| 刘侠 刘剑仑 蒋奕 唐玮 杨华伟 韦薇△.串联亲和标签LOX蛋白慢病毒表达载体的构建及在乳腺癌细胞中的表达[J].,2015,15(29):5613-5616 |
| 串联亲和标签LOX蛋白慢病毒表达载体的构建及在乳腺癌细胞中的表达 |
| Construction of the Lentivirus Expression Vector Carrying TandemAffinityPurification Tag for Lysyl Oxidase and Its Expression in Breast Cancer Cell |
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| DOI： |
| 中文关键词: 串联亲和纯化 标签 赖氨酰氧化酶 慢病毒载体 相互作用蛋白质 |
| 英文关键词: Tandemaffinity purification Tag Lysyl oxidase Lentivirus vector Interaction protein |
| 基金项目:国家自然科学基金项目（81160321）；广西自然科学基金项目（2013GXNSFAA019235） |
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| 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Strep
Ⅱ/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 StrepⅡ/FLAG tag.Methods:To gain the vector of LOX-SF recombination protein by polymerase chain reaction, adopting
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 tandemaffinity purification (TAP) method and it was expressed stably in MDA-MB-231 cells by anti-Tag.Conclusion:The successfully
expression and purification of LOX fusion with StrepⅡ /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. |
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