负载ERRα基因片段慢病毒构建及表达  

Construction,expression and preliminary function of lentivirus gene encoding ERRα

作　　者：王海彬[1] 周驰[1] 刘锋[1] 曾展鹏[1] 

机构地区：[1]广州中医药大学第一附属医院关节中心,广东广州510405

出　　处：《中国公共卫生》2012年第9期1194-1197,共4页Chinese Journal of Public Health

基　　金：国家自然科学基金(30973761);广东省自然科学基金(8151040701000016);广东省中医药管理局基金(2009216)

摘　　要：目的构建编码人雌激素相关受体α(hERRα)的慢病毒载体,并测定其滴度,观察hERRα基因在体外的表达情况及其对人肺癌细胞A549增殖的影响。方法采用PCR扩增hERRα基因片段,插入转移载体pW-PXLD,用磷酸钙法将pWPXLD-hERRα、psPAX2和pMD2.G共转染293T细胞包装慢病毒,浓缩后测定病毒滴度,再将病毒感染A549细胞,用蛋白印迹方法测定感染病毒的A549细胞中hERRα的表达。选择合适滴度的病毒感染A549细胞,采用cell-titer的方法检测感染病毒的A549细胞的活力。结果测序结果显示成功构建了重组质粒pWPXLD-hERRα,测定浓缩后的病毒滴度为1.8×108TU/mL,蛋白印迹方法检测到慢病毒感染的A549细胞中hERRα呈阳性表达;cell-titer glo检测结果表明过表达hERRα能促进细胞增殖。结论成功构建了负载hERR基因片段的慢病毒,hERRα能在感染的A549细胞中高效表达,并促进感染病毒的A549细胞增殖。Objective To construct a lentiviral vector encoding human estrogen related receptor alpha （hERRα） gene and to study the influence of lenti-ERRα on A549 cell prolifertation. Methods A fragment of the hERRα gene was amplified with PCR and was inserted into the plasmid pWPXLD. The three plasmids （pWPXLD-hERRα, psPAX2, and pMD2G） were cotransfected the virus packaging cell line 293T using the Ca3 （PO4）2 method. Then the cell supernatant was harvested and the virus titration was determined after concentration. Also the expression of hERRα in human lung cancer cell line A549 cells infected by the lentivirus was determined with western blot. The effect of ERRa overexpression on the A549 cell growth was detected by Cell Titer Glo kit. Results DNA sequencing demonstrated that the recombinant plasmid pWPXLD-Herr was successfully constructed. The titration of the concentrated lentivirus was 1.8 × 108 TU/mL. Western blot assay proved positive for the hERRa. Cell-Titer Glo results revealed that overexpression of hERRα in A549 cells up-regulated the cell viability. Conclusion Lentivirus encoding hERRcx was successfully constructed and hERRα could express in A549 cells. The overexpression of hERRα in A549 cells up-regulates the cell viability.

关 键 词：人雌激素相关受体(hERR) 慢病毒载体 细胞增殖 

分 类 号：R373[医药卫生—病原生物学]