论文部分内容阅读
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 a triple-transgenic mouse model of hepatitis C virus (HCV) NS3 / 4A serine protease with Tet-On regulatory system and Cre / loxP knockout system. Methods: The Lap-LC-1 double transgenic mouse liver-specific expression of Cre recombinase and Tet-on system-regulated liver-specific expression of luciferase (Luc ) Mice. The offspring mice were screened by PCR and screened the mice in which three NS3 / 4A, Lap, LC-1 and other transgene fragments were positive in the genome. One week after induced by doxycycline (Dox), the three positive NS3 / 4A / Lap / LC-1 mice were examined for the expression of luc gene in vivo bioluminescence imaging system (BLI) In vivo Cre recombinase, HCV NS3 / 4A serine protease expression status. Results: After induced by Dox, the BLI results of NS3 / 4A / Lap / LC-1 mice showed that there was a strong luminescence signal only in the liver of mice, which indicated that the reporter gene Luc was specifically and efficiently expressed in the hepatocytes of these mice. As a result, Cre recombinase and NS3 / 4A protease were specifically expressed only in the induced mouse hepatocytes. CONCLUSIONS: A three-transgenic mouse model expressing the HCV NS3 / 4A serine protease under the dual regulation of the Tet-On regulatory system and the Cre / loxP knockout system was established. In order to further investigate the interaction of the HCV NS3 / 4A serine protease with the host after HCV infection Of the mechanism, as well as anti-NS3 / 4A serine protease-specific inhibitors laid the foundation.