作　　者： ; ; ;

机构地区： 广东医科大学

出　　处： 《传染病信息》 2018年第6期532-536,共5页

摘　　要： 目的建立高嗜肝性重组8型腺相关病毒(recombinant adeno-associated virus 8, rAAV8)介导的C基因型多重耐药乙型肝炎病毒(multidrug resistant hepatitis B virus, MDR HBV)稳定复制表达小鼠模型。方法分别构建rAAV8-1.3倍C基因型多重耐药型(HBV逆转录酶区含有rtL180M+S202G+M204V+N236T位点)和野生型HBV重组质粒,然后包装纯化并筛选高滴度重组病毒(rAAV8-1.3HBV-C-MDR和rAAV8-1.3HBV-C-WT),将病毒经尾静脉注射至6～8周龄的C57BL/6小鼠,建立C基因型多重耐药型(实验组,n=6)和野生型(对照组,n=6)HBV稳定复制表达小鼠模型,监测病毒复制和抗原表达至9周并于第9周末处死小鼠,取肝脏组织进行HE染色和免疫组化染色,观察肝组织病理改变并分析HBsAg和HBcAg的表达。结果小鼠注射重组病毒后第2、3、5、7、9周,血清HBV DNA表达较稳定,实验组和对照组小鼠血清HBV DNA波动范围分别为3.67～4.06 lg IU/ml和4.36～5.11 lg IU/ml。血清HBsAg和HBeAg在观察期间均高表达,第2周实验组和对照组血清HBsAg和HBeAg OD值分别为3.501±0.230和2.989±0.250,3.967±0.230和3.384±0.230。2组小鼠肝组织未见明显的炎性细胞浸润及组织结构异常,但可检测到HBsAg和HBcAg蛋白表达。结论利用高嗜肝性rAAV8载体携带1.3倍C基因型MDR HBV基因组体内转导C57BL/6小鼠,成功建立了稳定复制并持续表达C基因型MDR HBV的小鼠模型,为后续评价抗MDR HBV药物疗效提供实验平台。 Objective To establish a mouse model stably replicating and expressing genotype C multidrug resistant hepatitis B virus(MDR HBV)mediated by recombinant adeno-associated virus8(rAAV8).Methods The recombinant adeno-associated virus plasmids containing1.3copies of wild or MDR HBV(HBV reverse transcriptase region included rtL180M+S202G+M204V+N236T site)genotypes C genome were constructed,then the recombinant virus was packaged and purified.High-titer recombinant viruses rAAV8-1.3HBV-C-MDR and rAAV8-1.3HBV-C-WT were screened and injected via the tail vein into C57BL/6mice at the age of6-8weeks,to establish mouse models model stably replicating and expressing genotype C MDR HBV(experimental group,n=6)and WT HBV(control group,n=6).Viral replication and antigen expression were monitored until9weeks after injection,then mice were sacrificed at the end of the ninth weeks.Liver tissue was sampled for hematoxylin-eosin(HE)staining and immunohistochemistry staining.The pathological changes of liver tissue were observed,the HBsAg and HBcAg expression in liver tissue was analyzed.Results At week2,3,5,7,9after recombinant virus injection,serum HBV DNA load maintained stable.The serum HBV DNA load of experimental group and control group fluctuated within3.67-4.06lg IU/ml and4.36-5.11lg IU/ml,respectively.While,serum HBsAg and HBeAg were both highly expressed during the observation period.The OD values of serum HBsAg and HBeAg in experimental group and control group at week2were3.501±0.230and2.989±0.250,3.967±0.230and3.384±0.230,respectively.No obvious infiltration of inflammatory cells or abnormal structure of liver tissue was observed,while HBsAg and HBcAg expression in the liver tissue were detected for both groups.Conclusions By in vivo transduction with recombinant virus rAAV8-1.3HBV-C-MDR,a C57BL/6mouse model that stably replicated and expressed genotype C MDR HBV is successfully established,providing an experimental platform for further evaluation of anti-MDR HBV drug efficacy.

关 键 词： 多重耐药突变 乙型肝炎病毒 病毒复制 抗原表达 小鼠模型

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