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【目的】制备非结构蛋白1α(non-structural protein 1α, NSP1α)多克隆抗体，为深入研究NSP1α蛋白功能提供物质基础，并为猪繁殖与呼吸综合征（porcine reproductive and respiratory syndrome, PRRS）防控靶点的选择提供新思路。【方法】以pCAGGS-NSP1α-HA质粒为模板，聚合酶链式反应扩增NSP1α基因，利用相关生物信息学在线软件分析PRRSV SD16毒株NSP1α基因和其他PRRSV毒株NSP1α基因序列同源性、蛋白的理化性质、氨基酸亲-疏水性、跨膜结构域、信号肽序列、磷酸化位点、二级和三级结构。通过EcoR I和Xho I酶切位点将NSP1α克隆至pET-28a-His原核表达载体。测序正确的pET-28a-NSP1α-His质粒转化至大肠杆菌E. coli BL21(DE3)表达感受态细胞，将诱导表达、纯化复性后的NSP1α蛋白免疫小鼠制备多克隆抗体，Western blot和间接免疫荧光试验（indirect immunofluorescence assay, IFA）鉴定制备的多克隆抗体的反应原性，通过Western blot和激光共聚焦分析NSP1α蛋白亚细胞分布。【结果】信息学分析结果表明，SD16毒株NSP1α蛋白有501个氨基酸，为不稳定的疏水性蛋白，其序列与HLJ毒株序列同源性最高，与I型PRRSV序列同源性最低；NSP1α蛋白无信号肽序列，无跨膜结构域，存在13个磷酸化位点；二级结构主要包含α-螺旋、延伸链和无规则卷曲。将获得的高纯度并且有活性的NSP1α蛋白进行小鼠免疫，检测其血清抗体效价可达1∶64 000。Western blot和IFA结果显示，制备的多克隆抗体特异性识别PRRSV NSP1α蛋白。Western blot和激光共聚焦结果显示，NSP1α蛋白主要定位在细胞核内。【结论】成功表达、纯化了PRRSV NSP1α蛋白并制备了鼠源多克隆抗体，该多克隆抗体与PRRSV NSP1α蛋白具有良好的反应活性和特异性。
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 protein physicochemical properties, amino acid hydrophilicity/hydrophobicity, transmembrane domains, signal peptide sequences, phosphorylation sites, secondary and tertiary structures of proteins were analyzed 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 protein after induced expression and purification was used to immunize mice to prepare polyclonal antibody against PRRSV NSP1α protein. The reactivity of the prepared polyclonal antibodies was identified using Western blot and indirect immunofluorescence assay(IFA). Finally, the subcellular distribution 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 structure 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 antibody has good reactivity and specificity with PRRSV NSP1α protein.
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基本信息:
DOI：10.16445/j.cnki.1000-2340.20250512.001
中图分类号:S852.651
引用信息:
[1]张铭芳,任佳慧,张亚慈,等.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(05):838-848.DOI:10.16445/j.cnki.1000-2340.20250512.001.
基金信息:
国家自然科学基金项目（32302871）; 河南省重点研发与推广专项（科技攻关）（232102110101）