论文部分内容阅读
目的建立调控因素存在下表达丙型肝炎病毒(HCV)NS3/4A丝氨酸蛋白酶的转基因小鼠。方法采用分子克隆技术构建可受Tet-On调控系统和Cre-LoxP基因敲除系统双重调控表达HCV NS3/4A丝氨酸蛋白酶的真核表达质粒PBI-Ⅲ/LoxP-Luc-PolyA-LoxP-NS3/4A。该重组质粒线性化后,经显微注射制备转基因小鼠。首建鼠及经PCR检测阳性的子代鼠与C57BL/6小鼠交配传代。选择部分F2鼠与已经稳定建系的转基因小鼠Lap杂交,利用在体生物发光成像系统(BLI)检测肝脏稳定表达荧光素酶(Luc)报告基因的经盐酸强力霉素(Dox)诱导的双转基因子代鼠,并选择性针对稳定表达系传代扩群。结果酶切鉴定和测序分析显示重组载体构建成功。经PCR检测得到6只转基因首建鼠,其中3只可繁殖传代并持续检测到阳性子代鼠。BLI结果显示,由其中1只首建鼠传代而来的不同F2鼠与Lap鼠杂交得到的部分双转基因鼠肝脏部位发光信号强烈,表明这些小鼠肝细胞内报告基因Luc特异高效表达。结论建立了转基因小鼠NS3/4A,并筛选到调控因素存在时转入外源基因特异稳定表达的转基因小鼠,为进一步建立严格调控型表达NS3/4A蛋白酶的小鼠模型奠定基础。
Objective To establish a transgenic mouse expressing hepatitis C virus (HCV) NS3 / 4A serine protease in the presence of regulatory factors. Methods The eukaryotic expression plasmid PBI-Ⅲ / LoxP-Luc-PolyA-LoxP-NS3 / 4A which can be regulated by Tet-On regulatory system and Cre-LoxP gene knockdown system was constructed by molecular cloning . After linearization of the recombinant plasmids, transgenic mice were prepared by microinjection. The first mouse and PCR-positive progeny mice were crossed with C57BL / 6 mice. Selected F2 mice were hybridized with transgenic mouse Lap that has been stably established, and double-stained with Doxy-induced Dox in the liver for stable expression of the luciferase (Luc) reporter gene by the in vivo bioluminescence imaging system (BLI) Transgenic progeny mice were passaged and expanded selectively for stable expression lines. Results Enzyme digestion and sequencing analysis showed that the recombinant vector was successfully constructed. Six transgenic mice were obtained by PCR. Three of them were passaged and the positive progeny mice were detected continuously. The results of BLI showed that some of the double transgenic mice from different F2 mice passaged from one of the first mice and the second mouse showed strong luminescence signals, indicating that luc gene was highly expressed in liver cells of mice. Conclusion The transgenic mouse NS3 / 4A was established and transfected into the transgenic mice stably expressing exogenous genes in the presence of regulatory factors, which laid the foundation for further establishment of a mouse model of tightly regulated NS3 / 4A protease expression.