A型塞内卡病毒VP2蛋白原核表达及其多克隆抗体的制备

本研究旨在表达A型塞内卡病毒(Senecavirus A,SVA)的衣壳蛋白VP2,并制备其多克隆抗体。以SVA GD01/2017分离株RNA为模板,RT-PCR扩增VP2基因序列,并克隆至原核表达载体pET-30a(+)中构建重组质粒pET-30a-SVA-VP2。经测序鉴定后,将重组质粒转化大肠杆菌Rosetta(DE3)感受态细胞,利用IPTG进行诱导表达。在非变性条件下,利用Ni-NTA琼脂糖树脂从菌体裂解液上清中纯化重组SVA VP2蛋白,并免疫新西兰大白兔制备多克隆抗体。利用Protein A Sepharose CL-4B树脂从兔血清中亲和层析纯化多克隆抗体,并对其进行间接免疫荧光分析。结果显示,重组SVA VP2蛋白以可溶性和包涵体两种形式在大肠杆菌Rosetta(DE3)感受态细胞内进行表达,分子质量约为47ku;制备的兔抗VP2蛋白多克隆抗体效价高达1∶64 000,该多克隆抗体仅识别SVA,与猪繁殖与呼吸综合征病毒、猪圆环病毒2型、脑心肌炎病毒、猪瘟病毒和猪伪狂犬病病毒等常见猪病原无交叉反应,表明制备的多克隆抗体具有良好的特异性。重组SVA VP2蛋白及其多克隆抗体的成功制备为猪塞内卡病毒病血清学检测方法的建立提供了良好的生物材料。

Prokaryotic Expression of the VP2 Protein of Senecavirus A and Preparation of Its Polyclonal Antibodies

The present study was aimed to express the VP2 protein of Senecavirus A (SVA) and prepare its polyclonal antibodies.The full-length coding sequence of the SVA VP2 gene was amplified by RT-PCR using the SVA RNA of GD01/2017 strain as the template.The amplicon was then cloned into the prokaryotic expression vector pET-30a(+) to construct a recombinant plasmid pET-30a-SVA-VP2.After verification by DNA sequencing,the plasmid was transformed into E.coli Rosetta (DE3) competent cells which were then induced by IPTG.The recombinant SVA VP2 protein was purified from the supernatant of the lysate of pET-30a-SVA-VP2 plasmid-transformed E.coli Rosetta (DE3) cells using the Ni-NTA agarose under native conditions.The purified VP2 protein was used to immunize the New Zealand White rabbits to prepare its polyclonal antibodies,which were then purified from the rabbit sera by affinity chromatography using the Protein A Sepharose CL-4B.The reactivity of the polyclonal antibodies with SVA was analyzed by an indirect immunofluorescence assay.The results showed that the recombinant SVA VP2 protein was expressed in E.coli Rosetta (DE3) cells in the form of both soluble and inclusion bodies with a molecular weight of about 47 ku,and that the titer of the rabbit anti-VP2 polyclonal antibodies was 1:64 000.The prepared polyclonal antibodies merely reacted with SVA and had no cross-reactivity with other common porcine pathogens,such as porcine reproductive respiratory and syndrome virus,porcine circovirus type 2,encephalomyocarditis virus,classical swine fever virus and pseudorabies virus.It indicated that the prepared polyclonal antibodies against SVA VP2 protein had good specificity.The successful preparations of recombinant SVA VP2 protein and its polyclonal antibodies provided valuable biomaterials for the establishment of serological detection method for SVA.