猪圆环病毒2b型表位嵌合病毒样颗粒在E.coli中的制备及鉴定

Production and Characterization of PCV2b Epitope Chimeric Virus-like Particles Expressed in E.coli

为了研究一种更为高效、廉价的猪圆环病毒2型(PCV2)亚单位疫苗,针对PCV2b型Cap基因核酸序列进行优化,将其编码中和表位226LKDPPLNP233的基因序列连接于Cap基因C端(Capc),并插入PE-Sumo载体,借助大肠杆菌表达系统对Capc蛋白进行表达;利用SDS-PAGE、Western blot、动态光散射和透射电镜观察等手段,对纯化后的目的蛋白活性及其形成的结构进行初步鉴定。结果显示,嵌合Capc基因片段成功插入PE-Sumo载体,并转化到大肠杆菌BL21(DE3)中;Sumo-Capc重组蛋白在26℃下诱导12 h能够以可溶形式大量表达;经纯化的Capc蛋白能够与PCV2单克隆抗体特异结合,并能够自组装形成直径约为18 nm的病毒样颗粒。综上,成功构建了能够表达PCV2b型中和表位嵌合Cap的大肠杆菌表达系统,纯化得到的Capc蛋白具有良好的免疫反应性,且能够自组装成病毒样颗粒。

To develop a high efficient and economic PCV2b subunit vaccine,the codons of PCV2 Cap gene were optimized for E.coli expression system,and the gene sequence of PCV2 neutralizing epitope 226LKDPPLNP233 was conjugated at its C-terminus(Capc).After the recombinant sequence was constructed on PE-Sumo vector,recombinant chimeric Capc was expressed in E.coli.By using SDS-PAGE,Western blot,Dynamic light scattering and TEM,the immune reactivity and the structure of Capc protein were preliminarily identified.The results showed PE-Sumo-Capc vector was successfully constructed and transferred into E.coli BL21(DE3);Sumo-Capc protein was mostly expressed in soluble form after 12 h induction under 26℃.The final purified Capc protein was able to specifically react with PCV2 Cap monoclonal antibody,and self-assembly into virus-like particles with the diameter around 18 nm.The current study successfully constructed an E.coli expression system for PCV2b neutralizing epitope chimeric Cap protein,which showed a good immunogenic reactivity and could self-assembly into virus-like particles.