血管内皮功能调节酶pCDH-Myc-UBR5分子克隆的构建  

Construction of a recombinant plasmid encoding vascular endothelial functional regulator,pCDH-Myc-UBR5

作　　者：赵海静 刘琪 金蕊 程龙 刘昱圻 陈韵岱 ZHAO Hai-Jing;LIU Qi;JIN Rui;CHENG Long;LIU Yu-Qi;CHEN Yun-Dai(Medical School of Chinese PLA,Beijing 100853,China;Institute of Biotechnology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100071,China;Senior Department of Cardiology,Sixth Medical Center,Chinese PLA General Hospital,Beijing 100048,China)

机构地区：[1]解放军医学院,北京100853 [2]中国人民解放军军事科学院军事医学研究院生物工程研究所,北京100071 [3]中国人民解放军总医院第六医学中心心血管病医学部,北京100048

出　　处：《中华老年多器官疾病杂志》2022年第5期361-365,共5页Chinese Journal of Multiple Organ Diseases in the Elderly

基　　金：国家自然科学基金(82070434);北京市科技新星计划(Z191100001119020)。

摘　　要：目的构建pCDH-Myc-UBR5重组质粒,并探究泛素蛋白连接酶E3组分N-识别蛋白5(UBR5)在调控血管生成方面的生物学功能。方法以人乳腺癌细胞(MCF7)的互补DNA(cDNA)为模板,将UBR5基因编码区序列分为前后两段,经聚合酶链式反应(PCR)扩增。两段扩增序列插入到真核表达载体pCDH-Myc中,通过菌液PCR及DNA测序鉴定插入片段。将重组质粒瞬时转染至人胚胎肾细胞(HEK293T)中,通过蛋白质印迹法检测Myc-UBR5蛋白的表达。为了验证UBR5蛋白的功能,通过免疫共沉淀实验检测UBR5与先天性角化不良1(DKC1)蛋白的相互作用。结果成功构建pCDH-Myc-UBR5重组质粒。经菌液PCR及DNA测序证实UBR5扩增序列成功插入到pCDH-Myc载体中,并在HEK293T中表达。免疫共沉淀实验发现UBR5与DKC1存在相互作用。结论通过分子克隆技术可成功构建pCDH-Myc-UBR5质粒并在细胞中正确表达。UBR5与血管生成调控蛋白DKC1存在相互作用。Objective To construct recombinant plasmid pCDH-Myc-UBR5 and then explore the biological function of ubiquitin protein ligase E3 component N-recognin 5(UBR5)in regulating angiogenesis.Methods Using the complementary DNA(cDNA)of Michigan cancer foundation-7(MCF7)cells as a template,we divided the UBR5 gene coding region into two segments and amplified them by polymerase chain reaction(PCR).Then the two amplified fragments were inserted into the eukaryotic expression vector pCDH-Myc.Bacterial liquid PCR and DNA sequencing were adopted to identify the inserted fragments.The recombinant plasmid was transiently transfected into human embryonic kidney cells 293 SV40 LT(HEK293 T)and the expression of Myc-UBR5 protein was detected by Western blotting.Co-immunoprecipitation(Co-IP)assay was employed to verify whether UBR5 interacts with dyskeratosis congenita 1(DKC1).Results The recombinant plasmid pCDH-Myc-UBR5 was successfully constructed.The amplified sequence of UBR5 was successfully inserted into pCDH-Myc vector and expressed in HEK293 T cells,which was confirmed by bacterial liquid PCR and DNA sequencing.The results of Co-IP assay indicated the interaction between UBR5 and DKC1.Conclusion The plasmid pCDH-Myc-UBR5 is successfully constructed by molecular cloning and expressed correctly in cells.UBR5 interacts with the angiogenic regulatory protein DKC1.

关 键 词：泛素蛋白连接酶E3组分N-识别蛋白5 重组质粒 分子克隆 先天性角化不良1蛋白 血管生成 

分 类 号：R34[医药卫生—基础医学]