目的:构建真核表达质粒pEGFP-Mfn2,并将其转染乳腺癌MCF-7细胞系,观察其在乳腺癌MCF-7细胞中的Mfn2基因表达情况及其对乳腺癌细胞MCF-7迁移能力的影响。方法:①以正常人骨骼肌为模板获得Mfn2基因片段,用限制性核酸内切酶XhoI、HidⅢ分别对pEGFP-N1和获得的Mfn2基因片段进行双酶切,并将酶切产物进行重组,最后对其行PCR、酶切和DNA测序鉴定。②用激光共聚焦显微镜、Western blot方法及划痕实验分别对转染后的乳腺癌细胞MCF-7中的Mfn2表达情况及迁移能力的影响进行检测。结果:①PCR、酶切和DNA测序结果均证实pEGFP-Mfn2重组质粒构建成功。②细胞转染48 h后共聚焦激光显微镜验下可见绿色荧光细胞中显影约占60%,Western blot方法检测到实验组中Mfn2基因明显过表达。空白对照组和空载体对照组的Mfn2基因表达情况无明显差别,二者皆证实转染成功。划痕试验观察到24 h和48 h后,实验组划痕区细胞明显少于空质粒对照组和空白对照组划痕区的细胞。结论:成功构建了pEGFP-Mfn2真核表达质粒,为研究Mfn2基因在体内外对肿瘤细胞的作用及相关机制奠定了基础。证实了Mfn2基因可以在乳腺癌MCF-7细胞系中高表达,并可以降低乳腺癌细胞MCF-7的迁移能力。 OBJECTIVE: To construct the eukaryotic expression plasmid pEGFP-Mfn2 and transfect it into breast cancer MCF-7 cell line to observe its Mfn2 gene expression in breast cancer MCF-7 cells and its effect on breast cancer MCF-7 migration Impact. Methods: ① The normal human skeletal muscle was used as a template to obtain Mfn2 gene fragment. Restriction endonucleases XhoI and HidⅢ were used to double-digest the pEGFP-N1 and Mfn2 gene fragments, respectively, and the digested products were recombined. Finally, The PCR, digestion and DNA sequencing identification. ② Confocal laser scanning microscopy, Western blot and scratch assay were used to detect the Mfn2 expression and migration ability in transfected breast cancer cells MCF-7. Results: ①PEGFP-Mfn2 recombinant plasmid was successfully constructed by PCR, enzyme digestion and DNA sequencing. ② 48 h after transfection, confocal laser microscopy showed that about 60% of green fluorescent cells were developed, and Mfn2 gene was overexpressed in the experimental group by Western blot. Mfn2 gene expression in the blank control group and empty vector control group no significant difference, both confirmed the successful transfection. Scratch test observed after 24 h and 48 h, the experimental group of cells in the scratch area was significantly less than empty plasmid control group and blank control group scratched area cells. Conclusion: The eukaryotic expression plasmid pEGFP-Mfn2 was successfully constructed, which laid the foundation for the study of the role of Mfn2 gene in tumor cells in vitro and in vivo and related mechanisms. Confirmed that Mfn2 gene can be highly expressed in breast cancer MCF-7 cell line, and can reduce the migration ability of breast cancer cell MCF-7.