生物技术通讯
生物技術通訊
생물기술통신
LETTERS IN BIOTECHNOLOGY
2015年
2期
159-163
,共5页
高同同%刘晨风%姜玉庭%王友亮%吴小红%曾扬%赵亚光%郭彦%于虹%赵光宇%高基民%孙世惠%周育森
高同同%劉晨風%薑玉庭%王友亮%吳小紅%曾颺%趙亞光%郭彥%于虹%趙光宇%高基民%孫世惠%週育森
고동동%류신풍%강옥정%왕우량%오소홍%증양%조아광%곽언%우홍%조광우%고기민%손세혜%주육삼
丙型肝炎病毒%转基因小鼠%Dox诱导%药物评价
丙型肝炎病毒%轉基因小鼠%Dox誘導%藥物評價
병형간염병독%전기인소서%Dox유도%약물평개
hepatitis C virus%transgenic mice%Dox induction%drug evaluation
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pTRE2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pTRE2-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-rtTA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-miR122评价该模型在药物评价中的应用。结果：获得了整合rtTA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-miR122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。
目的：構建一種可調控肝細胞特異性錶達丙型肝炎病毒（HCV）全基因的小動物模型。方法：將9.6 kb的HCV全長基因JFH1插入Tet-on繫統效應錶達載體pTRE2中，併在HCV全基因3'耑插入丁型肝炎病毒（HDV）覈酶序列，從而構建轉基因載體pTRE2-JFH1（HCV）；將該轉基因載體線性化後顯微註射穫得整閤有HCV全基因的TRE2-HCV首建鼠；將該暘性鼠與本室保存的Alb-rtTA轉基因小鼠雜交，穫得肝細胞白蛋白啟動子調控HCV全基因錶達的Tet-on-Alb-HCV雙轉基因小鼠；在彊力黴素（Dox）誘導後通過PCR、Western印跡、免疫組化等方法鑒定轉基因小鼠HCV基因整閤及HCV蛋白在肝組織中的錶達；實時定量PCR檢測小鼠血清及肝組織中的HCV滴度；用HCV反義小分子藥物anti-miR122評價該模型在藥物評價中的應用。結果：穫得瞭整閤rtTA基因和HCV全長基因的雙轉基因小鼠；Dox誘導下，該轉基因小鼠可在肝組織長期特異性錶達HCV全長基因，小鼠血清中可檢測到HCV的RNA；分子藥物anti-miR122可降低血清中的HCV滴度。結論：構建瞭可調控肝組織特異性錶達HCV全基因的轉基因小鼠模型，該小鼠模型可應用于HCV藥物篩選和評價研究。
목적：구건일충가조공간세포특이성표체병형간염병독（HCV）전기인적소동물모형。방법：장9.6 kb적HCV전장기인JFH1삽입Tet-on계통효응표체재체pTRE2중，병재HCV전기인3'단삽입정형간염병독（HDV）핵매서렬，종이구건전기인재체pTRE2-JFH1（HCV）；장해전기인재체선성화후현미주사획득정합유HCV전기인적TRE2-HCV수건서；장해양성서여본실보존적Alb-rtTA전기인소서잡교，획득간세포백단백계동자조공HCV전기인표체적Tet-on-Alb-HCV쌍전기인소서；재강력매소（Dox）유도후통과PCR、Western인적、면역조화등방법감정전기인소서HCV기인정합급HCV단백재간조직중적표체；실시정량PCR검측소서혈청급간조직중적HCV적도；용HCV반의소분자약물anti-miR122평개해모형재약물평개중적응용。결과：획득료정합rtTA기인화HCV전장기인적쌍전기인소서；Dox유도하，해전기인소서가재간조직장기특이성표체HCV전장기인，소서혈청중가검측도HCV적RNA；분자약물anti-miR122가강저혈청중적HCV적도。결론：구건료가조공간조직특이성표체HCV전기인적전기인소서모형，해소서모형가응용우HCV약물사선화평개연구。
Objective: To establish a transgenic mouse model in which inducible hepatitis C virus(HCV) ex?pressed 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 immunohisto?chemistry assay. HCV virus titers in serum and liver were also detected by quantitative real time PCR. The appli?cation 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 produc?tion could be detected in liver and serum for 3 months. The HCV virus titer in serum decreased after the admin?istration of anti-miR122. Conclusion: The inducible and liver specific HCV expression mouse model was generat?ed successfully, which could be applied for the evaluation of HCV candidate vaccines and drugs.
