中国兽医杂志2025，Vol.61Issue(9)：44-52,9.DOI:10.20157/j.cnki.zgsyzz.2025.09.006
猪氨基肽酶N受体结合区Fc融合表达及其病毒结合活性分析
Fc-fusion Expression of Porcine Aminopeptidase N Receptor-Binding Region and Analysis of Its Viral Binding Activity
摘要
Abstract
To investigate the viral receptor-binding functional regions of porcine aminopeptidase N(pAPN)and their binding activity with transmissible gastroenteritis virus(TGEV)and porcine epidemic diarrhea virus(PEDV),this study employed splicing by overlap extension PCR(SOE-PCR)to divide the extracellular domain of pAPN into five segments and fuse them with the Fc fragment.Yeast secretion expression vectors(pPICZαA-pAPN-Fc)were constructed for fusion expression.The binding abilities of the five Fc fusion proteins to PEDV and TGEV,as well as their effects on viral nucleocapsid protein(N)gene transcription and replication,were analyzed at the molecular and cellular levels to identify key functional regions involved in virus-receptor interaction.The results showed that all five fusion proteins—pAPN1-Fc,pAPN2-Fc,pAPN3-Fc,pAPN4-Fc,and pAPN5-Fc—were efficiently secreted and expressed in Pichia pastoris X33 and specifically bound to antisera against native pAPN protein.Enzyme-linked immunosorbent assay(ELISA)analysis revealed that pAPN2-Fc,pAPN3-Fc,pAPN4-Fc,and pAPN5-Fc displayed varying degrees of binding activity with PEDV and TGEV,with stronger binding to TGEV than PEDV.Among them,pAPN2(574-678 aa)and pAPN3(669-773 aa)exhibited the strongest binding.Real-time fluorescence quantitative reverse transcription polymerase chain reaction(qRT-PCR)and immunofluorescence confocal assays confirmed that pAPN2-Fc and pAPN3-Fc effectively inhibited the transcription of PEDV and TGEV N gene mRNA and suppressed viral replication.These results indicate that the pAPN extracellular region spanning amino acids 574-773 can bind both PEDV and TGEV and inhibit N gene expression and viral replication,suggesting it may serve as a shared critical region for virus-receptor interaction.This study provides a theoretical basis for understanding the infection mechanisms of PEDV and TGEV and for the design of antiviral agents.关键词
猪氨基肽酶N/肠道冠状病毒/Fc融合表达/病毒结合活性Key words
porcine aminopeptidase N/intestinal coronavirus/Fc-fusion expression/viral binding activity分类
农业科技引用本文复制引用
郭宏伟,刘妍,赵绪永,龚婷,马辉,刘薇,郑鸣..猪氨基肽酶N受体结合区Fc融合表达及其病毒结合活性分析[J].中国兽医杂志,2025,61(9):44-52,9.基金项目
河南省科技攻关项目(222102110158) （222102110158）
河南省自然科学基金面上科学基金项目(252300420187) （252300420187）
河南省重点研发专项项目(251111110700) （251111110700）
