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肿瘤坏死因子alpha的拮抗剂是治疗多种炎症性自身免疫疾病的首选,但抗体类拮抗物因副作用明显而使用受限,尤其是机体内抗抗体的产生,严重影响治疗效果和药物代谢。而肽类物除免疫原性低之外,和小分子相比也有更低的毒性和更强的靶标特异性。使用7肽和12肽两种M13噬菌体展示库筛选TNFα拮抗肽,以分析7肽和12肽分别作为TNFα拮抗肽的亲和性与功能性。经过3~4轮的筛选和验证,得到2条7肽序列和2条12肽序列。利用ELISA方法检测合成肽与TNFα结合的亲和性,编号632的7肽亲和性最强,Kd=138nmol/L;编号636的12肽亲和性稍差,Kd=8.59μmol/L。InsightⅡ软件分别分析632肽和636肽与TNFα二聚体结合,发现632肽与TNFα二聚体结合更加稳定,并且在细胞水平上632肽拮抗TNFα活性功能比636肽更强,有632肽存在的条件下TNFα诱导的L929细胞生存率上升了3倍,而636肽的作用只有2倍。7肽比12肽更适合作为TNFα拮抗肽。
Tumor necrosis factor alpha antagonists are the first choice for the treatment of a variety of inflammatory autoimmune diseases. However, the use of antibody antagonists due to obvious side effects is limited, especially the production of anti-body antibodies in the body, seriously affecting the therapeutic effect and drug metabolism. Peptides, besides their low immunogenicity, have lower toxicity and stronger target specificity than small molecules. TNFα antagonist peptides were screened using both the 7 peptide and 12 peptide M13 phage display libraries to analyze the affinity and functionality of the 7 and 12 peptides as TNFα antagonist peptides, respectively. After 3 to 4 rounds of screening and verification, two 7-mer peptide sequences and two 12-mer peptide sequences were obtained. The binding affinity of the synthesized peptide to TNFα was detected by ELISA. The affinity of the 7 peptide of No.632 was the strongest, Kd = 138 nmol / L. The 12 peptide of No 636 had a slightly lower affinity and Kd = 8.59 μmol / L. Insight II software separately analyzed the binding of 632 peptide and 636 peptide to TNFα dimer and found that 632 peptide binds to TNFα dimer more stably and that 632 peptide antagonizes TNFα activity more strongly than 636 peptide at the cellular level with 632 peptide present TNFα-induced L929 cell survival rate increased by 3 times, while the role of 636 peptide only 2 times. 7 peptide is more suitable than 12 peptide as a TNFα antagonist peptide.