重组anti-CD20 scFv-CD80-CD28-ζ非复制型逆转录病毒构建.docVIP

重组anti-CD20 scFv/CD80/CD28/ζ非复制型逆转录病毒构建 　【摘要】 目的:构建pLNCX/anti-CD20 scFv/CD80/CD28/ζ的重组真核表达载体，转染PA 317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PA 317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NIH 3T3细胞株，用PCR、流式细胞术检测目的基因在NIH 3T3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-CD20scFv/CD80/CD28/ζ的成功构建；采用PCR方法能够从逆转录病毒感染的NIH 3T3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NIH 3T3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。 【关键词】 单链抗体 逆转录病毒 PA 317细胞 基因表达 NIH 3T3细胞 Abstract: 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. Key words: single chain fragment; retrovirus; PA 317 cells; gene expression; NIH 3T3 cells 机体抗肿瘤免疫主要是细胞免疫，而肿瘤细胞一般不表达或低表达B7分子及其他黏附分子，不能与CD28相互作用，从而难以诱导肿瘤特异性细胞免疫，使肿瘤逃避宿主免疫杀伤作用。为此，我们利用anti-CD20单链抗体（scFv）、CD80穿膜蛋白及T细胞活化信号CD28-CD3ζ构建表达pLNCX/anti-CD20scFv/CD80/CD28/ζ的非复制型高滴度逆转录病毒，为以后该逆转录病毒转染原代T淋巴细胞从而制备CD20靶向性嵌合锚定T细胞奠定基础。 1 材料和方法 1.1 材料 含anti-CD20 scFv/CD80的pLNCX质粒由华盛