利用细菌展示技术系统地筛选猪圆环病毒2型衣壳蛋白的线性抗原区域

Systematic mapping the linear antigenic domains of the porcine circovirus 2b Cap protein by bacterial display technology

为了系统的筛选位于猪圆环病毒2型（PCV2）Cap蛋白上的抗原优势区域,本研究将缺少N端核定位信号的Cap蛋白基因分成7个连续重叠的DNA片段cap（1~7）,然后分别克隆于细菌展示载体APEx中,IPTG诱导重组蛋白片段表达于细菌内膜外侧,采用流式细胞仪检测各片段和抗PCV2 Cap蛋白的多克隆抗体的结合能力。结果表明,cap（1~7）均可以和抗该Cap蛋白的多抗结合,并且cap6的结合能力最强。将cap6片段进一步分为连续不重叠的3段（cap6-1、cap6-2、cap6-3）,以同样的方法鉴定抗原表位。结果显示,cap6-2与抗PCV2 Cap蛋白的多抗结合能力最强。此外,应用western blot的方法对细菌展示结果进行了验证。本研究利用细菌展示技术以及流式细胞仪快速的筛选了PCV2b Cap蛋白上的抗原优势区域,cap（1~7）片段均包含抗原表位,并且cap6是Cap蛋白上的抗原优势区域,cap6-2可能是抗原优势表位。

The Cap protein encoded by porcine circovirus type 2 （PCV2） plays an important role in the host immune response and therefore becomes the important target for development of diagnostic reagents against PCV2. To systematically map the linear B cell antigenic domains of the PCV2b Cap protein, in this study, Cap gene without nuclear localization signal sequence was truncated into seven sequential and overlapping fragments of cap （1-7）. All fragments were expressed on the inner membrane of the Escherichia coli and the binding ability of these fragments cap （1-7） to the polyclonal antibody against PCV2b Cap protein was identified by flow cytometry. The result showed that all fragments of cap （1-7） were able to react with the polyclonal antibody, and the binding ability of cap6 was the strongest. Then cap6 was further truncated into three sequential and non-overlapping fragments （cap6-1, cap6-2 and cap6-3）, and the location of the antigenic epitope was identified. The result showed that cap6-2 （aa183-aa193） had the strongest binding ability with the polyclonal antibody. We conclude that there are antigenic epitopes in all of the fragments of cap（1-7）, and the cap6 （aa166-aa207） is the major antigenic domain of the PCV2b Cap. cap6-2 （aa183-aa193） may be a dominant antigenic epitope.