IL-12和粒细胞-巨噬细胞集落刺激因子真核表达载体的构建及在肝癌细胞中的表达

Construction of the eukaryotic expression vectors of interleukin-12 and granulocyte-macrophage colony-stimulating factor and their expression in hepatoma cells

目的构建小鼠IL-12和粒细胞-巨噬细胞集落刺激因子(GM-CSF)真核表达载体PBI-CMV3-IL-12和PBICMV3-GM-CSF,转染H22肝癌细胞,检测IL-12和GM-CSF在肝癌细胞中的表达。方法 Trizol法提取小鼠肝脏总RNA,反转录成c DNA,以含有Bam HⅠ和HindⅢ酶切位点的特异性引物扩增得到IL-12和GM-CSF编码序列,将所得产物和PBI-CMV3空载体进行双酶切、回收、连接后转化至DH5α中,挑取单克隆菌落进行质粒提取、酶切鉴定及测序分析,对构建的表达载体进行鉴定。将构建好的质粒分为空载组、PBI-CMV3-IL-12组、PBI-CMV3-GM-CSF组和共转染组,分别转染至H22肝癌细胞中,荧光定量PCR和Western Blot检测细胞中IL-12和GM-CSF mRNA及蛋白表达水平。计量资料多组间比较采用单因素方差分析,进一步两两比较采用Dunnett's T3法。结果成功构建了PBI-CMV3-IL-12和PBI-CMV3-GM-CSF真核表达载体,荧光定量PCR检测结果显示,4组间IL-12和GM-CSF mRNA相对表达量差异均有统计学意义(F值分别为522、163,P值均<0.001);其中IL-12 mRNA表达水平比较,PBI-CMV3-IL-12组、共转染组较空载组均显著升高(P值均<0.05),GM-CSF mRNA相对表达水平比较,PBI-CMV3-GM-CSF组、共转染组较空载组均显著升高(P值均<0.05)。Western Blot结果显示,目的蛋白可以与抗体结合并在膜上产生特异性条带,条带大小和灰度分析结果显示,IL-12和GM-CSF蛋白的表达趋势与mRNA表达水平一致。结论成功构建了IL-12和GM-CSF真核表达载体,且可在H22肝癌细胞中高效表达。

Objective To construct the eukaryotic expression vectors of interleukin-（-1）2（ IL-12） and granulocyte-macrophage colony-stimulating factor（ GM-CSF）,PBI-CMV3-IL-12 and PBI-CMV3-GM-CSF,and the expression of IL-12 and GM-CSF in hepatoma cells after H22 hepatoma cells are transfected with such vectors. Methods The Trizol method was used to extract total liver RNA from mice,which was then reversely transcribed into c DNA. The coding sequences of IL-12 and GM-CSF were obtained by amplification using specific primers containing Bam HI and Hind III restriction sites. Double enzyme digestion was performed for the products and PBI-CMV3 empty vectors,and then the digested products were transformed into DH5α after being recycled by gel extraction kit and connected by T4 DNA ligase. Monoclonal bacterial colonies were selected and plasmid extraction,enzyme digestion,and DNA sequencing were performed for the identification of the expression vector constructed. The constructed plasmids were divided into empty vector group,PBI-CMV3-IL-12 group,PBI-CMV3-GM-CSF group,and co-transfection group. These plasmids were transfected into H22 hepatoma cells,and quantitative real-time PCR and Western blot were used to measure the mRNA and protein expression of IL-12 and GM-CSF in cells. A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the Dunnett＇s T3 method was used for further comparison between two groups. Results The eukaryotic expression vectors,PBI-CMV3-IL-12 and PBI-CMV3-GM-CSF,were successfully constructed. Quantitative real-time PCR showed that there were significant differences in the mRNA expression of IL-12 and GM-CSF between the four groups（ F = 522 and 163,both P〈0. 001）. Compared with the empty vector group,the PBI-CMV3-IL-12 group and the co-transfection group had a significant increase in the mRNA expression of IL-12（ both P〈0. 05）. Compared with the empty vector group,the PBI-CMV3-GM-CSF group and the co-transfection group had a significant increase in the mRNA expression of GM-CSF（ both P〈0. 05）. Western blot showed that the interest protein could bind to antibody and produce a specific band in the membrane,and an analysis of band size and gray value showed that the protein expression of IL-12 and GM-CSF had a similar trend as the mRNA expression of IL-12 and GM-CSF. Conclusion The eukaryotic expression vectors of IL-12 and GM-CSF are successfully constructed,and they are highly expressed in H22 hepatoma cells.