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目的构建含小鼠血管内皮生长因子(mVEGF)的重组慢病毒表达载体,包装成病毒颗粒后感染NS-1小鼠骨髓瘤细胞株,以便进一步探索VEGF在骨髓瘤病理生理机制中的作用。方法聚合酶链反应法扩增mVEGF基因,克隆入含嘌呤霉素抗性的pCDH慢病毒表达载体,构建出表达mVEGF的慢病毒表达载体pCDH-mVEGF;采用磷酸钙法将慢病毒系统三质粒pCDH-mVEGF、psPAX2、pMD2.G共转染293FT细胞包装病毒,分别收集转染后48 h和72 h病毒上清并感染靶细胞NS-1,初次感染72 h后开始采用嘌呤霉素筛选稳定株,筛选2周后采用ELISA法检测稳定株细胞培养上清中mVEGF的表达,建立出稳定高表达mVEGF的NS-1小鼠骨髓瘤细胞株。结果成功构建重组慢病毒表达质粒pCDH-mVEGF,并包装成慢病毒颗粒,感染NS-1细胞株后获得靶基因的稳定高表达。结论成功构建出含mVEGF的慢病毒表达载体pCDH-mVEGF,慢病毒系统能有效介导目的基因在NS-1小鼠骨髓瘤细胞株中稳定表达,病毒包装成功并能有效感染NS-1细胞,为进一步探索VEGF在骨髓瘤病理生理机制中的作用奠定了基础。
Objective To construct a recombinant lentiviral vector containing mouse vascular endothelial growth factor (mVEGF) and inject it into a cell line of NS-1 murine myeloma cells after being packaged into virosomes to further explore the role of VEGF in the pathophysiology of myeloma. Methods The mVEGF gene was amplified by polymerase chain reaction and cloned into puromycin-resistant pCDH lentiviral vector. The lentiviral vector pCDH-mVEGF expressing mVEGF was constructed. The lentiviral vector pCDH -mVEGF, psPAX2, pMD2.G were co-transfected 293FT cell packaging virus were collected 48 h and 72 h after the virus supernatant and infected with the target cell NS-1, 72 h after the initial infection began using puromycin screening stable strains After 2 weeks of selection, the expression of mVEGF in stable cell culture supernatant was detected by ELISA and a NS-1 mouse myeloma cell line stably expressing mVEGF was established. Results The recombinant lentiviral plasmid pCDH-mVEGF was successfully constructed and packaged into lentivirus particles. The stable and highly expressed target gene was obtained after infection with NS-1 cells. Conclusion The lentiviral vector pCDH-mVEGF containing mVEGF was successfully constructed. The lentiviral vector can effectively induce the target gene to express stably in the NS-1 mouse myeloma cell line. The virus was successfully packaged and effectively infected NS-1 cells. Which laid the foundation for further exploring the role of VEGF in the pathophysiology of myeloma.