PAX2慢病毒表达载体的制备及鉴定  

Construction and identification of lentiviral expression vector carrying PAX2

作　　者：李里[1,2] 吴玉斌[2] 宫亮[3] 

机构地区：[1]辽宁医学院,辽宁医学院附属第一医院儿科,辽宁锦州121001 [2]中国医科大学附属盛京医院小儿肾脏风湿免疫科 [3]辽宁医学院附属第一医院耳鼻喉咽喉科

出　　处：《现代预防医学》2013年第15期2859-2862,共4页Modern Preventive Medicine

基　　金：辽宁医学院青年科技启动基金项目(F2011Z005);沈阳市科学技术计划项目(F10-205-1-29)

摘　　要：目的构建大鼠PAX2慢病毒表达载体,以期在大鼠肾脏中高效、稳定表达。方法设计引物引入AgeⅠ酶切位点,使用PCR方法从质粒pcDNA3.1-PAX2中扩增小鼠PAX2基因的编码区序列,对所扩增出的目的片段回收纯化。用In-Fusion技术将AgeⅠ内切酶消化后目的片段交换连接入AgeI酶切的pGC-FU载体,构建PAX2慢病毒表达载体pGC-FU-PAX2。酶切验证并测序正确后,将质粒pGC-FU-PAX2与慢病毒辅助包装载体共转染293T细胞,Western-blot验证PAX2在转染293T细胞中表达。结果通过PCR扩增获得了PAX2基因,将PAX2克隆到慢病毒转移质粒pGC-FU中,并在293T细胞中包装产生慢病毒颗粒。结论成功构建了PAX2慢病毒表达载体,为进一步从分子水平探讨PAX2功能奠定了基础。OBJECTIVE To construct a lentiviral expression vector carrying PAX2 and obtain PAX2 efficient and stable expression in rats’ kidney. METHODS The DNA fragment of PAX2 coding sequence was amplified by PCR with designed primer from the plasmid of pcDNA3.1-PAX2, and then subcloned into pGC-FU vector with InFusion technique to generate the lentiviral expression vector, pGC-FU-PAX2. The positive clones were screened by PCR and the correct PAX2 was confirmed by sequencing. Recombinant lentiviruses were produced in 293T cells following the cotransfection of pGC-FU-PAX2 , and packaging plasmids of pHelper 1.0 and pHelper 2.0. PAX2 protein expression in 293T cells was detected by Western blot. RESULTS The lentiviral expression vector carrying PAX2 was successfully constructed and PAX2 protein expression was detected in 293T cells. CONCLUSION The recombinant lentiviruse pGC-FU- PAX2 is successfully produced and it can establish a foundation for our study on the molecular function of PAX2.

关 键 词：PAX2基因 慢病毒载体 293T细胞 

分 类 号：R329.23[医药卫生—人体解剖和组织胚胎学]