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目的筛选与白细胞介素-13(interleukin-13,IL-13)具有较强结合能力的功能小肽,并检测其对气道上皮细胞HBE16分泌黏蛋白(mucin,MUC)5AC的影响。方法以重组人IL-13为靶蛋白,采用固相包被法对噬菌体随机展示十二肽库进行亲和筛选,并进行测序;将获得的与IL-13具有高亲和力的小肽偶联到嗜孔蓝蛋白(keyhole limpethemocyanin,KLH)载体上,制备KLH偶联肽K1、K3、K4,通过夹心ELISA法检测3种偶联肽对IL-13的封闭作用;通过细胞试验检测3种偶联肽对HBE16细胞分泌MUC5AC的影响。结果经6轮亲和富集筛选,获得了3个与IL-13具有较高亲和力的噬菌体克隆,插入的十二肽编号依次为P1、P3、P4;3种偶联肽K1、K3、K4对IL-13均具有明显的封闭作用;经K1+IL-13、K3+IL-13和K4+IL-13处理的HBE16细胞中MUC5AC基因mRNA的转录水平和蛋白表达水平均明显低于IL-13对照组(P<0.01)。结论经偶联肽封闭后,可减少IL-13刺激的气道上皮细胞HBE16中MUC5AC的分泌,为慢性气道炎症黏液高分泌的防治提供了新的思路和手段。
Objective To screen small peptides with high binding capacity to interleukin-13 (IL-13) and to detect the effects of mucin (MUC) 5AC on HBE16 in airway epithelial cells. Methods Recombinant human IL-13 was used as the target protein, and the phage displayed by the phage display library was affinity screened by solid phase method and sequenced. The obtained small peptide with high affinity to IL-13 KLH coupling peptides K1, K3 and K4 were prepared on keyhole limpethemocyanin (KLH) vector. The blocking effect of three kinds of coupling peptides on IL-13 was detected by sandwich ELISA. Three kinds of coupling were detected by cell assay Effect of Peptides on MUC5AC Secretion by HBE16 Cells. Results Three prokaryotic clones with high affinity to IL-13 were screened by six rounds of affinity enrichment. The inserted dodecyl peptides were P1, P3 and P4 in sequence. Three kinds of coupling peptides K1, K3 and K4 The mRNA and protein expression of MUC5AC in HBE16 cells treated with K1 + IL-13, K3 + IL-13 and K4 + IL-13 were significantly lower than that of IL- 13 control group (P <0.01). Conclusion After the coupling peptide is blocked, the secretion of MUC5AC in IL-13-stimulated airway epithelial cells can be decreased, which provides a new way of prevention and treatment of mucus hypersecretion in chronic airway inflammation.