小鼠血管内皮生长因子慢病毒载体的构建及其在NS-1小鼠骨髓瘤细胞株中的表达  

Construction of Lentiviral Vector Containing Murine Vascular Endothelial Growth Factor Gene and Its Expression in NS-1 Murine Myeloma Cell Line

作　　者：杨金荣[1,2] 曾科[1,2] 李军[1] 牛挺[1,2] 

机构地区：[1]四川大学华西医院血液科及血液学研究室,成都610041 [2]四川大学生物治疗国家重点实验室,成都610041

出　　处：《华西医学》2013年第7期998-1002,共5页West China Medical Journal

摘　　要：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 murine vascular endothelial growth factor（mVEGF） lentiviral expression vector,package it into lentiviral particles and infect NS-1 cell line,to obtain the mVEGF expression in NS-1 cells,in order to build a foundation for further research into the pathophysiologic role of VEGF in multiple myolema.Methods The target gene mVEGF was amplified from the pCDNA3.1-mVEGF by PCR,and then it was cloned into lentiviral expression plasmid pCDH.Human embryonic kidney 293FT cells were co-transfected with the three plasmids（pCDH-mVEGF,psPAX2,pMD2.G） by means of calcium phosphate method.Forty-eight h and 72 h later,the viral supernatant was collected and used to infect NS-1 cells.Seventy-two h after infection,the infected NS-1 cells were selected by puromycine,and the selection lasted for 2 weeks.Then the mVEGF expression in the cell culture supernatant was detected by ELISA.Results The recombinant lentiviral vector pCDH-mVEGF was constructed,and was successfully packaged into lentiviral particles.The NS-1 cells were infected and mVEGF was stably expressed in a high level in NS-1 cells.Conclusions The lentiviral vector pCDH-mVEGF has been constructed and packaged into lentiviral particles which successfully has infected the NS-1 cells and mediated the stable expression of target gene mVEGF in NS-1cells.Therefore,it provides a basis for further research on the pathophysiologic role of VEGF in multiple myeloma.

关 键 词：小鼠血管内皮生长因子 慢病毒载体 NS-1细胞株 多发性骨髓瘤 

分 类 号：R733.3[医药卫生—肿瘤]