猪繁殖与呼吸综合征病毒非结构蛋白1α多克隆抗体的制备及应用

Preparation and Application of Nonstructural Protein 1 α (nsp1α) Polyclonal Antibody of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

以真核质粒pcDNA3.1-flag-nsp1α为模板,利用PCR扩增出nsp1α基因片段,构建原核重组载体pET32a-nsp1α,转化到大肠杆菌BL21(DE3)感受态,1mmol/LIPTG在37℃诱导表达6h,成功获得以包涵体形式存在的重组蛋白,能够与小鼠抗His标签单克隆抗体反应;包涵体复性后作为抗原免疫新西兰大白兔,3次免疫后20d获得兔多抗血清。通过间接ELISA、Westernblot、间接免疫荧光、免疫组化等试验获得了高效价的兔多抗血清,该血清能够特异性地识别真核质粒表达的nsp1α以及PRRSVBJ4株。该试验成功表达了pET32a-nsp1α重组蛋白,并制备了高效价、高特异性的兔抗nsp1α多克隆抗体。

Using eukaryotic plasmid pcDNA3.1-flag-nsp1α as a template,nsp1α gene fragment was amplified by PCR.The prokaryotic recombinant vector pET32a-nsp1α was constructed and transformed the pET32a-nsp1α into receptor state of Escherichia coli cells BL21 (DE3) to express the nsp1α by adding1 mmol/L IPTG at 37 ℃.The recombinant protein nsp1α was obtained in the form of inclusion body,which could react with His tag monoclonal antibody.The inclusion bodies after renaturation was used as antigen to immunize New Zealand white rabbit,rabbit polyclonal antiserum was obtained on the 20^th day after three times of immunization.The results of indirect ELISA,Western blot,indirect immunofluorescence,immunohistochemistry showed that the rabbit polyclonal antibody with high titer were obtained,which could specifically identify nsp1α expressed by eukaryotic plasmid and BJ-4 strains of PRRSV.The recombinant protein pET32a-nsp1α was successfully expressed,high-specificity rabbit anti-nsp1α polyclonal antibody with high tier was prepared.