人白细胞介素12双亚基共表达pVAX1-IRES-hIL12载体的构建与表达  

Construction of human interleukin 12 double subunits co-expressing pVAX1-IRES-hIL12 vector and its expression

作　　者：王伟[1,2] 高江平[2] 阎瑾琦[1] 肖毅[1,2] 任杰[1,2] 王宇[1] 江乐[1] 于继云[1] 

机构地区：[1]军事医学科学院基础医学研究所,北京100850 [2]解放军总医院泌尿外科,北京100853

出　　处：《军医进修学院学报》2010年第6期600-603,共4页Academic Journal of Pla Postgraduate Medical School

基　　金：国家"863"计划项目(2007AA02Z451);国家自然科学基金项目(30772002;30840094)~~

摘　　要：目的构建下游可以共表达人白细胞介素12(hIL12)双亚基的双顺反子真核表达载体pVAX1-IRES-hIL12。方法通过搭桥PCR获得人白细胞介素12P35及P40双亚基的融合基因P35-F2A-P40,插入DNA疫苗载体pVAX1-IRES的下游,瞬时转染293-T细胞,ELISA检测融合基因的表达。结果酶切鉴定和序列分析表明融合基因与设计完全一致,融合基因在体外细胞培养液检测中获得分泌表达。结论该载体的成功构建可以为肿瘤基因疫苗研制提供免疫增效载体。Objective To construct the bicistronic eucaryotic expression vector of human interleukin 12（hIL12）pVAX1-IRES-hIL12 co-expressing the hIL12 double subunits.Methods The fusion gene P35-F2A-P40,amplified by overlap extension PCR,was inserted into the downstream of the eukaryotic expression vector pVAX1-IRES.Then the recombinant plasmid pVAX1-IRES-hIL12 was transfected to 293T cells,and its expression was detected by ELISA.Results Enzyme digestion and sequence analysis showed that the bicistronic eucaryotic expression vector pVAX1-IRES-hIL12 was successfully constructed.The expression of recombinant plasmid was detected by ELISA,suggesting that the fusion genes can express in 293T cells.Conclusion Successful construction of the vector can provide an effective immune tool for the development of anti-tumor vaccine.

关 键 词：白细胞介素12 基因融合 真核细胞 IRES 

分 类 号：R392.11[医药卫生—免疫学]