Rspo1-EGFP重组腺相关病毒载体的构建

Construction of Recombinat Adeno-assosiated Virus Vector Containing a Fusion Rspo1-EGFP Gene

本文通过构建携带Rspo1-EGFP融合基因的重组腺相关病毒载体,包装出含有目标基因的重组腺相关病毒。首先利用PCR从cDNA文库中把Rspo1基因扩增出来,插入到pcDNA6-EGFP中EGFP的上游形成融合基因Rspo1-EGFP。采用AAVHelper-free包装系统,将融合基因用PCR方法从质粒pcDNA6-Rspo1-EGFP上扩增出来,亚克隆到AAV表达质粒pAAV-MCS多克隆位点中,构建出重组质粒pAAV-Rspo1-EGFP。重组质粒与包装质粒pAAV-RC和辅助质粒pHelper磷酸钙法共转染AAV-293细胞制备重组病毒rAAV-Rspo1-EGFP。重组病毒感染AAV-HT1080细胞,荧光显微镜检测病毒介导的目标基因表达,流式细胞技术测定病毒滴度。结果:酶切鉴定和测序确定重组病毒载体pAAV-Rspo1-EGFP构建成功。病毒感染的细胞中检测到绿色荧光,表明重组病毒包装成功并介导融合基因在宿主细胞里表达,病毒滴度达107VP/mL。本研究成功包装出具有侵染性的重组腺相关病毒AAV-Rspo1-EGFP,为今后利用腺相关病毒载体进行Rspo1相关的体内体外研究提供了实验基础。

In this paper, recombinant Adeno-assosiated virus （AAV） vector was constructed containing a fusion Rspol-EGFP protein. Rspol gene was first amplified from a library clone by PCR and then inserted into plasmid pcDNA6-EGFP, fusing with the enhanced green fluorescent protein （EGFP） gene downstream. The EGFP-1abeled Rspol was then subcloned into plasmid pAAV-MCS. Recombinant plasmid pAAV-Rspo1-EGFP was co-transfected into AAV-293 cells with pAAV-RC and pHelper for recombinant AAV packaging. Expression of fusion Rspo1-EGFP protein mediated by AAV was detected in the AAV-infected HT1080 cells under fluorescent microscope, while viral titering was measured by flow cytometry （FCM）. Results showed that the recombinant plasmid pAAV-Rspo1-EGFP was verified by double digestion and sequence analysis. The detected green fluorescence indicated that recombinant AAV was successfully packaged and mediating gene expression, with viral titer as high as 107 VP/mL. This research provided references for further experiments of Rspol analysis.