可调控肝脏特异性表达HCV全基因转基因小鼠模型的建立  

Construction of an Inducible and Liver Specific HCV Expression Mouse Model

作　　者：高同同 刘晨风[2] 姜玉庭 王友亮[3] 吴小红[2] 曾扬 赵亚光[2] 郭彦[2] 于虹[2] 赵光宇[2] 高基民[1] 孙世惠[2] 周育森[1,2] 

机构地区：[1]温州医科大学检验医学院,生命科学学院,浙江温州325035 [2]军事医学科学院微生物流行病研究所,病原微生物国家重点实验室,北京100071 [3]军事医学科学院生物工程研究所,北京100071

出　　处：《生物技术通讯》2015年第2期159-163,共5页Letters in Biotechnology

基　　金：国家高技术研究发展计划(2007AA02Z151);国家"十一五"传染病重大专项(2009ZX10004401);国家"十二五"传染病重大专项(2012ZX10004502)

摘　　要：目的:构建一种可调控肝细胞特异性表达丙型肝炎病毒(HCV)全基因的小动物模型。方法:将9.6 kb的HCV全长基因JFH1插入Tet-on系统效应表达载体p TRE2中,并在HCV全基因3'端插入丁型肝炎病毒(HDV)核酶序列,从而构建转基因载体p TRE2-JFH1(HCV);将该转基因载体线性化后显微注射获得整合有HCV全基因的TRE2-HCV首建鼠;将该阳性鼠与本室保存的Alb-rt TA转基因小鼠杂交,获得肝细胞白蛋白启动子调控HCV全基因表达的Tet-on-Alb-HCV双转基因小鼠;在强力霉素(Dox)诱导后通过PCR、Western印迹、免疫组化等方法鉴定转基因小鼠HCV基因整合及HCV蛋白在肝组织中的表达;实时定量PCR检测小鼠血清及肝组织中的HCV滴度;用HCV反义小分子药物anti-mi R122评价该模型在药物评价中的应用。结果:获得了整合rt TA基因和HCV全长基因的双转基因小鼠;Dox诱导下,该转基因小鼠可在肝组织长期特异性表达HCV全长基因,小鼠血清中可检测到HCV的RNA;分子药物anti-mi R122可降低血清中的HCV滴度。结论:构建了可调控肝组织特异性表达HCV全基因的转基因小鼠模型,该小鼠模型可应用于HCV药物筛选和评价研究。Objective： To establish a transgenic mouse model in which inducible hepatitis C virus（HCV） expressed in liver effectively. Methods： The plasmid pTRE2-JFH1（HCV） was constructed which contains the 9.6 kb full-length JFH1 genome（HCV） and an HDV ribozyme sequence located at the 3＇ end of the JFH1 genome. The transgenic mouse model integrated JFH1 was generated after pronuclear injection of the transgenic construct pTRE2-JFH1（HCV）. The double transgenic Tet-on-Alb-HCV mouse was generated from a cross between the Alb-rtTA transgenic mouse and the TRE2-HCV positive mouse. After Dox induction, the integration and expression of HCV in liver of double transgenic Tet-on-Alb-HCV mouse was detected by PCR, Western blot and immunohistochemistry assay. HCV virus titers in serum and liver were also detected by quantitative real time PCR. The application of this model in anti-HCV drug evaluation with anti-miR122 was also investigated. Results： The double transgenic mouse model integrated HCV and rtTA was generated. After Dox induction, HCV replication and production could be detected in liver and serum for 3 months. The HCV virus titer in serum decreased after the administration of anti-miR122. Conclusion： The inducible and liver specific HCV expression mouse model was generated successfully, which could be applied for the evaluation of HCV candidate vaccines and drugs.

关 键 词：丙型肝炎病毒 转基因小鼠 Dox诱导 药物评价 

分 类 号：Q78[生物学—分子生物学]