p62基因过表达慢病毒载体的构建及表达  

Construction and expression of lentiviral vector of p62 gene over-expression

作　　者：张春[1] 苏丛 伍婷 朱立雨 Zhang Chun;Su Cong;Wu Ting;Zhu Liyu(Dept of Infectious Diseases,The Chaohu Affiliated Hospital of Anhui Medical University,Chaohu 238000;Dept of Infection Management,The First Affiliated Hospital of Anhui Medical University,Hefei 230022;Dept of Infectious Diseases,The First Affiliated Hospital of Anhui Medical University,Hefei 230022;Anhui Center for Surveillance of Bacterial Resistance,Hefei 230022;Institute of Bacterial Resistance,Anhui Medical University,Hefei 230022)

机构地区：[1]安徽医科大学附属巢湖医院感染病科,巢湖238000 [2]安徽医科大学第一附属医院感染管理科,合肥230022 [3]安徽医科大学第一附属医院感染病科,合肥230022 [4]安徽省细菌耐药性监控中心,合肥230022 [5]安徽医科大学细菌耐药研究所,合肥230022

出　　处：《安徽医科大学学报》2024年第3期398-402,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:82100017);安徽省卫生健康科研项目(编号:AHWJ2022b076)。

摘　　要：目的构建p62基因过表达慢病毒载体及其表达系统,并在人单核细胞白血病细胞(THP-1)中稳定表达,为在细胞水平研究p62基因的作用提供途径与方法。方法采用聚合酶链式反应(PCR)扩增p62基因片段,将扩增产物连接至线性化pcDNA3.1-Flag-PCDH10真核表达慢病毒载体;PCR鉴定重组质粒,将构建成功的重组质粒和包装质粒共转染至人胚胎肾细胞293(HEK 293T);用重组慢病毒感染人单核细胞白血病(THP-1)细胞,氨苄霉素筛选阳性细胞克隆株;Western blot和实时荧光定量聚合酶链反应(RT-qPCR)检测高表达p62基因的THP-1细胞株(过表达组)和转染不含p62基因空质粒(对照组)的THP-1细胞株;感染肺炎克雷伯菌(Klebsiella Pneumoniae,K.p.)后,RT-qPCR检测THP-1细胞中肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β和趋化因子1(Cxcl1)表达水平。结果通过PCR成功获得p62基因片段并连接到pcDNA3.1-Flag-PCDH10病毒载体上,并且PCR检测显示p62-pcDNA3.1-Flag-PCDH10重组质粒构建成功;挑选慢病毒感染THP-1细胞后抗氨苄青霉素细胞株,Western blot检测结果显示药筛存活的THP-1细胞较对照组细胞P62蛋白表达增多(P<0.001),且RT-qPCR检测显示p62 mRNA表达增高(P<0.001),说明成功构建高表达P62蛋白的THP-1细胞株;感染K.p.后,高表达P62的THP-1细胞中TNF-α、IL-1β和Cxcl1表达水平增高(P<0.01)。结论经三质粒包装系统可以成功构建p62-pcDNA3.1-Flag-PCDH10慢病毒载体及高表达P62蛋白THP-1细胞株,为后续p62基因的研究提供了基础。Objective To construct lentiviral vector of p62 gene over-expression,and stably express p62 gene in human monocytic leukemia cells 1(THP-1),and to provide a way to study the role of p62 gene at the cellular level.Methods The p62 gene fragment was amplified by polymerase chain reaction(PCR),and the amplified product was ligated to the linearized pcDNA3.1-Flag-PCDH10 lentiviral vector.After identifying with PCR,the PCR product was cotransfected with the packaging plasmid into human embryonic kidney cells 293(HEK 293T).THP-1 cells were infected with recombinant lentivirus.Positive cell clones were screened by ampicillin.Western blot and real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)were used to detect THP-1 cell lines with high p62 expression(overexpression group)and THP-1 cell lines transfected with empty plasmid without p62 gene(control group).The expression levels of TNF-α,IL-1β and Cxcl1 after K.p.infection were detected by RT-qPCR.Results The p62 gene fragment was successfully obtained by PCR and ligated to pcDNA3.1-Flag-PCDH10 vector.PCR confirmed that p62-pcDNA3.1-Flag-PCDH10 recombinant plasmid was constructed successfully.Ampicillin-resistant cell lines were selected after lentiviral infection of THP-1 cells.The results of Western blot analysis showed that the THP-1 cells with drug sieve survival increased the expression of P62 protein compared with the control cells(P<0.001),and RT-qPCR analysis showed that the relative mRNA expression of p62 increased(P<0.001).THP-1 cells with high expression of P62 were successfully constructed.The levels of TNF-α、IL-1β and Cxcl1 from THP-1 cells with high expression of P62 significantly increased after infection with K.p.(P<0.01).Conclusion P62-pcDNA3.1-Flag-PCDH10 vector and THP-1 cells with high expression of P62 can be successfully constructed by three-plasmid packaging system,which provides a basis for the study of p62.

关 键 词：P62 慢病毒载体构建 三质粒包装系统 THP-1细胞 肺炎克雷伯菌 过表达 

分 类 号：R363[医药卫生—病理学]