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采用RT-PCR技术从感染齿兰环斑病毒贵州分离物(ORSV-GZ)的苋色藜叶片中扩增出该病毒的运动蛋白(MP)基因。测序结果表明:该MP基因全长912 bp,编码303个氨基酸残基。构建原核表达载体pET32a(+)-MP,将重组质粒转化大肠杆菌BL21(DE3),在25℃以1.0 mmol/L IPTG诱导表达重组蛋白基因。SDS-PAGE分析表明,重组蛋白分子量约为54 kDa,与预测相符合。以该重组蛋白为抗原免疫新西兰大白兔,制备的抗体效价达1:25600。该多抗与ORSV病叶汁发生特异性免疫反应,而与其他5种同属或不同属病毒叶汁无血清学交叉反应。本实验为进一步研究该蛋白的结构和功能奠定基础。
The motility protein (MP) gene of this virus was amplified from the leaves of Guizhou shrub (ORSV-GZ) infected with Toxoplasma gondii by RT-PCR. The sequencing results showed that the MP gene was 912 bp in length and encoded 303 amino acid residues. The prokaryotic expression vector pET32a (+) - MP was constructed and the recombinant plasmid was transformed into E. coli BL21 (DE3). The recombinant protein was induced by 1.0 mmol / L IPTG at 25 ℃. SDS-PAGE analysis showed that the molecular weight of the recombinant protein was about 54 kDa, which was consistent with the prediction. New Zealand white rabbits were immunized with the recombinant protein as antigen, and the titer of the antibody was 1: 25600. The polyclonal antibody and ORSV leaf juice specific immune response, but with the other five belong to the same or different virus leaf serum serological cross-reaction. This experiment lays the foundation for further study of the structure and function of this protein.