Li Qing-chun, Liang Shu-juan, Wang Xue-jing, Liu Yan-yan, Wang Huan-qin, Zhang Su-hua
Department of Immunology in Weifang Medical College, Key Lab for Immunology in Universities of Shandong, Weifang 261042, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2009 Jul;25(7):603-5.
To establish a hepatoma specific murine IL-1beta (mIL-1beta) expression vector operated by AFP promoter and analysis its expression in H22 cell.
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 pIRES2-EGFP to establish the novel hepatoma specific vector p(afp)IRES2-EGFP. Full length of murine IL-1beta was amplified through RT-PCR by pfu DNA polymerase followed by cloning to establish the recombinant pIRES2-EGFP-mIL-1beta expression vector verified through PCR, restriction enzyme assay, DNA sequencing and cell transfection. p(afp)IRES2-EGFP-mIL-1beta was tranfected into H22 hepatoma cells and YAC-1 lymphoma cells in a transient transfection system mediated by jetPEI. Expression of the vector was observed under fluorescent microscope 48 h after transfection. Expression level of mIL-1beta was detected by RT-PCR.
A 537 bp chimeric AFP promoter and ECMV was yield and inserted to establish a novel hepatoma specific vector p(afp)IRES2-EGFP proved by restriction enzyme assay, DNA sequencing and transfection. Full length murine IL-1beta was then amplified and cloned to establish the recombinant expression vector p(afp)IRES2-EGFP-mIL-1beta verified through repeated clony PCR, restriction enzyme assay by EcoR I and Xho I, DNA sequencing and transfection. Purified p(afp)IRES2-EGFP-mIL-1beta 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 p(afp)IRES2-EGFP-mIL-1beta can specifically express target gene within the murine hepatoma cells. Simutaneously, the expression level of mIL-1beta was markedly elevated in H22/mIL-1beta in RT-PCR assay.
We successfully prepared a hepatoma specific expression vector named p(afp)IRES2-EGFP-mIL-1beta that could expression high level of murine IL-1beta in a transient transfection system.
构建由甲胎蛋白（AFP）启动子调控的肝癌特异性小鼠白细胞介素-1β（mIL-1β）表达载体，并分析其在H22细胞中的表达情况。
通过重叠延伸PCR（SOE-PCR）制备由最小AFP启动子和巨细胞病毒增强子（ECMV）组成的嵌合调控序列。将该序列插入pIRES2-EGFP中替换传统的增强子和启动子，构建新型肝癌特异性载体p(afp)IRES2-EGFP。用pfu DNA聚合酶通过RT-PCR扩增小鼠IL-1β全长，随后克隆构建重组pIRES2-EGFP-mIL-1β表达载体，经PCR、酶切鉴定、DNA测序及细胞转染验证。在jetPEI介导的瞬时转染系统中，将p(afp)IRES2-EGFP-mIL-1β转染至H22肝癌细胞和YAC-1淋巴瘤细胞。转染48小时后在荧光显微镜下观察载体的表达情况。通过RT-PCR检测mIL-1β的表达水平。
获得一条537 bp的AFP启动子与ECMV的嵌合序列，并插入构建了新型肝癌特异性载体p(afp)IRES2-EGFP，经酶切鉴定、DNA测序及转染验证。随后扩增并克隆小鼠IL-1β全长，构建重组表达载体p(afp)IRES2-EGFP-mIL-1β，经多次克隆PCR、EcoR I和Xho I酶切鉴定、DNA测序及转染验证。用jetPEI将纯化的p(afp)IRES2-EGFP-mIL-1β瞬时转染至H22细胞和YAC-1细胞，仅在H22细胞表面可见明亮的绿色荧光，表明p(afp)IRES2-EGFP-mIL-1β能在小鼠肝癌细胞内特异性表达目的基因。同时，RT-PCR检测显示H22/mIL-1β中mIL-1β的表达水平显著升高。
我们成功制备了一种肝癌特异性表达载体p(afp)IRES2-EGFP-mIL-1β，其在瞬时转染系统中可高水平表达小鼠IL-1β。
