新型假型逆转录病毒载体的构建  

Construction of a new type of pseudotyped murine leukemia retrovirus vector(VSV-G/MuLV)

作　　者：陈勇兵[1] 陈如坤[2] 陈力[2] 吴明[2] 施立[1] 杨文涛[1] 钱永跃[1] 

机构地区：[1]苏州大学附属第二医院胸心外科,215004 [2]浙江大学附属第二医院胸心外科

出　　处：《江苏医药》2004年第9期644-646,F003,i001,共5页Jiangsu Medical Journal

基　　金：苏州大学青年基金;浙江省医药卫生科学研究基金(2000A110)

摘　　要：目的 将疱疹性口炎病毒壳G糖蛋白(VSV-G)用于构建新型假型逆转录病毒载体(VSV-G/MuLV)。方法 利用三质粒系统一过性转染293T/17细胞,通过磷酸钙共沉淀法产生VSV-G/MuLV;一过性转染后的病毒上清转导包装细胞系293/GPC以产生VSV-G/MuLV生产细胞系。G418筛选未成功转导的细胞,经筛选后的293/GPG细胞生产VSV-G/MuLV;用病毒上清转染NIH3T3细胞,并采用G418克隆筛选法测定病毒滴度。结果 感染后的NIH3T3细胞在G418的筛选培养下,有大量克隆形成。经计数和计算,VSV-G/MuLV病毒上清滴度为6×106。结论 成功构建了VSV-G/MuLV;该型病毒转染效率高,可直接转导大多数细胞,在有效监控条件下,VSV-G/MuLV作为载体可用于基因治疗。Objective A new type of pseudotyped MuLV(Murine leukemia retrovirus, MuLV) vector(VSV-G/MuLV)was constructed using the VSV-G(G glycoprotein of the vesicular stomatitis virus). Methods VSV-G/MuLV was produced from transient transfection of 293T/17 cell line with 3 plasmids system with the method of calcium phosphate precipitation; The producer cell 293/GPG for VSV-G/MuLV was generated; The titer of VSV-G/MuLV was determined by the means of G418 filtration. Results Clones of NIH3T3 transfected with VSV-G/MuLV were formed and the virus titer of VSV-G/MuLV was 6×106. Conclusion The new vector VSV-G/MuLV which was successfully constructed would be transduced into most of cells and be used for gene therapy in the future.

关 键 词：LV 转导 VSV 逆转录病毒载体 转染 细胞 一过性 克隆 筛选 质粒 

分 类 号：R735[医药卫生—肿瘤] R540.45[医药卫生—临床医学]