重组anti－CD20 scFv／CD80／CD28/ζ非复制型逆转录病毒的构建及其在Jurkat细胞株中的表达  

CONSTRUCTION OF RECOMBINANT RETROVIRUSES EXPRESSING ANTI-CD20 SCFV/CD80/CD28/ζ GENE AND EXPRESSION IN JURKAT CELL LINE

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

机构地区：[1]温州医学院附属第一医院血液内科 [2]医学科学研究所,浙江温州325000 [3]美国华盛顿大学Bio-Rad实验室主任

出　　处：《中国应用生理学杂志》2008年第3期343-347,共5页Chinese Journal of Applied Physiology

基　　金：浙江省自然科学基金资助项目(302781)

摘　　要：目的:构建表达anti-CD20 scFv/CD80/CD28/ζ重组非复制型逆转录病毒,转染Jurkat细胞株使其表达目的蛋白。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,收获上清液获非复制型逆转录病毒,感染NI H3T3细胞株,用PCR、流式细胞术检测目的基因在NI H3T3细胞的表达情况,确定病毒滴度。挑取高滴度的包装细胞株收获病毒,感染Jurkat细胞,经G418筛选细胞,用RT-PCR检测目的基因表达情况。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;用PCR能够从逆转录病毒感染的NI H3T3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NI H3T3细胞中表达目的蛋白;经RT-PCR,能够从转染的Jurkat细胞株中扩增出1条与目的基因大小一致的DNA片段。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Jurkat细胞中表达目的蛋白。Aim： To construct recombinant retroviruses expressing anti-CD20 scFv/CD80/CD28/ζ gene and detect its expression in Jurkat cells. Method： CD28- ζ cDNA were amplified from plasmids pBULLET and inserted into pLNCX vector that contained anti-CD20 scFv/CD80 gene. The recombinant plasmids were transfected into PA 317 cells. Retroviruses were harvested from culture medium of PA 317 cells. Then NIH 3T3 were transfected with retroviruses. Objective gene expression was determined by PCR and FACS. Jurkat cells were transfected with high titer of retroviruses and resistant clones were obtained by G418 selection. Objective mRNA was determined by RT PCR. Results： The recombinant eukaryotic vector was constructed successfully by PCR and enzyme digestion analysis and objective gene was amplified from NIH 3T3 cells transfected with retroviruses by PCR; FACS showed that objective protein could be expressed in NIH 3T3 cells. Objective gene was amplified from Jurkat cells transfected with retroviruses by RT PCR. Conclusion： Recombinant retrovirus expressing anti-CD20 scFv/CD80/CD28/ζ gene was successfully constructed and objective protein could be expressed in Jurkat 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 JURKAT细胞 

分 类 号：R73-3[医药卫生—肿瘤]