医学论文人白细胞介素12双亚基共表达pVAX1IREShIL12载体的构建与表达.docVIP

医学论文：人白细胞介素12双亚基共表达pVAX1-IRES-hIL12载体的构建与表达 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12 P35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。 关键词：白细胞介素12；基因融合；真核细胞；IRES　文献标识码：A　　 文章编号：1005-1139(2010)06-0600-04 Construction of human interleukin 12 double subunits co-expressing pVAX1-IRES-hIL12vector and its expressionWANG Wei1,2,GAO Jiang-ping2,YAN Jin-qi1,XIAO Yi1,2,REN Jie2,WANG Yu1,JIANG Le1,YU Ji-yun11Institute of Basic Medical Sciences,Academy of Military Medical Sciences,Beijing 100850,China;2Department of Urology,Chinese PLA General Hospital,Beijing 100853,ChinaCorresponding author:YU Ji-yun.Email:yujyun@126.comAbstract: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,wasinserted into the downstream of the eukaryotic expression vector pVAX1-IRES.Then the recombinant plasmid pVAX1-IRES-hIL12was transfected to 293T cells,and its expression was detected by ELISA.Results Enzyme digestion and sequence analysis showedthat the bicistronic eucaryotic expression vector pVAX1-IRES-hIL12 was successfully constructed.The expression of recombinantplasmid was detected by ELISA,suggesting that the fusion genes can express in 293T cells.Conclusion Successful construction ofthe vector can provide an effective immune tool for the development of anti-tumor vaccine.Key words:hIL12;Gene Fusion;Eukaryotic Cells;IRESIL12是肿瘤和病毒免疫基因治疗的主要候选基因。由于hIL12是由分子量为40kD(P40)和35kD(P35)两个亚基构成的异源二聚体分子，发挥生物学效应需要P40和P35的共同作用，本实验研究将P40和P35两个亚基用自体切割肽F2A序列串联连接后克隆入真核表达载体pVAX1-IRES中，F2A由蛋白切割酶furin识别位点及来源于手足口病毒的2A序列连接构成[1]，构建成功的双亚基共表达质粒pVAX1-IRES-hIL12能够更接近hIL12基因的生物学作用模式，显著提高了hIL12的分泌型表达。因此，我们在本研究室已构建的双顺反子真核表达载体pVAX1-IRES的基础上，将人IL12融合基因插入载体IRES序列的下游，构建一个下游可以表达免疫协同增效分子的双顺反子真核表达载体pVAX1-IRES-hIL12，在基