高嗜肝性8型重组腺相关病毒体内转导法制备乙型肝炎病毒持续感染小鼠模型  

Establishment of Hepatitis B Virus(HBV) Chronic Infection Mouse Model by in vivo Transduction with a Recombinant Adeno-associated Virus 8 Carrying 1.3 Copies of HBV Genome(rAAV8-1.3HBV)

作　　者：董小岩[1] 尉迟捷[2] 王刚[2] 田文洪[2,3] 陆月[2,4] 张风卫[2,4] 王文[2] 王岳[2] 谭文杰[2] 吴小兵[2] 

机构地区：[1]复旦大学生命科学学院遗传学研究所遗传工程国家重点实验室,上海200433 [2]中国疾病预防控制中心病毒病预防控制所病毒基因工程国家重点实验室,北京100052 [3]吉林大学生命科学学院,长春130012 [4]温州医学院浙江省医学遗传学重点实验室,温州325035

出　　处：《病毒学报》2010年第6期425-431,共7页Chinese Journal of Virology

基　　金：肝炎相关肝癌生物治疗新途径新制品的研究和应用(No.2008ZX10002-023);病毒基因工程国家重点实验室开放课题(2008-S-0003);新药创制科技重大专项(No.2009ZX09102-237)资助

摘　　要：用高嗜肝性的重组8型腺相关病毒(Recombinant adeno-associated virus type8,rAAV8)载体携带1.3拷贝乙型肝炎病毒(Hepatitis B virus,HBV)基因组(ayw亚型)体内转导法,建立持续表达HBV抗原的C57BL/6小鼠模型。首先,制备并纯化了携带1.3拷贝HBV基因组(ayw亚型)的重组8型腺相关病毒(rAAV8-1.3HBV);将rAAV8-1.3HBV以剂量2×10e11vg/只注射C57BL/6小鼠(Viralgenome,vg);在不同时间点实施尾静脉采血,采用ELISA方法监测血清中HBsAg和HBeAg的水平及动力学变化;10周后处死小鼠,取血液、肝组织样本,提取基因组DNA,荧光定量PCR检测HBVDNA拷贝数;利用鉴定HBVDNA环化形式的特异性引物进行PCR扩增以检测肝组织中环化的HBVDNA,并检测HBV抗原特异性的免疫组化和肝脏病理变化。结果显示,注射rAAV8-1.3HBV的3只C57BL/6小鼠第1周开始在血液中检测到HBsAg和HBeAg的表达,并持续至第10周均为阳性,其中HBsAg的表达水平经历了一个上升-下降-再上升的过程(注射后第4周时最低,第6周后维持较高水平),而HBeAg表达水平则持续阳性且比较稳定。荧光定量PCR结果显示,3只小鼠10周后血清中HBV DNA的拷贝数分别为4.2×103、3.6×103、2.5×103copies/mL,肝脏中则分别为8.0×106、5.7×106、2.6×106copies/g肝组织。在3只小鼠肝组织中均检测到环化HBV DNA,提示AAV8载体携带的线性HBV DNA成功回复成环化HB VDNA。免疫组化分析显示3只小鼠肝脏中均存在HBsAg和HBcAg表达;体内转染10周后肝脏组织切片的HE染色分析显示未见明显的炎性细胞浸润及组织结构异常。结果表明,本研究利用高嗜肝性重组8型腺相关病毒载体携带1.3拷贝乙型肝炎病毒基因组(ayw亚型)体内转导C57BL/6小鼠,成功地建立了HBV病毒在肝内稳定复制并持续表达HBV抗原的小鼠模型,为进一步研究HBV慢性持续感染的机制与应用于药物以及疫苗评价打下了基础。In this report,we developed a HBV infection model in C57BL/6 mouse line by in vivo injection of a recombinant adeno-associated virus 8 vector carrying 1.3 copies of HBV genome（ayw subtype）（rAAV8-1.3HBV）.We firstly prepared and purified the rAAV8-1.3HBV and then injected it into three C57BL/6 mice with the dose of 2×10e11vg,respectively.HBsAg and HBeAg were assayed in sera collected at different time points post injection.Ten weeks post injection,the three mice were sacrificed and blood and liver tissue were taken for assay.Copies of HBV DNA were detected by real time PCR and the way of HBV DNA replication was identified by PCR.Subsequently,detection of HBV antigen by immunohistochemistry and pathology analysis of liver tissue of mice were performed.The results suggested that expression of HBsAg and HBeAg lasted for at least 10 weeks in mice sera.Among mice injected with rAAV8-1.3HBV,HBsAg levels were showed an ＇increasing-decreasing-increasing＇ pattern（the lowest level at the 4th week post injection）,while HBeAg levels were kept high and relatively stable.HBV DNA copies were 4.2×103,3.6×103,2.5×103copies/mL in sera and 8.0×106,5.7×106,2.6×106 copies/g in hepatic tissues of three mice,respectively.We found that the linear 1.3 HBV DNA in the rAAV8-1.3HBV could self form into circular HBV genome and replicate in livers of HBV transfected mice.HBsAg and HBcAg were both positive in liver tissue of mice injected with rAAV8-1.3HBV and no obvious pathological characters were found in liver of mice injected with rAAV8-1.3HBV.In conclusion,we successfully developed a HBV chronic infection model in C57BL/6 mouse line by in vivo transduction with the recombinant virus rAAV8-1.3HBV,in which HBV genes could be continuously expressed and replicated over 10 weeks,and paved a way for further characterization of the human chronic hepatitis B virus infection and evaluation of vaccine and anti-HBV agents.

关 键 词：动物模型 乙型肝炎病毒 AAV8 持续感染 

分 类 号：R373.2[医药卫生—病原生物学] Q78[医药卫生—基础医学]