与猪PD-1和PD-L1互作表位多肽的筛选及其免疫调节功能

Screening of Epitope Peptides Binding to Porcine PD-1 and PD-L1 and their Immune Regulatory Function

本试验旨在筛选和鉴定与猪程序性死亡因子1(PD-1)及其配体(PD-L1)相互作用的表位多肽,为阻断猪PD-1/PD-Ls通路逆转机体的免疫功能提供新策略。根据已解析人与鼠的PD-1与PD-L1相互作用的关键氨基酸位点信息,分析猪PD-1与PD-L1蛋白相互结合的关键氨基酸位点,在关键氨基酸位点处设计系列表位多肽,固相合成法合成表位多肽;分离自然感染猪圆环病毒2型(PCV2)仔猪的外周血单个核细胞(PBMC),检测表位多肽与猪重组蛋白PD-1、PD-L1和PBMC的结合能力,选取能结合猪PD-L1和PBMC的表位多肽pPD-15,检测其对猪PBMC增殖的影响,分析其作为佐剂免疫后对小鼠猪瘟病毒(CSFV)抗体水平的影响,最后,高效液质联用色谱法(HPLC-MS)检测表位多肽pPD-15的纯度和氨基酸序列正确性。结果显示,表位多肽pPD-15能结合猪PD-L1和PBMC;增殖试验显示,pPD-15组M1的平均荧光强度(74.20%)比空白对照组M1的平均荧光强度(4.37%)提高了69.83%,表明表位多肽pPD-15可促进猪PBMC的增殖;动物免疫试验显示,pPD-15作为佐剂可以提高CSFV抗体水平;HPLC-MS结果显示,合成的表位多肽pPD-15纯度高,氨基酸序列正确。本试验证实,表位多肽pPD-15具有提高体内外免疫力的能力,为研制新型免疫调节佐剂提供了科学依据。

This study aimed to screen and identify the epitopes that bind to porcine PD-1 and PD-L1,and to provide a new strategy for blocking porcine PD-1/PD-Ls pathway and thus reversing immune function.Based on the information on the key amino acid sites of the interaction between human and mouse PD-1 and their ligands,the key amino acid sites for the mutual binding of porcine PD-1 and PD-L1 proteins were identified,and a series of epitope peptides containing key amino acid sites were designed and synthesized by solid-phase synthesis.PBMC of piglets naturally infected with PCV2 were isolated and the binding ability of FITC-labeled epitope polypeptides to porcine recombinant protein PD-1,PD-L1 and PBMC was assessed.The epitope polypeptide pPD-15 that binds to porcine PD-L1 and PBMC was selected and analyzed for its effect on the proliferation of porcine PBMC.The effect of pPD-15 as an adjuvant on the antibody level of mice after immunization was examined.The purity and amino acid sequence correctness of epitope polypeptide pPD-15 was determined by HPLC-MS.The results showed that the epitope polypeptide pPD-15 bound to porcine PD-L1 and PBMC.The proliferation test showed that the average fluorescence intensity of M1 in the pPD-15 group(74.20%)was 69.83%higher than that in the control group(4.37%),indicating that the epitope polypeptide pPD-15 could promote the proliferation of porcine PBMC.Animal immune test showed that pPD-15 as adjuvant improved the level of CSFV antibody.The results of HPLC-MS showed that the synthesized epitope polypeptide pPD-15 had high purity and correct amino acid sequence.Thus,the epitope polypeptide pPD-15 has the ability to improve the immunity in vivo and in vitro,and provides a basis for the development of a new immune regulatory adjuvant.