抗TSLP全人源单链抗体筛选与初步鉴定  

Selection and identification of full human scFv against TSLP

作　　者：朱建光 袁青[2] 黄黎[2] 徐文峰[2] 年四季[2] 

机构地区：[1]咸宁市中心医院,咸宁437100 [2]泸州医学院基础医学院,泸州646000

出　　处：《中国免疫学杂志》2014年第12期1662-1665,1669,共5页Chinese Journal of Immunology

基　　金：四川省青年基金(2012JQ0018);泸州市科技局项目(2013LZLY-K77);四川省卫生厅项目(130259)

摘　　要：目的:表达TSLP蛋白,从全人源单链抗体文库中筛选得到抗TSLP单链抗体。方法:扩增TSLP cDNA,将目的基因与表达载体pET101/D-TOPO连接,转化大肠杆菌BL21,IPTG诱导表达并对表达产物进行纯化鉴定。以生物素化的TSLP蛋白为抗原从前期构建的全人源抗体文库中采用噬菌体展示技术筛选抗TSLP全人源单链抗体(scFv)。结果:扩增的TSLP cDNA片段大小为423 bp左右。表达的TSLP蛋白大小为26 kD左右,Dot blot及Western blot鉴定显示表达的蛋白正确,为TSLP目的蛋白。以生物素化的TSLP蛋白为抗原,采用噬菌体展示技术对全人源抗体文库进行3轮富集,通过ELISA检测有35%的scFv与TSLP有结合特性。将筛选的与TSLP结合能力强的单链抗体进行了Western blot鉴定和测序。结论:成功筛选到抗TSLP全人源单链抗体。Objective：Expression of protein TSLP and selection of full human anti-TSLP single chain Fv （ scFv）.Methods：The cDNA of TSLP was amplified.The amplified target gene and the expression vector pET 101/D-TOPO were ligated , and then transformed into E.coli BL21.The protein was induced to expression by IPTG and purified and identified.The biotinylated TSLP protein was used as antigen to select of human TSLP scFv from a constructed human scFv library by phage display .Results： The size of amplified cDNA of TSLP was about 423 bp,and that of expressed protein was about 26 kD.Dot blot and Western blot results showed that the expressed protein was correct.The constructed human scFv library was enriched for three rounds using biotinylated TSLP as antigen by phage display.ELISA results showed that 35% scFvs had binding activity with TSLP.The scFvs with good binding activity were selected and identified by Western blot and sequencing.Conclusion： The full human scFvs against for TSLP were selected suc-cessfully.

关 键 词：TSLP 人源单链抗体 筛选 

分 类 号：R392.11[医药卫生—免疫学]