可调控性uPA诱导表达系统的构建及功能鉴定

The establishment of the u PA inducible expression system

目的构建可调控性尿激酶型纤溶酶原激活物(urokinase-type plasminogen activator,u PA)诱导表达系统并进行相关鉴定,可为进一步构建可诱导型肝脏人源化u PA-SCID动物模型奠定基础。方法可调控性u PA诱导表达系统即在强力霉素(doxycycline,Dox)诱导下通过tet-on系统调控u PA表达。为此,需要构建两个重组质粒,分别为p LNHXO1O2-Alb-GLUC-FMN2A-rt TA和p LNHXO5O6-TRE2-u PA-IRES-Zs Green,前者引入Gaussia荧光素酶(gaussia enzyme fluorescent element,Gluc)与四环素反式激活因子(reverse tetracycline transactivator,rt TA)偶联表达,以便于监测rt TA表达水平;后者同时偶联表达Zs Green,以便于用荧光显微镜观察基因表达。重组逆转录病毒载体进行表达功能鉴定后,将构建质粒与病毒包装衣壳蛋白VSV-G质粒共转染GP2-293包装细胞系后获得具有感染能力的病毒颗粒,利用该病毒感染NIH/3T3细胞,并用G418筛选出阳性细胞;期间各取部分细胞,使用PCR方法鉴定其基因组内是否含有相应基因转录调控本。结果成功构建p LNHXO1O2-Alb-GLUC-FMN2A-rt TA和p LNHXO5O6-TRE2-u PA-IRES-Zs Green克隆。功能鉴定显示,p LNHXO1O2-Alb-GLUC-FMN2A-rt TA克隆可表达rt TA;p LNHXO5O6-TRE2-u PA-IRES-Zs Green克隆转染Hep G-Tet-on细胞,加Dox可诱导u PA表达。包装病毒感染NIH/3T3细胞后通过G418筛选获得单细胞克隆,通过PCR方法鉴定表明其基因组内含有相应转录本,从而成功构建u PA诱导表达稳定细胞株。结论本研究初步建立了可调控性u PA诱导表达系统,从而为可诱导肝损的u PASCID转基因小鼠的构建及在此基础上的肝脏人源化动物模型的建立奠定了基础。

Objective To establish uPA inducible expression system using recombinant retroviral system for the further construction of inducible uPA-SCID animal model. Methods The Inducible expression system need to construct two plasmids： p LNHXO1O2-Alb-GLUC-FMN2A-rtTA and p LNHXO5O6-TRE2-uPA-IRES-ZsGreen respectively. Both plasmids were based on retroviral vector p LNHX,Albumin promoter gene（ Alb） and rtTA gene or uPA gene and ZsGreenwere obtained by PCR reaction and inserted into p LNHX. The Gaussia enzyme fluorescent element（ GLUC） was used to monitor rtTA expression in p LNHXO1O2-Alb-GLUC-FMN2A-rtTA,and the ZsGreen for uPA expression monitoring in p LNHXO5O6-TRE2-uPA-IRES-ZsGreen. The correct constructed plasmids were transfected into packaging cell line GP2-293 to gain recombinant viral particles. NIH /3T3 cells were infected with these viral particles and selected with G418. Gene expression in the surviving cells was confirmed by the PCR method. Results The recombinant retroviral vectors harbouring target genes were successfully cloned. The rtTA gene in p LNHXO1O2-Alb-GLUC-FMN2A-rtTA was expressed,and uPA can be induced to express in p LNHXO5O6-TRE2-uPA-IRES-ZsGreen by doxycycline（ Dox） when the plasmid transfected into the HepG-Tet-on cell. The constructed recombinant two retroviral vectors were transfected into GP2-293 packaging cells respectively to gain infectious viral particles. Then,NIH /3T3 cells were infected with these viral particles and singlecell clones which stably expressed the transgenes were successfully established. Conclusion This study primarily established uPA inducible expression system,it laid a foundation for the murine model of inducible liver damage,and provided a novel technical platform for further building the liver humanised murine models for viral hepatitis studying.