IL-4、IL-13蛋白表达及抗IL-4、IL-13人源单链抗体的筛选

摘要

Objective:Expression of IL-4 and IL-13 proteins and selection of human scFvs against IL-4 and IL-13 by phage display.Methods:The mRNA of PBMC from healthy volunteer was extracted and cDNA of IL-4 and IL-13 were synthesized by RT-PCR,and the proteins of IL-4 and IL-13 were expressed and identified.The scFvs against IL-4 and IL-13 were selected from a non-immune human scFvs library by phage display with biotinylation IL-4 and IL-13 protein.Results:The size of cDNA of amplified IL-4 was 280 bp,and that of expressed fusion protein was about 27 kD.The size of cDNA of amplified IL-13 was 252 bp,and that of expressed fusion protein was about 25 kD.The human scFvs library was screened for three rounds by ribosome display,and about 37％ scFvs has binding activities with IL-4 and about 27％ scFvs has binding activities with IL-13.Four scFvs with good binding activities with IL-4 or IL-13 were selected and identified by Western blot.Conclusion:The scFvs against IL-4 and against IL-13 were selected successfully.%目的:表达IL-4和IL-13蛋白,从人源单链抗体文库中分别筛选抗IL-4和抗IL-13单链抗体.方法:采用RT-PCR从健康志愿者外周血单核细胞(PBMC) mRNA中扩增IL-4和IL-13 cDNA;构建硫氧还蛋白融合表达载体,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IL-4和IL-13为抗原从前期构建的人源抗体文库中采用噬菌体展示技术分别筛选抗IL-4和抗IL-13人源单链抗体(scFv).结果:扩增的IL-4 cDNA大小为280 bp,表达的融合蛋白大小为27 kD左右.扩增的IL-13 cDNA大小为252 bp,表达的融合蛋白大小为25 kD左右.分别以生物素化的IL-4和IL-13蛋白为抗原,采用噬菌体展示技术对人源抗体文库进行3轮富集后,分别有大约37％的scFvs与IL-4有结合特性,有约27％的scFvs与IL-13有结合特性.筛选了4株分别与IL-4和IL-13结合能力强的单链抗体进行了Westem blot鉴定和测序.结论:成功筛选到抗IL-4和抗IL-13人源性单链抗体.