人p65和IκBα基因真核表达载体的构建及在永生化滑膜细胞中的表达  

Construction and expressions of eukaryotic expression vectors of human p65 and IκBα gene in MH7a cells

作　　者：程欣[1] 王立峰[1] 张健[1] 刘娜[1] 苏金[1] 于江天[1] 刘新平[1] 

机构地区：[1]第四军医大学基础部生物化学与分子生物学教研室,陕西西安710033

出　　处：《第四军医大学学报》2006年第19期1729-1732,共4页Journal of the Fourth Military Medical University

基　　金：国家重点基础研究和开发计划(2002CB513007);国家自然科学基金(30300319)

摘　　要：目的构建人p65和IκBα基因的真核表达载体并检测其在人永生化滑膜细胞MH7a中的表达.方法应用RT-PCR方法从体外培养的HEK293细胞mRNA中扩增出p65和IκBα基因的编码区,克隆至pMD18T载体并测序,然后亚克隆至真核表达载体pcDNA3.1myc/His(-)A,酶切鉴定正确后以脂质体法瞬时转染MH7a细胞,WesternBlot检测p65和IκBα的表达.结果测序证实PCR扩增得到的p65和IκBα基因编码区序列正确;脂质体法转染MH7a细胞后检测到预期目的蛋白的表达.结论成功构建了p65和IκBα的真核表达载体并使其在人永生化滑膜细胞中表达.AIM ： To construct the eukaryotic expression vectors of human p65 and IκBα gene and to testify their expressions in human immortalized rheumatoid arthritis synoviocytes-MH7a cells. METHODS： Human p65 and IκBα gene coding sequences were amplified from HEK293 mRNA by RT-PCR. The PCR products were cloned into pMD18T vectors, sequenced, then subcloned into eukaryotic expression vector pcDNA3.1 myc/His（ - ） A. After restriction enzyme digestion, these recombined plasmids were transiently transfected into MHTa cells subsequently with lipofectin to detect the expressions of p65 and IκBα by Western Blot. RESULTS： Sequencing showed that the eukaryotic expression vectors of p65 and IκBα were successfully constructed. Their expressions in MH7a cells were confirmed. CONCLUSION： The eukaryotic expression vectors containing p65 and IκBα are constructed and they can express p65 and IκBα in human immortalized rheumatoid arthritis synoviocytes-MH7a cells.

关 键 词：NF-κB P65 IΚBΑ 类风湿性关节炎 逆转录聚合酶链反应 

分 类 号：R382.31[医药卫生—医学寄生虫学]