论文部分内容阅读
目的:为了研究赖氨酰氧化酶(Lysyl Oxidase,LOX)及其相互作用蛋白质在乳腺癌中的功能,构建带StrepⅡ/FLAG串联亲和标签的重组LOX蛋白慢病毒表达载体并在乳腺癌细胞MDA-MB-231中表达。方法:设计引物通过聚合酶链式反应获得带StrepⅡ/FLAG串联亲和标签的LOX蛋白(LOX-SF)的亲本质粒,双酶切后鉴定测序克隆至GV303表达载体,连同慢病毒包装质粒共同转染293T得到GV303/LOX-SF慢病毒,将其转染MDA-MB-231细胞,使用荧光定量PCR和蛋白质印迹实验对细胞中重组蛋白LOX-SF进行检测。结果:通过串联亲和纯化获得LOX-SF重组蛋白,使用标签抗体成功鉴定到LOX-SF重组蛋白在MDA-MB-231细胞内稳定表达。结论:GV303/LOX-SF的构建,使带StrepⅡ/FLAG融合标签的LOX蛋白在MDA-MB-231中成功表达及纯化,为筛选和研究LOX及其相互作用蛋白在乳腺癌细胞内的功能奠定了实验基础。
OBJECTIVE: To study the function of Lysyl Oxidase (LOX) and its interacting proteins in breast cancer, a recombinant LOX lentiviral vector with StrepⅡ / FLAG tandem affinity tag was constructed and expressed in breast cancer cells MDA -MB-231. Methods: The primers of LOX-SF with StrepⅡ / FLAG tandem affinity tag were designed by polymerase chain reaction (PCR). After double enzyme digestion, the recombinant plasmid was cloned into GV303 expression vector and co-transfected with lentiviral packaging plasmid 293T cells were infected with GV303 / LOX-SF lentivirus and transfected into MDA-MB-231 cells. The recombinant protein LOX-SF was detected by real-time PCR and Western blotting. Results: LOX-SF recombinant protein was obtained by in-line affinity purification. The recombinant protein LOX-SF was stably expressed in MDA-MB-231 cells using labeled antibody. CONCLUSION: The construction of GV303 / LOX-SF enables the expression and purification of LOX protein with StrepⅡ / FLAG fusion tag in MDA-MB-231, and to screen and study the function of LOX and its interacting proteins in breast cancer cells The experimental basis.