非洲猪瘟病毒P30蛋白的原核表达及其多克隆抗体的制备

Prokaryotic expression of African swine fever virus P protein and preparation of polyclonal antibody

利用原核表达系统表达纯化重组非洲猪瘟病毒P30蛋白,并进一步制备抗P30重组蛋白的多克隆抗体,为非洲猪瘟病毒诊断试剂盒的研发提供实验材料和理论依据。根据GenBank中非洲猪瘟结构蛋白P30序列,经密码子优化后合成相应的基因序列,构建重组表达质粒pET-32a-P30并转化至大肠杆菌Rosetta感受态细胞中,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达纯化后获得重组P30蛋白,并制备抗P30的多克隆抗体。结果表明:16℃、0.5 mmol/L IPTG诱导表达12 h获得质量浓度为0.6 mg/mL以上的重组P30蛋白,Western blot证实原核表达的P30具有较好的免疫原性,ELISA检测纯化后的多克隆抗体效价高达1∶5120000,且具有良好的抗原特异性。

The purpose of this study is to express and purify the recombinant African swine fever virus P30 protein by the prokaryotic expression system,and further prepare the polyclonal antibody against the recombinant P30 protein,so as to provide experimental materials and theoretical basis for the development of diagnostic kit for African swine fever virus.According to the sequence of ASF structural protein P30 in GenBank,the corresponding gene sequences were synthesized by codon optimization.In addition,the recombinant expression plasmid pET-32 a-p30 was constructed and transformed to E.coli Rosetta competent cell.After induction and purification by isopropyl-β-D-thiogalactoside(IPTG),the recombinant P30 protein was obtained,and the polyclonal antibody against P30 was prepared.The result showed that the recombinant P30 protein with a concentration of more than 0.6 mg/mL was obtained after being induced at 16℃and 0.5 mmol/LIPTG for 12 h.Western blot result showed that it had good immunogenicity.ELISA test indicated that the titer of the purified polyclonal antibody was as high as 1∶520000,and it had good antigen specificity.