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目的根据细胞因子间协同作用特点,构建人白细胞介素-2(IL-2)与干扰素(IFN)β的融合基因,并用癌胚抗原(CEA)基因启动子调控其在结肠癌细胞株中的靶向表达。方法分别用原核及真核表达载体,检测融合蛋白的表达情况。PCR、RT-PCR法扩增得到CEA启动子、IL-2 cDNA、IFNβ cDNA,应用原核表达质粒pGEX-5X-1、真核表达质粒pcDNA3．1／HisA、绿色荧光蛋白质粒pEGFP-C1,构建出原核表达质粒和真核表达质粒;SDS-PAGE电泳检测原核表达;用脂质体法将真核表达质粒转入结直肠癌细胞株 Lovo(CEA高表达)、HT-29(CEA低表达)和对照Hela细胞(CEA阴性),转染后分别以流式细胞仪、荧光显微镜、Western印迹等方法检测肿瘤细胞凋亡情况及蛋白表达情况。结果融合基因可在大肠杆菌及肿瘤细胞内表达融合蛋白;对肿瘤细胞有明显的杀伤作用;由CEA启动子调控的融合基因在Lovo细胞的表达高于HT-29细胞,在CEA阴性的Hela细胞中几乎不表达。结论 CEA启动子可靶向性调控融合基因在肿瘤细胞的表达,具有协同作用的细胞因子融合蛋白可能有更强的抗肿瘤作用。
Objective To construct a fusion gene of human interleukin-2 (IL-2) and interferon (IFN) β according to the synergistic effect between cytokines and to use the CEA gene promoter to regulate its expression in colon cancer cell lines Targeted expression. Methods Prokaryotic and eukaryotic expression vectors were used to detect the expression of the fusion protein. CEA promoter, IL-2 cDNA and IFNβ cDNA were amplified by PCR and RT-PCR. The prokaryotic expression plasmid pGEX-5X-1, the eukaryotic expression plasmid pcDNA3.1 / HisA, and the green fluorescent protein plasmid pEGFP-C1 were constructed The prokaryotic expression plasmids and eukaryotic expression plasmids were obtained. The prokaryotic expression was detected by SDS-PAGE electrophoresis. The eukaryotic expression plasmids were transfected into Lovo (CEA overexpression), HT-29 (CEA overexpression) And control Hela cells (CEA negative). After transfection, the apoptosis and protein expression of tumor cells were detected by flow cytometry, fluorescence microscope and Western blot respectively. Results The fusion gene was expressed in Escherichia coli and tumor cells. The fusion gene was significantly inhibited in tumor cells. The expression of fusion gene regulated by CEA promoter in Lovo cells was higher than that in HT-29 cells. In CEA negative Hela cells In almost no expression. Conclusion The CEA promoter can regulate the expression of fusion gene in tumor cells. Synergistic cytokine fusion protein may have a stronger anti-tumor effect.