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目的构建应用于体内表达和研究的小鼠IL-1β肝癌细胞特异性表达载体,观察其在小鼠HE-PA1-6细胞中的表达水平。方法分离BALB/c小鼠外周血淋巴细胞,LPS刺激后提到总RNA,RT-PCR扩增IL-1β基因,与pLIVETM连接构建pLIVE-IL-1β重组表达载体,并通过菌落PCR、限制性酶谱分析、DNA序列分析进行鉴定。利用TransITTM将pLIVE-IL-1β转染至Hepa1-6肝癌细胞,RT-PCR分析IL-1β的表达水平。结果从小鼠腹腔巨噬细胞中RT-PCR扩增得到一822bp的片段,纯化的PCR产物与pLIVETM同时经XhoⅠ和NheⅠ双酶切后连接,菌落PCR鉴定获得多个阳性克隆,经质粒XhoⅠ+NheⅠ限制性酶谱分析,酶解出822bp的条带,DNA序列分析证实重组载体中的DNA序列与GeneBank登录的小鼠IL-1β基因一致。将该载体转染Hepa1-6细胞,RT-PCR证实,与转染pLIVETM的对照细胞相比,IL-1β的表达水平明显升高。结论成功构建了小鼠IL-1β肝癌组织特异性表达载体pLIVE-IL-1β,该载体能够在小鼠肝癌细胞中稳定高效表达IL-1β。
Objective To construct a specific expression vector for mouse IL-1β hepatocellular carcinoma cells used in vivo expression and study, and to observe its expression level in mouse HE-PA1-6 cells. Methods BALB / c mouse peripheral blood lymphocytes were isolated and total RNA was extracted after LPS stimulation. IL-1β gene was amplified by RT-PCR and ligated with pLIVETM to construct pLIVE-IL-1β recombinant vector. Zymogram analysis, DNA sequence analysis. PLIVE-IL-1β was transfected into Hepa 1-6 hepatoma cells by TransITTM, and the expression level of IL-1β was analyzed by RT-PCR. Results A 822bp fragment was amplified by RT-PCR from mouse peritoneal macrophages. The purified PCR product was ligated with pLIVETM by digestion with XhoⅠand NheⅠ, and several positive clones were obtained by colony PCR. The positive clones were identified by XhoⅠ + NheⅠ Restriction enzyme digestion analysis revealed a 822bp band, and DNA sequence analysis confirmed that the DNA sequence in the recombinant vector was identical to the mouse IL-1β gene registered in GeneBank. The vector was transfected into Hepa 1-6 cells and RT-PCR confirmed that the expression level of IL-1beta was significantly increased compared to the control cells transfected with pLIVETM. Conclusion The tissue-specific expression vector pLIVE-IL-1β of murine IL-1β hepatocellular carcinoma was successfully constructed, which can stably and efficiently express IL-1β in mouse hepatoma cells.