pLIVE-IL-1β表达载体的构建及在Hepa1-6细胞的表达

Establishment of the Recombinant Murine IL-1β Vector pLIVE-IL-1β and Its Expression in Hepa1-6 Cells

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 establish a murine IL-1β expression vector for in vivo assay and analysis its specific expression in Hepal-6 hepatoma ceils. Methods Total RNA was prepared from peripheral blood monocytes （PBMCs） of BALB/C mouse treated with LPS（ 5 g/ml） ,full length 1L-1β gene（ 822bp） was obtained through RT-PCR ,purified PCR product digested with Xho Ⅰ and Nhe Ⅰ was cloned into pLIVETM to construct the recombinant murine IL-1β expression vector for in vivo research. Positive recombinant vector was verified through colony PCR,restriction enzyme assay（ Xho Ⅰand Nhe Ⅰ ） and DNA sequencing. Purified pLIVE-IL-1β plasmid was transfected into Hepa1-6 hepatoma cells using TransITTM. The expression level of IL-1β was detected by RT-PCR 48h after transfection and compared with cells with pLIVE. Results A 822bp gene segment were amplified from total RNA isolated from PBMCs treated with LPS which was compatible to the sequence expected. Purified PCR product and pLIVETM digested with Xho Ⅰ and Nhe Ⅰ were ligated together to create the recombinant IL-1β expression vector pLIVE-IL-1β. When screening by colony PCR, it was showed that we got several positive clones,further assay by restriction enzyme assay indicated that positive clones gave rise to a 822bp DNA fragment which was finally proved to be identical to the full length murine IL-1β reported by GeneBank. The recombinant vector were then transfeeted into Hepa1-6 hepatoma cells,48h after transfection, RT-PCR indicated that the expression level of IL-1β was significantly enhanced compare with cells transfected with pLIVETM. Conclusion The authors successfully constructed a murine IL-1β expression vector pLIVE-IL-1β for in vivo research, the recombinant vector could stably express high level of IL-1β in murine hepatoma cells.