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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。
OBJECTIVE: To construct the recombinant eukaryotic expression vector pafpIRES2-EGFP-mIL-1β of mouse IL-1β (mIL-1β) regulated by mouse AFP promoter and to detect its expression in eukaryotic cells. Methods: The chimeric gene containing mouse AFP minimal promoter and cytomegalovirus (CMV) enhancer region (ECMV) was obtained by overlap extension PCR (SOE-PCR) and cloned into pIRES2-EGFP vector to construct hepatocellular carcinoma cell-specific Sex expression vector pafpIRES2-EGFP. The mouse IL-1β gene was amplified by RT-PCR and cloned into pafpIRES2-EGFP for enzyme digestion and DNA sequencing. The transfected cells were transfected into H22 cells and YAC-1 cells respectively by jetPEI method. The expression of mIL-1β was analyzed by reverse transcription polymerase chain reaction (RT-PCR). Results: AFP and ECMV chimeric genes with a length of 537bp were obtained by SOE-PCR. After cloning, restriction analysis and DNA sequence analysis confirmed that the mouse hepatoma cell-specific expression vector pafpIRES2-EGFP was successfully constructed. The mouse IL-1β gene was amplified by PCR and cloned into pafpIRES2-EGFP. The recombinant murine IL-1β eukaryotic expression vector pafpIRES2-EGFP-mIL-1β was obtained by AFP promoter. Can express specifically and specifically in mouse hepatoma cells, and the level of mIL-1β in transfected cells is obviously increased by RT-PCR. CONCLUSION: The eukaryotic expression vector pafpIRES2-EGFP-mIL-1β was successfully constructed and was able to express IL-1β specifically in mouse hepatoma cells.