肿瘤基因疫苗免疫增效载体pVAX1-IRES-GM/B7的构建与表达  

Cloning and expression of fusion gene of human GM-CSF and B7.1 in bicistronic eukaryotic vector pVAX1-IRES

作　　者：刘宁[1] 阎瑾琦[2] 冉多良[1] 贾锐[2] 张亮[2] 王浩[2] 于继云[2] 

机构地区：[1]新疆农业大学动物医学学院,乌鲁木齐830052 [2]军事医学科学院基础医学研究所,北京100850

出　　处：《军事医学科学院院刊》2008年第3期249-252,共4页Bulletin of the Academy of Military Medical Sciences

基　　金：国家“863”计划专项课题(2007AA02Z451);国家自然科学基金课题(30772002)

摘　　要：目的:构建上游可以表达肿瘤抗原复合物,下游表达人GM-CSF和B7.1免疫协同增效分子的双顺反子真核表达载体pVAX1-IRES-GM/B7。方法:通过搭桥PCR获得人GM-CSF和B7.1融合基因,插入已构建好的DNA疫苗载体pVAX1-IRES的下游,瞬时转染293T细胞,通过流式细胞术和间接免疫荧光检测融合基因的表达。此外,还在载体上游插入肿瘤抗原复合物,进一步验证该载体对肿瘤抗原的表达情况。结果:酶切鉴定和序列分析表明,GM/B7融合基因与设计完全一致,在体外细胞检测中获得表达,而且上游的抗原基因也可以顺利表达。结论:该载体的构建成功可以为肿瘤基因疫苗研制提供免疫增效载体。Objective：To construct a bicistronic eukaryotic expression vector pVAX1- IRES-GM/B7 to express the fusion gene of human GM-CSF and B7.1. Methods： The fusion gene was amplified by the overlap extension PCR, and inserted into the downstream of the eukaryotic expression vector pVAX1-IRES. Then the recombinant plasmid pVAX1- IRES- GM/B7 was transfected to the 293T cells, and the expression was detected by FACS and IMF. In addition, the recombinant tumor antigen was cloned into the upstream of the vector pVAX1- IRES-GM/B7 and the expression of the recombinant antigen gene was also detected in the 293T cells. Results： The enzyme digestion and sequence analysis showed that the bicistronic eukaryotic expression vector pVAX1- IRES-GM/B7 was constructed successfully. The expression of recombinant plasmid was demonstrated by FACS and IMF. It was shown that the fusion genes of GM/B7 and the recombinant antigen gene could be expressed in the 293T cells. Conclusion： The results provide necessary basis for the research on effective multitarget anti-tumor cancer vaccine in the future.

关 键 词：GM-CSF B7.1 基因融合 IRES 真核表达 癌症疫苗 抗原 肿瘤 

分 类 号：R730.3[医药卫生—肿瘤]