中文摘要：

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。

英文摘要：

Objective： In order to screening and research the function of lysyl oxidase （LOX） and its interaction proteins in MDA-MB-231 invasive breast cancer cell line, using tandem affinity purification by gateway to lentiviral vector caring recombination LOX fusion with Strep II/FLAG tag. Methods： To gain the vector of LOX-SF recombination protein by polymerase chain reaction, adopt- ing overlap PCR to construct the fusion gene through a chimeric primer, then the recombination vector GV303/LOX-SF was constructed by the fusion gene and GV303 were digested. To packaging lentivirus vector from 293T cells using three-plasmid transient transfections, then MDA-MB-231 cells were transfection by recombinant lentivirus vector, The cell line stably expression of fusion protein preliminary detected by realtime fluorescence quantitative PCR and Western Blot. Results： The LOX-SF recombination protein was gained through the tandem affinity purification （TAP） method and it was expressed stably in MDA-MB-231 cells by anti-Tag. Conclusions： The success- fully expression and purification of LOX fusion with Strep II/FLAG tag in MDA-MB-231 cells because of the recombinant vector GV303/LOX-SF was constructed, laying the foundation to further study the protein-protein interactions of LOX in breast cancer cell lines.