魔芋花叶病毒衣壳蛋白的原核表达和多克隆抗体制备

采用小RNA测序技术对甘肃省榆中县疑似感染病毒病的当归(Angelica sinensis(Oliv.)Diels]样品进行测序鉴定,发现样品中含有魔芋花叶病毒(Konjac mosaic virus,KoMV),通过反转录PCR(RT-PCR)扩增KoMV衣壳蛋白(capsid protein,CP)的cp基因,克隆的KoMV cp基因连接原核表达载体pET-28a(+),导入E.coli Rosetta^TM(DE3)诱导表达蛋白,在Ni-NTA重力柱层析作用下纯化CP融合蛋白,并以此作为抗原免疫新西兰大耳白兔制备多克隆抗体。序列分析表明:KoMV cp基因片段大小为840 bp,编码280个氨基酸的外壳蛋白;与GenBank已注册同源性较高的KoMV分离物相比,核苷酸序列相似性为85.58%~99.41%,氨基酸序列相似性为89.32%~99.29%;KoMV的病毒种群分布存在明显的区域性和寄主差异。SDS-PAGE显示,不同温度诱导下KoMV CP融合蛋白在E.coli Rosetta^TM(DE3)中均以包涵体的形式大量表达,融合蛋白分子量为36 ku。间接ELISA和Western blot检测结果显示,制备的多克隆抗体效价达到32 000,能够与感染KoMV的当归叶片和纯化蛋白特异性结合,具有良好的特异性。本研究成功制备了当归KoMV CP融合蛋白的多克隆抗体IgG,为开发KoMV的血清学检测技术及CP蛋白的功能研究奠定了基础。

Prokaryotic expression of Konjac mosaic virus capsid protein and preparation of polyclonal antibody

The samples of Angelica sinensis(Oliv.)Diels suspected to be infected with virus disease in Yuzhong County, Gansu Province were sequenced and identified by small RNA sequencing. It was found that Konjac mosaic virus(KoMV) was contained in the sample, and the cp gene of KoMV capsid protein was amplified by RT-PCR. The cloned KoMV cp gene connected to the prokaryotic expression vector pET-28a (+), and introduced into E.coli Rosetta^TM (DE3) to induce expression protein. The CP fusion protein was purified by Ni-NTA gravity column chromatography and immunized New Zealand white-eared rabbits with this protein as antigen to prepare polyclonal antibodies. Sequence analysis showed that the size of KoMV cp gene fragment was 840 bp, encoding a coat protein of 280 amino acids. Compared with KoMV isolates registered in GenBank, the nucleotide sequence similarity was 85.58%-99.41%, and the amino acid sequence similarity was 89.32%-99.29%. There were obvious regional and host differences in the distribution of KoMV virus populations. SDS-PAGE showed that the fusion protein of KoMV CP was expressed in large quantities in the form of inclusion bodies in E.coli Rosetta^TM (DE3) induced by different temperatures, and the molecular weight of the fusion proteins was 36 ku. The results of indirect ELISA and Western blot showed that the prepared polyclonal antibody had a titer of 32 000, which could specifically bind to the leaves of Angelica infected with KoMV and purified protein, showing good specificity. In this study, the polyclonal antibody IgG of KoMV CP fusion protein was successfully prepared, which laid a foundation for the development of serological detection technology of KoMV and the functional research of CP protein.