IL-31重组腺病毒载体的构建及鉴定  

Construction and identification of IL-31 recombinant adenoviral plasmid

作　　者：黄俊琼[1] 张海峰[2] 章春花[2] 胡永林[1] 

机构地区：[1]遵义医学院附属医院检验科,贵州遵义563003 [2]苏州大学医学院细胞与分子生物学教研室,江苏苏州215123

出　　处：《临床和实验医学杂志》2008年第6期17-19,共3页Journal of Clinical and Experimental Medicine

摘　　要：目的构建并鉴定人白细胞介素31(IL-31)重组腺病毒Ad-IL-31,为下一步IL-31功能研究奠定基础。方法以pGEM-T-IL-31为模板扩增IL-31基因,将其克隆到腺病毒穿梭质粒pAdTrack-CMV,PmeⅠ线性化后与腺病毒骨架质粒共转化BJ5183细菌,获得重组腺病毒质粒pAd-IL-31,经PacⅠ线性化后转染QBI-293A细胞,荧光显微镜下观察细胞内荧光,测定病毒效价,逆转录-聚合酶链反应(RT-PCR)和蛋白质印迹(westernblot)分析细胞内IL-31信使核糖核酸(mRNA)和蛋白质的表达。结果经双酶切及基因扩增仪鉴定,重组穿梭质粒和重组腺病毒质粒均见500bp插入片段,测序结果和基因库中的基因序列一致;重组病毒效价为2.6×108pfu/ml;重组病毒感染后,QBI-293A细胞中有IL-31mRNA和蛋白质表达。结论成功构建IL-31重组腺病毒载体,并在QBI-293A细胞中表达,为进一步研究IL-31的功能奠定了基础。Objective To construct and identify human IL -31 recombinant adenovirus. Methods IL -31 gene was amplified from recombinant plasmid pGEM -T -IL-31 and cloned into the shutde plasmid pAdTrack -CMV. The recombinant shuttle plasmid was digested by Pine 1 , followed by homologous recombination with bone plasmid pAdEasy -1 in BJ5183. Recombinant adenovirus was obtained after being packaged in QBI-293A cells, then identified by RT - PCR and western blot. The titer of virus was analyzed with fluorescent counter. Results In recombinant shuttle plasmid and recombinant adenovirus, a fragment of 500bp detected by RT- PCR and digested by enzyme was identical with that included in Genbank. The titer of recombinant adenovirus reached 2.6 × 10^8 pfu/ml. IL -31 mRNA and protein were expressed in QBI --293A cells infected by recombinant adenovirus. Conclusion The recombinant adenovirus containing IL - 31 gene was constructed suceessfully. IL - 31 protein could be expressed stably and effectively. This will be helpful for further study of the function of IL -31.

关 键 词：IL-31 重组腺病毒 构建 表达 

分 类 号：R392[医药卫生—免疫学]