Max二聚化蛋白1（Mad1）真核表达载体的构建及其对胃癌细胞增殖和迁移能力的影响

目的：构建Max二聚化蛋白1 (Max dimerization protein 1,Mad1) 的真核表达载体，研究Mad1对胃癌细胞增殖和迁移能力的影响。方法：利用DNA重组技术将Mad1基因克隆至pEGFP-N1载体,构建成pEGFP-N1-Mad1重组真核表达载体。经酶切和测序鉴定后，采用脂质体转染技术将重组质粒瞬时转染人胃癌AGS细胞， RT-PCR及Western blot检测Mad1基因和蛋白的表达。荧光显微镜观察Mad1在AGS细胞内的定位情况。CCK-8和Transwell实验研究Mad1对胃癌AGS细胞增殖和迁移能力的影响。结果：成功构建携带Mad1基因的真核表达载体pEGFP-N1-Mad1。将重组质粒瞬时转染AGS细胞后，RT-PCR和Western blot检测到Mad1 基因和蛋白的表达。Mad1基因表达产物定位于AGS细胞核中。CCK-8和Transwell实验结果显示，转染Mad1的AGS细胞与转染空载体的AGS细胞、及正常AGS细胞相比，细胞增殖活力和迁移能力明显降低。 结论：成功构建了真核表达载体pEGFP-N1-Mad1,研究表明Mad1可以抑制胃癌AGS细胞的增殖和迁移。

Construction of eukaryotic expression vector containing Max dimerization protein 1 and its effect on human gastric cancer cell proliferation and migration

Objective: To construct the recombinant eukaryote expression vector containing Max dimerization protein 1 (Mad1) gene and detect its effect on gastric cancer cell proliferation and migration. Methods: The Mad1 gene was cloned into pEGFP-N1 expression vector by recombining DNA technology. The recombinant vector was identified by restriction enzyme analysis and nucleotide sequence determination. The eukaryotic expression plasmid pEGFP-N1-Mad1 was transiently transfected into AGS cells. Expression of Mad1 gene and protein was identified by RT-PCR and Western blot, respectively. The location of Mad1 protein was detected by fluorescence microscope. The proliferation and migration capacity of AGS cells were examined by CCK-8 and Transwell assay, respectively. Results: The Mad1 gene was successfully cloned to the eukaryote expression vector pEGFP-N1. Expression of Mad1 gene and protein was confirmed by RT-PCR and Western blot. After transfection, Mad1 could be detected in the nucleus of AGS cells. CCK-8 and transwell experimental results show that the proliferation and migration ability of pEGFP-N1-Mad1 transfected cells were deteriorated significantly compared to empty vector transfected AGS cells and normal AGS cells. Conclusion: The new recombinant expression vector pEGFP-N1-Mad1 was constructed and expressed successfully in AGS cells. Mad1 could inhibit the proliferation and migration of gastric cancer cells.