人EPO基因重组腺病毒载体的构建及表达  

Construction and expression of a recombinant adenovirus with hEPO

作　　者：陈邦党[1] 马依彤[1] 马翔[1] 杨毅宁[1] 刘芬[1] 

机构地区：[1]新疆医科大学第一附属医院心脏中心,新疆乌鲁木齐830054

出　　处：《细胞与分子免疫学杂志》2010年第3期223-226,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30760263);新疆医科大学一附院奖励基金(2007YFYO1)

摘　　要：目的:制备表达人促红细胞生成素基因(hEPO)的重组腺病毒。方法:将hEPO基因亚克隆到穿梭质粒pAdTrack-CMV上,采用细菌内同源重组"两步转化法"构建携带hEPO基因的重组腺病毒载体。获得的重组腺病毒载体线性化后转染HEK293细胞,包装、扩增出重组腺病毒,氯化铯梯度离心法进行纯化,PCR和电镜负染鉴定重组腺病毒,荧光显微镜观察绿色荧光蛋白(GFP)的表达并检测重组腺病毒的滴度。继而用纯化的重组腺病毒感染HeLa细胞,测定转染效率,以Western blot检测hEPO蛋白的表达。结果:成功构建了携带hEPO基因的重组腺病毒载体pAd-hEPO,PCR、透射电镜和绿色荧光鉴定证实病毒包装成功,并且具有感染力,病毒的滴度可达1.8×1010pfu/mL。Western blot检测表明RAd-hEPO感染的HeLa细胞中hEPO基因能够有效表达。结论:制备的RAd-hEPO可成功表达hEPO基因,为后续开展RAd-hEPO治疗缺血性心脏病的研究奠定基础。AIM：To construct the adenovirus vector containing human erythropoietin gene（hEPO） and to detect its expression in HeLa cells.METHODS：hEPO gene was subcloned into the shuttle plasmid pAdTrack-CMV and then a two-step transformation procedure was employed to construct a recombinant adenoviral plasmid with hEPO.The recombinant adenoviral plasmid with hEPO was digested with Pac I and then transfected into HEK293 cells to package recombinant adenovirus particles.The recombinant adenovirus containing hEPO gene was identified by PCR and transmission electron microscopy and was purified by cesium choride density centrifugation.The viral titer was checked by GFP.HeLa cells were infected by the recombinant adenovirus and the transcription and expression of hEPO gene were analyzed by RT-PCR and Western blot.RESULTS：Recombinant adenovirus vector pAdEasy-hEPO was constructed and a recombinant adenovirus was obtained by transfecting HEK293 cells with pAdEasy-hEPO.The high expression of green fluorescence protein expression in HEK293 and HeLa cell lines was observed under fluorescent microscope.PCR and electron microscopy test showed that a recombinant adenovirus RAd-hEPO was successfully constructed and the titer of the recombinant adenovirus reached 1.8×1010 pfu/mL.The expression of hEPO gene in the infected HeLa cells was confirmed by Western blot.CONCLUSION：The recombinant adenovirus RAd-hEPO can be successfully expressed in the infected HeLa cells,which lays the foundation for further research into therapy for ischemic heart diseases.

关 键 词：人促红细胞生成素 同源重组 表达 重组腺病毒 

分 类 号：Q782[生物学—分子生物学]