靶向髓样分化蛋白—2特异性抗炎多肽的优化

Study on the optimization of specific anti-inflammatory peptides targeting to myeloid differentiation protein-2

目的对原始多肽即靶向髓样分化蛋白-2（MD-2）拮抗多肽T6、T11 进行突变多肽库的构建、筛选和功能鉴定,以获得具有更高抗炎活性的多肽.方法：以前期合成的可阻断MD-2 与内毒素（LPS） 结合但效率不高的原始多肽T6 和T11 序列为基础,每次随机突变相邻的3~5 个氨基酸,保留剩余氨基酸,并同时在肽链的氨基端和羧基端引入额外的两个氨基酸,构建大容量的噬菌体突变肽库;再用全长的MD-2 蛋白进行靶向淘选获得特异结合的多肽.最后用细胞实验和动物实验验证所获特异多肽的抗炎效果：①用获得的多肽200 μg/ml 分别孵育巨噬细胞和人单核白细胞2 h,再用内毒素（LPS）1 μg/ml 刺激6 h.以单纯细胞培养液培养的细胞和LPS 刺激6 h 的细胞作对照.② C57 小鼠腹腔注射获得的多肽200 μg/ml, 1 h 后腹腔注射LPS 400 μg/ml,6 h 后取血.以腹腔注射生理盐水1 ml 或LPS 的小鼠作对照.每组5 只小鼠.采用ELISA 法测定各组细胞培养上清和各组小鼠血清的肿瘤坏死因子α（TNF-α）和白介素6（IL-6）的表达水平.结果：成功构建出原始多肽T6 和T11 序列为基础的突变肽库,用全长的MD-2 蛋白进行靶向淘选获得特异结合多肽A8、H2、F5、H5、H9,通过细胞实验筛选得到有抑炎效果的多肽2 条（A8、H2）,可以明显抑制LPS 所致细胞分泌TNF-α 和IL-6 水平的增高.动物实验发现只有1 条多肽（H2）可以明显抑制LPS 所致小鼠血清TNF-α 和IL-6 水平的增高.结论：成功构建了突变肽库,并从中筛选出一条具有高效抗炎效应的多肽.

Objective: To obtain higher anti-inflammatory activity of the peptides through constructing mutation peptides library based on the original peptides [myeloid differentiation protein-2(MD-2) antagonism peptides T6 and T11], and screening and identifying its function. Method: T6 and T11 were synthesized previously, but their effect of blocking the combination of MD-2 and lipopolysaccharide(LPS) was weak. In this study, based on the sequence of T6 and T11, every adjacent 3 to 5 amino acids were mutated randomly, and the rest of the amino acids were kept intact, at the same time two additional amino acids were added at the amino terminus and carboxyl terminus of the peptide chain, as to gain greater joint surface and to construct a mutation peptides library of phage with greater capacity. A total length of the MD-2 protein was targeted to screen peptides from the library. Finally, their anti-inflammatory effect was verified through cellular and animal experiments. 1 Different peptides(200 ug/ml) were used to stimulate macrophages(RAW264.7) and monocytes(THP-1) for 2 hours, and then the cells were stimulated with LPS(1 ug/ml) for 6 hours. Cells cultured in culture medium or LPS were used to serve as controls. 2 C57 mice were given different peptides(200 ug/ml) introperitoneally, and they received introperitoneal injection of LPS(400 ug/ml) 1 hour later. Blood was sampled 6 hours after the injection of LPS. The mice with injection of normal saline or LPS served as controls. The levels of the tumor necrosis factor –a(TNF-a) and interleukin-6(IL-6) in the supernant of cells or serum of mice were determined by ELISA. Results: A bank of mutant peptide library of phages was constructed successfully. Targeting the total length of the MD-2 protein, five specific polypeptide(A8, H2, F5, H5, H9) were obtained. Two peptides(A8, H2) were found to have inflammation-suppression effect in cell experiments, and they can alleviate the increase in TNF-a and IL-6 contents in the supernant induced by LPS stimulation. Only one peptide(H2) was found to have the ability of inhibiting the increase of serum TNF-a and IL-6 content induced by LPS. Conclusions: A mutant peptide library is constructed successfully, and from it, a peptide with high effect of antiinflammatory is obtained by sereening.