重组anti-CD20 scFv/CD80/CD28/ζ非复制型逆转录病毒的构建  

Research on recombinant retroviruses entegrated with anti-CD20 scFv/CD80 /CD28/ ζ gene

作　　者：胡永仙[1] 俞康[1] 谭映霞[2] 沈志坚[1] 梁彬[1] 钱红兰[1] 单大名[3] 

机构地区：[1]温州医学院第一附属医院血液内科,浙江温州325000 [2]温州医学院第一附属医院肿瘤与移植免疫研究所 [3]美国华盛顿大学Bio-Rad实验室

出　　处：《温州医学院学报》2007年第5期433-435,439,共4页Journal of Wenzhou Medical College

基　　金：浙江省自然科学基金资助项目(302781);温州市科技合作交流项目(H2006B02)

摘　　要：目的:构建pLNCX/anti-CD20 scFv/CD80/CD28/ζ的重组真核表达载体,转染PA317细胞株,包装制备重组非复制型逆转录病毒。方法:采用DNA重组技术把pBULLET上的CD28-ζcDNA插入到已含anti-CD20 scFv/CD80的真核逆转录病毒载体pLNCX质粒上,转染PA317细胞株,经G418筛选,收获上清液获非复制型逆转录病毒,将病毒感染NIH3T3细胞株,用PCR、流式细胞术检测目的基因在NIH3T3细胞中的表达情况,确定病毒滴度。结果:经酶切及测序鉴定均证实pLNCX/anti-CD20 scFv/CD80/CD28/ζ的成功构建;采用PCR方法能够从逆转录病毒感染的NIH3T3细胞中扩增出一条与目的基因大小一致的DNA片段;流式细胞术检测显示该目的基因能够在NIH3T3细胞中表达目的蛋白。结论:成功构建高滴度表达anti-CD20 scFv/CD80/CD28/ζ非复制型逆转录病毒,并能在NIH 3T3细胞中表达目的蛋白。Objective： To construct recombinant eukaryotic expression vectors of pLNCX/anti-CD20scFv/CD80/CD28/ζ,transfect into PA 317 cells and package recombinant retroviruses. Methods：CD28-ζcDNA were amplified from plasmids pBULLET and inserted into pLNCX vectors that contained anti-CD20 scFv/ CD80 gene. The recombinant plasmids were transfected into PA 317 cells and resistant clones were obtained by G418 selection. Retroviruses were harvested from culture medium of PA 317 cells. Then NIH 3T3 were transfected with retroviruses. After G418 selection, objective gene expression was determined with PCR and FACS. Results： The recombinant eukaryotic vector was constructed successfully with PCR, enzyme digestion analysis and objective gene was amplified from NIH 3T3 cells transfected with retroviruses with PCR; FACS showed that objective protein could be expressed in NIH 3T3 cells. At the same time viral titer was estimated. Conclusion： Recombinant retroviruses expressing anti-CD20 scFv/CD80/CD28/ζ gene are successfully constructed and objective protein can be expressed in NIH 3T3 cells.

关 键 词：单链抗体 逆转录病毒 PA 317细胞 基因表达 NIH 3T3细胞 

分 类 号：Q46[生物学—生理学]