分泌型人IL-1β表达载体的构建及在H7402细胞中的表达  

Recombinational expression of classical pathway secreted human IL-1β in hepatoma H7402 cells

作　　者：肖伟玲[1] 林亚杰[2] 牟东珍[1] 孙萍[1] 梁淑娟[1] 

机构地区：[1]潍坊医学院免疫学重点实验室,山东潍坊261042 [2]山东大学齐鲁医院,济南250012

出　　处：《山东大学学报（医学版）》2008年第2期119-122,共4页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金资助课题(30772497);山东省中青年科学家科研奖励基金课题(2004BSB14074);教育部科学研究项目重点项目(205090);山东省卫生高层次人才1020工程专项资金资助课题

摘　　要：目的构建经典途径分泌型人白细胞介素1β(shIL-1β)重组表达载体,检测其在H7402肝癌细胞中的表达。方法采用SOE方法将人红细胞生成素(EPO)信号肽序列和编码人白细胞介素(hIL-1β)成熟蛋白的基因序列拼接形成融合基因,与pIRES2-EGFP质粒重组构建shIL-1β真核表达载体pIRES2-EGFP-shIL-1β,利用jetPEI方法将其稳定转染H7402细胞(H7402/shIL-1β),荧光显微镜及RT-PCR检测融合基因的表达水平,间接ELISA方法测定培养上清hIL-1β分泌水平。结果荧光显微镜下观察可见H7402/shIL-1β细胞发出明亮绿色荧光,RT-PCR检测显示pIRES2-EGFP-shIL-1β能够在H7402细胞中稳定表达融合基因mRNA,同时细胞培养上清中hIL-1β表达水平明显上升。结论成功构建了经典途径分泌的hIL-1β重组表达载体,该载体可在H7402细胞中稳定分泌生物活性的hIL-1β。Objective To construct a classical pathway expressing secretory human interleukin 1 beta （shIL-1β） recombinant vector and to analyze its expression level in H7402 hepatoma cells. Methods The total RNA was isolated from LPS stimulated healthy donor PBMCs, and the full length human interleukin 1 beta（hIL-1β） gene was obtained by RT-PCR. The human EPO signal peptide gene was prepared by primer overlapping extension, and fusion gene encoding both the EPO signal peptide and mature IL-1β protein was synthesized through gene splicing by over lap extension（SOE）. The product was directly cloned into plRES2-EGFP to construct the recombinant eukaryotic expression vector plRES2-EGFP-shIL-1β which was verified by PCR, restriction enzyme assay（ BamH I and EcoR I） and DNA sequencing. Purified plRES2-EGFP-shIL-1β was stably transfected into the H7402 hepatoma cells by jetPEI, then the expression of the recombinant vector was verified under fluorescence microscopy, and the level of fusion gene mRNA was determined by RT-PCR and of hlL- 1β in cellular supematant was assessed by EIJSA. Results The expression vector pIRKS2-EGFP-shIL-1β that could induce the expression of hIL-1β was successfully prepared through the conventional protein secretory pathway. After the recombinant vector was transfected and selected with G418, the human H7402/shIL-1β cells could effectively express the fusion gene mRNA, and the active hIL-1β in the cellular supematant was also significantly increased compared with the 1-17402/mock cells. Conclusion The recombinant plRES2-EGFP-shIL-1β vector that could express hIL-1β was successfully established through the conventional protein secretory pathway.

关 键 词：人白细胞介素1β 分泌表达 肝癌细胞 信号肽 

分 类 号：R392.12[医药卫生—免疫学]