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目的:构建小鼠经典分泌途径IL-1β真核表达载体,转染至Hepa1-6细胞,并测定其分泌表达水平。方法:引物延伸获得小鼠AFP信号肽序列(sAFP),RT-PCR扩增获得小鼠IL-1β成熟蛋白基因序列(mIL-1β),SOE方法将两段序列拼接形成融合基因,与pIRES2-EGFP质粒重组构建分泌型真核表达载体pIRES2-EGFP-smIL-1β,并将其转染至Hepa1-6细胞,RT-PCR检测融合基因的表达水平,Western blot检测细胞内mIL-1β蛋白的表达,ELISA法测定培养上清及细胞裂解液中mIL-1β水平。结果:经SOE法拼接获得的融合基因与GenBank中登录的序列一致,RT-PCR检测显示该载体能够在Hepa1-6细胞中表达融合基因mRNA,细胞培养上清中mIL-1β的分泌水平明显上升,而胞质中的mIL-1β无明显变化。结论:成功地构建了经典途径分泌的mIL-1β重组表达载体,该载体可以在Hepa1-6细胞培养上清中有效分泌具有生物活性的mIL-1β。
OBJECTIVE: To construct the eukaryotic expression vector of murine classic secretory pathway IL-1β and transfect it into Hepa1-6 cells. The secretion level of IL-1β was determined. Methods: The mouse AFP signal peptide sequence (sAFP) was obtained by primer extension and the mouse IL-1β mature protein gene sequence (mIL-1β) was obtained by RT-PCR. The SOE method was used to construct a fusion gene by two- EGFP plasmid was used to construct the secreting eukaryotic expression vector pIRES2-EGFP-smIL-1β and transfected into Hepa1-6 cells. The expression of fusion gene was detected by RT-PCR and the expression of mIL-1β protein was detected by Western blot The level of mIL-1β in culture supernatant and cell lysate was determined by ELISA. Results: The fusion gene obtained by splicing by SOE was consistent with the sequence registered in GenBank. RT-PCR showed that the vector could express fusion gene mRNA in Hepa1-6 cells and the secretion level of mIL-1β in cell culture supernatant increased obviously , While the mIL-1β in the cytoplasm did not change significantly. CONCLUSION: The mIL-1β recombinant expression vector secreted by the classical pathway has been successfully constructed, which can effectively secrete the bioactive mIL-1β in the Hepa 1-6 cell culture supernatant.