重组人DcR3腺相关病毒的构建和表达  

Construction and expression of recombinant human DcR3 adeno-associated virus

作　　者：王槐志[1] 黎万玲[2] 董家鸿[1] 倪兵[2] 吴玉章[2] 

机构地区：[1]第三军医大学西南医院全军肝胆外科研究所,重庆400038 [2]第三军医大学基础医学部全军免疫学研究所,重庆400038

出　　处：《免疫学杂志》2006年第5期487-490,共4页Immunological Journal

基　　金：国家自然科学基金项目(30400418);重庆市自然科学基金重点项目(CSTC;2005BA5004)资助

摘　　要：目的克隆人DcR3基因于腺相关病毒(adeno-associatedvirus,AAV)载体pAAV-IRES-hrGFP中,以获得高感染滴度及纯度的重组人DcR3腺相关病毒,为将其用于感染移植肝,研究其对大鼠移植肝的保护作用打下基础。方法用基因重组的方法构建含全长人DcR3基因的重组腺相关病毒骨架质粒,利用脂质体法将腺相关病毒载体系统共转染入病毒包装细胞AAV-293细胞中,合成重组DcR3腺相关病毒,经PEG/氯仿纯化,用SDS-PAGE鉴定其纯度,扫描电镜观察病毒颗粒形态,倍比稀释法测定重组病毒的感染滴度。并用重组病毒感染COS-7细胞鉴定DcR3的表达。结果正确构建组装重组人DcR3腺相关病毒,纯化后病毒感染滴度为1.1×1010U/mL。在重组DcR3腺相关病毒感染的COS-7细胞上清液中检测到了DcR3的表达。结论成功构建了重组人DcR3腺相关病毒,为下一步研究DcR3对大鼠移植肝的保护作用打下基础。Objective To clone DcR3 gene into AAV vector pAAV-IRES-hrGFP and obtain high titre and purity recombinant human DcR3 AAV for infecting allograft liver and studying its protective effect on allograft liver in the further experiment. Methods Framework plasmid of recombinant DcR3 AAV was constructed by gene recombination. AAV vector system was cotransfected into AAV-293 ceils with lipo-fectamine2000. Recombinant DcR3 AAV was produced in AAV-293 ceils and purified by PEG/chloroform. The purity of recombinant DcR3 AAV was verified by SDS-PAGE. These virus particles were observed by scanning electron microscope. The titre of the virus particles was detected by multiple proportion dilution. DcR3 expression in COS-7 ceils which were infected by recombinant DcR3 AAV was verified by Western blotting. Results Recombinant human DcR3 AAV was successfully constructed and its fitre reached 1.1×10^10 U/L after purification. DcR3 expression in cultivation supernatant of COS-7 ceils which were infected by recombinant DcR3 AAV was successfully detected. Conclusion Recombinant human DcR3 AAV is successfully constructed, which will benefit our further study on protection effect of DcR3 to rat liver graft.

关 键 词：基因 DCR3 腺相关病毒 

分 类 号：R392[医药卫生—免疫学]