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

作　　者：刘影 陶思锐 舒金琪 查银河 舒建洪[1,2] LIU Ying;TAO Sirui;SHU Jinqi;ZHA Yinhe;SHU Jianhong(College of Life Sciences and Medicine,Zhejiang Sci-Tech University,Hangzhou,310018,China;Zhejiang Sci-Tech University Shaoxing Academy of Biomedicine,Shaoxing 312000,China)

机构地区：[1]浙江理工大学生命科学与医药学院,杭州310018 [2]浙江理工大学绍兴生物医药研究院,绍兴312000

出　　处：《浙江理工大学学报（自然科学版）》2021年第5期691-696,共6页Journal of Zhejiang Sci-Tech University(Natural Sciences)

基　　金：浙江省重点研发计划项目(2019C02043);企业委托横向课题(20040918-J)。

摘　　要：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.

关 键 词：非洲猪瘟病毒 P30基因 原核表达 多克隆抗体 

分 类 号：Q71[生物学—分子生物学]