果蝇硒蛋白dSelK基因重组腺病毒构建与表达  

Establishment of the Recombinant Adenovirus Vector for Drosophila Selenoprotein dSelK and Its Soluble Expression

作　　者：娄虹[1] 许鑫[1] 潘子瑶[1] 张保华[1] 贲松彬[1] 陈长兰[2] 

机构地区：[1]辽宁大学生命科学院,辽宁沈阳110036 [2]辽宁大学药学院,辽宁沈阳110036

出　　处：《生物技术》2014年第2期28-31,共4页Biotechnology

基　　金：国家自然基金项目(31371085);辽宁省教育厅资助项目(2009A310);辽宁省科学技术计划项目(2010225036);辽宁大学国家自然基金预资助和博士启动项目共同资助

摘　　要：目的:利用AdEasy腺病毒载体系统构建果蝇硒蛋白dSelK基因重组腺病毒并在AD-293细胞中包装扩增。方法:PCR扩增dSelK目的片段连接到穿梭载体pShuttle-CMV上,用电转化法将线性化的pShuttle-CMV-dSelK穿梭载体转入含腺病毒骨架质粒AdEasy1的BJ5183大肠杆菌电感受态细胞中,构建重组腺病毒载体AdEasy-dSelK,线性化后转染AD-293细胞进行包装,并传代扩增,TCID50法测病毒滴度,Western blot鉴定。结果:成功构建腺病毒载体AdEasy-dSelK,经AD-293细胞包装扩增得到重组腺病毒,滴度为107.5。Western blot鉴定AdEasy-dSelK成功表达。结论:成功构建重组腺病毒载体pAdEasy-dSelK,为硒蛋白功能研究奠定基础。Objective： To construct recombinant adenovirus vector for Drosophila Selenoprotein dSelK and amplify the adenovirus vector in AD293 ceils. Method： dSelK gene are amplified through PCR, then subcloned into the shuttle plasmid pShuttle - CMV. The recombinant plasmid pShuttle - CMV - dSelK linearized by Pme I , plasmid pShuttle - CMV - dSelK was electroporated into E. coli BJ5183 cells. The plasmid AdEasy - dSelK was digested with Pac I and transfected into AD - 293 cells to package the adenovirus, followed by identification of the recombinant adenovirus. The titer of recombinant adenovirus was calculated by TCIDS0 method. The recombinant adenovirus vector was identified by Western blot. Result： The recombinant adenoviral vector for Drosophila Selenoprotein dSelK was constructed successful- ly, and used it to transfected AD - 293 cfells to take the virus amplificated and passaged. To calculated the titer of recombinant adenovirus was 107. 5. The dSelK protein was identified by Western blot. Conclusion： The recombinant adenoviral vector containing Drosophila Seleno- protein dSelK gene was successfully constructed, it will settle the foundation for subsequent selenoproteins functions research.

关 键 词：硒蛋白 dSelK 腺病毒 病毒滴度 

分 类 号：Q786[生物学—分子生物学]