非洲猪瘟病毒CP204L基因的原核表达与单抗制备Prokaryotic Expression and Purification of ASFV CP204L Gene and Its Mabs Preparation
张赫;杭天宇;刘春羽;尹坤;温永俊;王凤雪;
摘要(Abstract)：
由非洲猪瘟病毒(ASFV)引起的非洲猪瘟(ASF)给我国养猪业带来了不可估量的经济损失,严重阻碍了我国养猪业的发展,研发ASFV快速诊断试剂是目前最重要的内容之一。CP204L基因编码ASFV结构蛋白p30。本研究以克隆ASFV的CP204L基因为基础,通过基因重组技术,加入His标签,将构建的重组质粒命名为pET-28a-CP204L。将重组质粒转化至大肠杆菌BL21(DE3)感受态细胞,37℃经1mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达6h,表达蛋白进行SDS-PAGE鉴定和Western Blot检测。重组蛋白纯化后免疫小鼠制备筛选单克隆抗体,Western Blot和IFA验证单抗的结合特异性。结果表明,重组的pET-28a-CP204L诱导后表达蛋白为30kD,以不可溶性包涵体形式存在;表达蛋白利用His标签进行纯化,获得纯化蛋白2mg,单克隆抗体筛选获得5株IgG亚型的ASFV p30蛋白的单抗,且均具有良好的结合活性。本研究为发展ASFV检测方法提供了基础。
关键词(KeyWords)： 非洲猪瘟病毒(ASFV);CP204L基因;原核表达;蛋白纯化;单抗
基金项目(Foundation): 内蒙古农业大学高层次人才引进项目（NDYB2018-2）和（NDGCC2016-22）~~
作者(Author): 张赫;杭天宇;刘春羽;尹坤;温永俊;王凤雪;
Email:
DOI: 10.13242/j.cnki.bingduxuebao.003883
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