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目的制备RPB5调节蛋白(RMP)抗体并研究RMP在60Coγ射线诱导的SMMC-7721肝癌细胞凋亡过程中的表达及功能。方法 RT-PCR检测HT-29、A549、Hep-2、SGC-7901、293T、HeLa、WI-38和SMMC-7721细胞株中RMP的表达;从质粒中PCR扩增得到人RMP基因并克隆至原核表达载体中,诱导表达后经亲和层析纯化获得融合蛋白GST-RMP/D1,以该蛋白作为抗原免疫家兔制备多克隆抗体;肝癌细胞SMMC-TT21、PFlaG-CMV4-SMMC-7721、PFlaG-CMV4-RMP-SMMC-7721经6 Gy 60 Coγ射线照射后,流式细胞术检测肝癌细胞凋亡的变化。结果RMP在以上细胞系中均有表达,但程度不同;SDS-PAGE鉴定示纯化蛋白为目的蛋白人RMP片段。Western blot结果显示,制备的多抗可与RMP蛋白特异性结合;肝癌细胞株经6 Gy 60Coγ射线照射后,流式细胞术结果表明RMP使肝癌细胞SMMC-7721的凋亡减少。结论 RMP在不同细胞株中均有表达且能减少肝癌细胞SMMC-7727的凋亡,为RMP用于肝癌的基因治疗奠定了基础。
Objective To prepare RPB5 regulatory protein (RMP) antibody and study the expression and function of RMP in 60Coγ-ray-induced SMMC-7721 hepatocellular carcinoma cell apoptosis. Methods The expression of RMP in HT-29, A549, Hep-2, SGC-7901, 293T, HeLa, WI-38 and SMMC-7721 cell lines was detected by RT-PCR. The human RMP gene was amplified from the plasmid and cloned The fusion protein GST-RMP / D1 was purified from the prokaryotic expression vector by affinity chromatography and then used as antigen to immunize rabbits to prepare polyclonal antibody. Liver cancer cells SMMC-TT21, PFlaG-CMV4-SMMC-7721, The changes of apoptosis in hepatocellular carcinoma cells were detected by flow cytometry after irradiation with 6 Gy 60 Coγ-rays. Results RMP was expressed in all of the above cell lines, but to a different degree. SDS-PAGE showed that the purified protein was the human RMP fragment. Western blot results showed that the prepared polyclonal antibody could specifically bind to RMP protein. The flow cytometry results showed that RMP decreased the apoptosis of hepatocellular carcinoma cell line SMMC-7721 after irradiation with 6 Gy 60Coγ-ray. Conclusion RMP is expressed in different cell lines and can reduce the apoptosis of hepatoma SMMC-7727 cells, which lays the foundation for the gene therapy of liver cancer by RMP.