TNF结合肽和TNFR封闭肽的筛选及其鉴定

Screening and Identification of TNF-α Binding Peptide and TNFR Blocking Peptide

目的筛选能与TNF-α结合的环肽(称为TNF结合肽)以及既能与TNFR-Ⅰ结合又不诱导TNFR相关生物学效应的环肽(称为TNFR封闭肽)。方法采用噬菌体随机肽库展示技术分别以hrTNF-α和hrTNFRⅠ为诱饵进行亲和筛选,ELISA方法进行噬菌体特异性鉴定,应用细胞毒抑制实验和RT-PCR技术等进行噬菌体的生物学效应鉴定。根据噬菌体展示肽的DNA序列合成环肽。结果对肽库进行3轮筛选后,噬菌体克隆具有良好的富集效果;5种TNF结合噬菌体克隆(5、6、7、32及38号克隆)和2种TNFR封闭噬菌体(X2、X4号克隆)分别能与TNF-α和TNFR特异性结合,并且均能显著抑制sTNF-α对L929细胞和U937细胞的杀伤作用,TNFR封闭噬菌体不仅本身无促进U937细胞IL-1βmRNA转录的作用,而且还能有效抑制sTNF-α的生物学效应;并且发现X4与32号、X4与38号联用的细胞毒抑制效应均较其单独作用的效果好,其中X4与38号联用的抑制率高达85.3%,显著高于各自单独的抑制作用(P<0.01)。选择32、38和X4号噬菌体克隆进行DNA测序并合成环肽。结论从噬菌体随机环肽库中筛选得到能够抑制sTNF-α生物学功能的TNF结合肽和TNFR封闭肽,为研制TNF相关的多肽类抗炎药物奠定了实验基础。

Objective To screen and identify both TNF-α binding peptide and TNFR blocking peptide and study their biological functions. Methods The soluble hrTNF-αand hrTNFR I were used respectively as a specific bait to screen phage peptide library. ELISA was used to identify the specificity of the selected phage clones. The inhibition test of eytotoxicity of TNF- α and RT-PCR were used to evaluate the biological functions of the selected clones. Results After three rounds of bio-panning, the selected phage clones showed better enrichment result. Five phage clones （No. 5 No. 6, No. 7, No. 32 and No. 38） and 2 phage clones （No. X2 and No. X4） were identified as the potent TNF-αbinding phages and TNFR blocking phages separately. Both phages could inhibit the cytotoxicity of L929 and U937 induced by sTNF obviously. TNFR blocking phages not only could inhibit the cytotoxicity induced by sTNF, but also had no effect to increase the level of IL-1β mRNA induced by TNF-α. The inhibition rate of cytotoxicity of the combination of clones No. X4 and No. 32, clones No. X4 and No. 38 was significantly higher than that of individual phage clones, among them the combination of clones No. 38 and No. X4 exerted higher inhibition rate （85.30 %） of cytotoxicity compared to clone No. 38 or No. X4 alone （P〈0.01）. This result indicated that TNF-α binding phages and TNFR blocking phages worked synergically. Clones No. 32, No. 38 and No. X4 were sequenced in order to determine the amino acid sequences of peptides, and the cycle peptides of clones No. 32, No. 38 and No. X4 were then synthesized. Conclusion The TNF-α binding peptide and TNFR blocking peptide obtained by phage display technique could block the biological activity of the TNF-α, which may provide an important clue for the development of novel TNF related anti-inflammatory peptide drug.