河南农业大学学报2025，Vol.59Issue(5)：838-848,11.DOI:10.16445/j.cnki.1000-2340.20250512.001
猪繁殖与呼吸综合征病毒非结构蛋白1α原核表达及其多克隆抗体的制备
Prokaryotic expression and preparation of polyclonal antibodies against PRRSV NSP1α protein
摘要
Abstract
[Objective]Preparation of polyclonal antibody against non-structural protein 1α(NSP1α)provides a material foundation for in-depth research on the NSP1α protein and offers a new insight for the prevention and control porcine reproductive and respiratory syndrome(PRRSV)infection.[Method]PRRSV NSP1α gene was amplified using pCAGGS-NSP1α-HA plasmid as the template,then the homology of NSP1α gene sequences between SD16 strain and other PRRSV strains,the pro-tein physicochemical properties,amino acid hydrophilicity/hydrophobicity,transmembrane domains,signal peptide sequences,phosphorylation sites,secondary and tertiary structures of proteins were ana-lyzed using bioinformatics online software.Then the polymerase chain reaction(PCR)products were purified and cloned into the pET-28a-His prokaryotic expression vector through EcoR I and Xho I.Next,the pET-28a-NSP1α-His plasmids were transformed into E.coli BL21(DE3).The NSP1α pro-tein after induced expression and purification was used to immunize mice to prepare polyclonal anti-body against PRRSV NSP1α protein.The reactivity of the prepared polyclonal antibodies was identi-fied using Western blot and indirect immunofluorescence assay(IFA).Finally,the subcellular distri-bution of the NSP1α protein was analyzed using Western blot and confocal microscopy.[Result]The results of information analysis showed that the NSP1α protein of PRRSV SD16 strain has 501 amino acids and is an unstable hydrophobic protein;Its sequence has the highest homology with the HLJ strain and the lowest homology with the type I PRRSV strain;NSP1α protein has no signal peptide sequence and transmembrane domain,but may contain 13 phosphorylation sites;The secondary struc-ture mainly includes alpha helices,extended chains,and random coil.The high-purity and active NSP1α protein was used to immunize BALB/c mice,and the serum antibody titer reached 1∶64 000.Western blot and IFA results showed that the prepared polyclonal antibody specifically recognized PRRSV NSP1α protein.And Western blot and confocal microscopy results showed that NSP1α protein is primarily localized in the cell nucleus.[Conclusion]The PRRSV NSP1α protein was expressed and purified,and the mouse derived polyclonal antibody was successfully prepared.The polyclonal anti-body has good reactivity and specificity with PRRSV NSP1α protein.关键词
猪繁殖与呼吸综合征病毒/非结构蛋白1α/原核表达/多克隆抗体/生物学特性Key words
porcine reproductive and respiratory syndrome virus/non-structural protein 1α/prokary-otic expression/polyclonal antibody/biological characteristics分类
农业科技引用本文复制引用
张铭芳,任佳慧,张亚慈,位雪丹,申艾娟,张昂克,段红..猪繁殖与呼吸综合征病毒非结构蛋白1α原核表达及其多克隆抗体的制备[J].河南农业大学学报,2025,59(5):838-848,11.基金项目
国家自然科学基金项目(32302871) （32302871）
河南省重点研发与推广专项(科技攻关)(232102110101) （科技攻关）
