CAJ | 학술논문

目的：构建人自噬相关基因LC3B的真核表达载体pEGFP-C1-LC3B，并鉴定其表达。方法：以乳腺文库为模板，PCR扩增LC3B全长基因，将其克隆至pEGFP-C1表达载体，得到pEGFP-C1-LC3B重组质粒，酶切和测序鉴定后，转染ZR75-1细胞，Western印迹检测真核细胞中GFP-LC3B融合蛋白的表达，用倒置荧光显微镜观察自噬诱导剂雷帕霉素处理和未处理ZR75-1细胞质中GFP-LC3B的分布。结果：Western印迹可见GFP-LC3B融合蛋白表达条带；在荧光显微镜下，ZR75-1细胞内可见绿色荧光，雷帕霉素刺激后有明显绿色荧光聚集体形成。结论：人自噬相关基因LC3B的真核表达载体pEGFP-C1-LC3B构建成功，为进一步研究自噬在乳腺癌细胞中的作用机制奠定了基础。
목적：구건인자서상관기인LC3B적진핵표체재체pEGFP-C1-LC3B，병감정기표체。방법：이유선문고위모판，PCR확증LC3B전장기인，장기극륭지pEGFP-C1표체재체，득도pEGFP-C1-LC3B중조질립，매절화측서감정후，전염ZR75-1세포，Western인적검측진핵세포중GFP-LC3B융합단백적표체，용도치형광현미경관찰자서유도제뢰파매소처리화미처리ZR75-1세포질중GFP-LC3B적분포。결과：Western인적가견GFP-LC3B융합단백표체조대；재형광현미경하，ZR75-1세포내가견록색형광，뢰파매소자격후유명현록색형광취집체형성。결론：인자서상관기인LC3B적진핵표체재체pEGFP-C1-LC3B구건성공，위진일보연구자서재유선암세포중적작용궤제전정료기출。
Objective: To construct an eukaryotic expression vector of human autophagy-related microtubule-asso?ciated protein 1 light chain 3B(LC3B) gene and to detect its expression. Methods: The coding sequence of full length LC3B was amplified from breast library by PCR and cloned into the pEGFP-C1 expression vector. The re?combinant plasmid was verified by restriction enzyme analysis and sequencing.The GFP-LC3B expression was de?tected by Western blotting. The fluorescent signals were detected by fluorescence microscope. Results: The full length LC3B expressed in ZR75-1 cells was shown by West blotting. Green fluorescent aggregates were observed by fluorescence microscoryin ZR75-1 cells transfected with LC3B and treated with rapamycin.Conclusion: Eukary?otic expression vector of human LC3B gene was constructed,which lays foundation for further research of autopha?gy in breast cancer.