基于天蚕素D的新型抗病毒多肽对猪繁殖呼吸综合征病毒的抑制作用

Novel cecropin D-derived peptide with inhibitory effect on porcine reproductive and respiratory syndrome virus entry

摘要:

由猪繁殖呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)引发的猪繁殖呼吸综合征(porcine reproductive and respiratory syndrome,PRRS)是目前危害养猪业的主要疾病之一。本研究旨在设计优化天然抗菌肽，筛选具有显著抑制PRRSV活性的抗病毒多肽，为PRRS防控制剂的研发提供新思路。以天蚕素D (cecropin D,CD)为母肽，通过氨基酸替换策略设计了CD-2、CD-3、CD-4这3个衍生物，利用固相合成技术合成CD及其衍生物，并使用质谱(mass spectrometry,MS)和反相高效液相色谱(reversed-phase high performance liquid chromatography,RP-HPLC)进行多肽的序列鉴定、纯化和纯度分析。通过圆二色谱研究多肽二级结构，使用CellTiter 96^® AQueous单溶液细胞增殖检测试剂盒测定多肽细胞毒性。通过蛋白免疫印迹(Western blotting,WB)、实时荧光定量逆转录聚合酶链反应(reverse transcription-quantitative polymerase chain reaction,RT-qPCR)以及间接免疫荧光(indirect immunofluorescence assay,IFA)研究多肽对PRRSV的抗病毒活性及作用阶段。MS以及RP-HPLC结果表明，CD及其衍生物被成功合成，且纯度高达95%。圆二色谱分析结果显示，CD衍生物在细胞膜模拟环境中表现出更稳定且丰富的α螺旋结构。细胞毒性测定结果表明，所有被测多肽在100 μg/mL的工作浓度下的细胞毒性可忽略。多肽抗PRRSV的作用阶段试验结果显示，衍生多肽在病毒侵入阶段的抗病毒活性较母肽显著增强，说明通过引入赖氨酸、色氨酸和苯丙氨酸，增强了母肽的阳离子性和疏水性，进而增强了衍生多肽在侵入阶段的抗PRRSV活性。本研究结果为抗病毒多肽的应用及结构优化提供了理论基础，并为PRRSV的防控提供了新的思路。

Abstract:

Porcine reproductive and respiratory syndrome (PRRS), caused by the porcine reproductive and respiratory syndrome virus (PRRSV), is one of the major diseases threatening the swine industry. This study aims to rationally design and optimize natural antimicrobial peptides to identify antiviral candidates with potent inhibitory activity against PRRSV, thereby establishing a foundation for the development of novel preventive and therapeutic agents targeting PRRS. In this study, with cecropin D (CD) as the parent peptide, three derivatives (CD-2, CD-3, and CD-4) were designed through amino acid substitutions. CD and derived peptides were obtained by solid-phase peptide synthesis. MS and reversed-phase (RP)-HPLC were employed for sequence identification, purification, and purity analysis. The secondary structures of the peptides were investigated by circular dichroism spectroscopy. CellTiter 96^® AQueous one solution cell proliferation assay was used to evaluate the cytotoxicity of the peptides. The inhibitory activities and mechanisms of the peptides against PRRSV were studied by Western blotting, RT-qPCR, and indirect immunofluorescence assay. The MS and RP-HPLC results showed that CD and derived peptides were successfully synthesized, with the purity reaching up to 95%. Circular dichroism analysis revealed that the CD derivatives exhibited more stable and abundant α-helices in a cell membrane-mimicking environment. The MTS assay indicated that all tested peptides at 100 μg/mL had negligible cytotoxicity. The experimental results of the action phase of the peptide against PRRSV demonstrated that the derived peptides significantly enhanced antiviral activities at the viral entry stage compared with the parent peptide. This enhancement was attributed to the introduction of lysine, tryptophan, and phenylalanine, which increased the hydrophobicity and positive charge of the peptides. These findings provide a theoretical basis for the application and structural optimization of antiviral peptides and may offer a new strategy for preventing and controlling PRRSV.