AFP启动子调控的小鼠IL-1β重组载体的构建及其在肝癌H22细胞中的表达

Establishment of AFP promoter operated murine IL-1β recombinant vector and its expression in H22 cells

目的:构建小鼠甲胎蛋白(AFP)启动子调控的小鼠IL-1β(mIL-1β)重组真核表达载体pafpIRES2-EGFP-mIL-1β,并检测其在真核细胞内的表达。方法:重叠延伸PCR技术(SOE-PCR)获得含小鼠AFP最小启动子及巨细胞病毒(CMV)增强子区(ECMV)的嵌合基因,将其克隆至pIRES2-EGFP载体,构建肝癌细胞特异性表达载体pafpIRES2-EGFP。RT-PCR扩增小鼠IL-1β基因,克隆至pafpIRES2-EGFP,进行酶谱分析及DNA序列测定。利用jetPEI法将其分别转染H22细胞和YAC-1细胞,倒置相差荧光显微镜下观察,RT-PCR分析mIL-1β表达水平的变化。结果:SOE-PCR方法获得长度为537bp的AFP和ECMV嵌合基因,克隆后经限制性酶切和DNA序列分析确认成功构建了小鼠肝癌细胞特异性表达载体pafpIRES2-EGFP。PCR扩增小鼠IL-1β基因后将其克隆至pafpIRES2-EGFP,获得AFP启动子调控的小鼠IL-1β重组真核表达载体pafpIRES2-EGFP-mIL-1β,细胞转染分析证实,该载体能够在小鼠肝癌细胞中特异性高效表达,RT-PCR检测可见转染细胞中mIL-1β水平明显升高。结论:成功构建真核表达载体pafpIRES2-EGFP-mIL-1β,该载体能够在小鼠肝癌细胞中特异性表达IL-1β。

AIM： To establish a hepatoma specific murine IL-1β（mIL-1β） expression vector operated by AFP promoter and analysis its expression in H22 cell. METHODS： The chimeric operating sequence composed of the minimal AFP promoter and CMV enhancer （ECMV） was prepared through SOE-PCR. The sequence was inserted to replace the conventional enhancer and promoter in plRES2-EGFP to establish the novel hepatoma specific vector Pafp IRES2-EG-FP. Full length of murine IL-1β was amplified through RT-PCR by pfu DNA polymerase followed by cloning to establish the recombinant pIRES2-EGFP-mIL-1β expression vector verified through PCR, restriction enzyme assay, DNA sequencing and cell transfection. Pafp IRES2-EGFP-mIL-1β was tranfected into H22 hepatoma cells and YAC-1 lymphoma cells in a transient transfection system mediated by jet- PEI. Expression of the vector was observed under fluorescent microscope 48 h after transfection. Expression level of mIL-1β was detected by RT-PCR. RESULTS ： A 537 bp chimeric AFP promoter and ECMV was yield and inserted to establish a novel hepatoma specific vector Pafp IRES2-EGFP proved by restriction enzyme assay, DNA sequencing and transfection. Full length murine IL-1β was then amplified and cloned to establish the recombinant expression vector Pafp IRES2-EGFP-mIL-1β verified through repeated clony PCR,restriction enzyme assay by EcoR I and Xho I, DNA sequencing and transfection. Purified Pafp IRES2-EGFP-mIL-1β was transiently transfected into H22 cells and YAC-1 cells by jetPEI, and bright green fluorescence was only seen on the surface of H22 cells, indicating that Pafp IRES2-EGFP-mIL-1β can specifically express target gene within the murine hepatoma cells. Simutaneously, the expression level of mIL-1β was markedly elevated in H22/mIL-1β in RT-PCR assay. CONCLUSION： We successfully prepared a hepatoma specific expression vector named Pafp IRES2-EGFP-mIL-1β that could expression high level of murine IL-1β in a transient transfection system.