Tet-on和Cre/loxP系统双重调控表达丙型肝炎病毒NS3/4A蛋白酶三转基因小鼠的建立

Establishment of Triple Transgenic Mice Conditionally Expressing Hepatitis C Virus NS3/4A Protease Simultaneously Controlled by Tet-on and Cre/loxP Systems

目的:建立Tet-On调控系统和Cre/loxP基因剔除系统双重调控表达丙型肝炎病毒(HCV)NS3/4A丝氨酸蛋白酶三转基因小鼠。方法:选择适龄并经鉴定的在Tet-on系统调控下肝脏特异性表达Cre重组酶的双转基因小鼠Lap/LC-1与在Tet-on系统调控下肝脏特异性表达萤光素酶(Luc)的双转基因小鼠Lap/NS3/4A交配,子代小鼠经PCR检测、筛选基因组中NS3/4A、Lap、LC-1等3个转基因片段均阳性的小鼠。三阳性的NS3/4A/Lap/LC-1小鼠经多西环素(Dox)诱导1周后,以在体生物发光成像系统(BLI)检测报告基因Luc的表达,免疫组化检测小鼠体内Cre重组酶、HCV NS3/4A丝氨酸蛋白酶的表达状况。结果:NS3/4A/Lap/LC-1小鼠经Dox诱导后,BLI结果显示仅在小鼠肝脏部位有强烈的发光信号,表明这些小鼠肝细胞内报告基因Luc特异高效表达;免疫组化结果证实Cre重组酶、NS3/4A蛋白酶仅在经诱导后的小鼠肝细胞中特异性表达。结论:建立了Tet-On调控系统和Cre/loxP基因剔除系统双重调控下表达HCV NS3/4A丝氨酸蛋白酶的三转基因小鼠模型,为进一步研究HCV NS3/4A丝氨酸蛋白酶在HCV感染后与宿主相互作用的机制,以及抗NS3/4A丝氨酸蛋白酶特异性抑制剂的筛选奠定了基础。

Objective： To establish triple-transgenic mice conditionally expressing hepatitis C virus（HCV） NS3/ 4A serine protease under tightly simultaneous control of Tet-on regulatory system and Cre/loxP knockout system. Methods： Lap/NS3/4A double transgenic mice liver-specific expressing the luciferase（Luc） under regulation of Tet-on system were mated with Lap/LC-1 double transgenie mice liver-speeifielly expressing the Cre recombinase under control of Tet-on system, and offsprings were detected and screened by PCR analysis of the NS3/4A, Lap, LC-1 fragment. Triple-fragment-positive mouse were induced with doxyeycline（Dox） for a week and then detected by in vivo bioluminescent imaging system（BLI） and immunohistochemistry. Results： BLI indicated that strong light signal could be detected only in the liver of NS3/4A/Lap/LC-1 triple transgenic mouse induced with Dox. Western blotting aslo showed that both the Cre recombinase and the NS3/4A protease only existed in the liver tissue, and immunohistochemistry staining further confirmed that the Cre recombinase located in the nucleolus and the NS3/4A protease distributed over the cytoplasm of the hepatocyte. Conclusion： Triple trausgenic mouse conditionally expressing HCV NS3/4A serine protease were successfully generated, which made a good tool for studying the interaction mechanisms of the NS3/4A protein and host, aslo and for screening and evaluation NS3/4A protease inhibitors.