡 ,取其脑组织一部分固定 ,石蜡包埋切片 ,用免疫组织化学法结合共聚焦显微镜检测组织中病毒抗原及HSP70的表达 ,一部分组织制匀浆液 ,用ELISA、免疫共沉淀方法分析病毒抗原和HSP70的表达及关系。结果 HV感染诱导了乳鼠脑组织神经细胞高表达热休克蛋白(HSP) 70 ,细胞内病毒抗原与HSP70有共定位关系 ;免疫共沉淀结果显示用HSP70mAb可以共沉淀感染乳鼠脑组织匀浆中的汉滩病毒核心抗原 (HV NP) ,用HV NPmAb 1A8可沉淀感染乳鼠脑组织匀浆中的HSP70 ,对照实验阴性 ,所用两种蛋白抗体间无交叉反应 ,因而HSP70与HV NP结合形成复合物 ,结果是特异的。结论 HV感染诱导HSP70的表达 ,后者与HV NP结合形成HSP70 病毒抗原肽复合物 ,该复合物可能在病毒感染和复制过程中发挥作用。

Objective To study the impact of viral infection on stress protein gene expression and investigate the possibility that heat shock proteins interact with viral components during viral assembly in the brain of suckling mice experimentally infected with Hantaan virus (HV). Methods New born mice less than 72 h of age were inoculated into brain with the 100 LD50 of Hantaan virus (strain 76 118). At the 8th day mice died from brain disease. Brains were prepared for tissue sections and extracts for the experiments. The Hantaan virus infection, HSP70 induction and their co localization in brain of infected suckling mice were demonstrated by immunohistochemical staining and laser scan co focal microscopic double labling. The possible intereaction between HV structural proteins and HSP70 was further analyzed by immunoprecipitation of infected tissue extracts with antibodies to either HSP70 or to viral nuclear capsule proteins HV NP by using SDS PAGE and Western blotting. Results HV infection induced an increased levels of HSP70 expression at the cytoplasma of neurons in the brain of suckling mice HV NP protein proved to be co localized with HSP70. Monoclonal antibody (mAb) to HSP70 could co immunoprecipitate the HV NP, and similarly mAb to HV NP could co immunoprecipitate HSP70 from virus infected tissue extracts, but not from mock infected tissue extracts. Antibody, which recognized one of these two proteins, did not cross react with the other. Thus, the association between HSP70 and HV NP appeared to be specific.Conclusions Hantavirus infection can directly induce HSP70 expression, leading to the formation of HV HSP 70 viral antigen peptide complex which plays some roles in the course of viral infection and replication.