融合蛋白mTSLPR-Ig的表达、纯化及其鉴定  

Expression,purification and identification of fusion protein mTSLPR-Ig

作　　者：张方[1] 施毅[1] 黄钢[2] 李子玲[1] 胡波[3] 宋勇[1] 

机构地区：[1]南京军区南京总医院呼吸内科,江苏南京210002 [2]第三军医大学分子遗传教研室,重庆400038 [3]南京军区联勤部卫生部,江苏南京210002

出　　处：《细胞与分子免疫学杂志》2011年第5期511-514,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：中国博士后科学基金项目(20070411050);江苏省博士后科研资助计划项目(0701023B)

摘　　要：目的:构建含小鼠TSLPR胞外段与小鼠Ig Fc段融合基因的腺病毒表达载体(Ad-mTSLPR-Ig),体外表达、纯化并鉴定可溶性融合蛋白mTSLPR-Ig。方法:运用PCR方法从小鼠胸腺cDNA文库中扩增mTSLPR胞外段基因,构建并鉴定mTSLPR胞外段与小鼠Ig Fc段融合基因的腺病毒表达载体(Ad-mTSLPR-Ig);将Ad-TSLPR-mIg转染COS-7细胞,收集细胞上清液;采用双抗体ELISA夹心法检测上清中融合蛋白表达;经蛋白A亲和层析,制备纯化可溶性融合蛋白mTSL-PR-Ig。结果:经测序证实,所构建表达载体的目的基因片段mTSLPR-Ig序列正确,成功包装出腺病毒表达载体Ad-mTSL-PR-Ig;ELISA法检测到上清中融合蛋白mTSLPR-Ig的表达;通过亲和层析法获得纯化的融合蛋白,经Western blot鉴定融合蛋白表达无误。结论:成功获得可溶性融合蛋白mTSLPR-Ig,为进一步研究其生物学特性奠定基础。AIM： To construct an adenovirus vector（Ad-mTSLPR-Ig） expressing the fusion protein of the extracellular domain of mouse TSLPR and the Fc fragment of mouse Ig,further purify and assess the soluble fusion protein mTSLPR-Ig.METHODS： The gene of mTSLPR extracellular domain was amplified from cDNA library of mouse thymus by PCR.Then an adenovirus vector（Ad-mTSLPR-Ig） expressing the fusion protein of mTSLPR and the Fc fragment of mouse Ig was constructed.After Ad-mTSLPR-Ig was transfected into COS-7 cells,the supernatants were collected.The fusion proteins in the supernatants were detected by double antibody sandwich ELISA.The soluble fusion proteins mTSLPR-Ig were purified by protein A affinity chromatography.RESULTS： The gene sequence of Ad-mTSLPR-Ig was confirmed by DNA sequencing,and the recombinat adenoviruses harboring mTSLPR-Ig were successfully obtained after the infection of Ad-mTSLPR-Ig.Fusion proteins mTSLPR-Ig in the supernatants were detected by ELISA assay.The purified fusion protein were obtained by affinity chromatography,and identified by Western blot.CONCLUSION： The soluble fusion proteins mTSLPR-Ig have been obtained successfully,which enables further study of its biological activity.

关 键 词：TSLPR 可溶性受体 TSLP 哮喘 

分 类 号：R392.12[医药卫生—免疫学]