猪圆环病毒2型病毒样颗粒的纯化及鉴定

为获得较高纯度的猪圆环病毒2型（porcine circovirus type 2，PCV2） Cap蛋白的病毒样颗粒（virus-like particles，VLPs）并对其进行结构和功能研究，本试验以大肠杆菌表达系统表达了PCV2重组Cap （PCV2 rCap）蛋白，通过硫酸铵分级沉淀、Sephacryl S-300 HR凝胶过滤层析、Capto Q离子交换层析、CsCl密度梯度离心等方法对其进行了纯化。通过Western blotting对纯化所得蛋白进行免疫反应性鉴定，透射电镜（TEM）观察纯化蛋白的粒径及形态，高效液相尺寸排阻色谱（HPSEC）检测其组分分布。结果显示，纯化后PCV2 rCap蛋白经SDS-PAGE检测，可见28 ku的目的蛋白带，灰度扫描法检测其纯度为97.53%。Western blotting结果显示，该处的特异性蛋白条带有明显的免疫反应性；TEM检测其为粒径17.35~19.24 nm的规则球形颗粒，表明所获得的PCV2 rCap蛋白在表达时能在菌体内自我装配成VLPs，且在纯化过程中没有被破坏而解聚，仍保持天然状态；HPSEC检测纯化样品中VLPs含量为92.67%。本试验结果为进一步研究PCV2 VLPs的空间结构及其免疫保护机制提供了参考依据。

Purification and Identification of Porcine Circovirus Type 2 Virus-like Particles

To obtain and analyze highly purified porcine circovirus virus type 2 (PCV2) virus-like particles(VLPs),the PCV2 recombinant Cap protein (PCV2 rCap) was expressed by Escherichia coli expression system,and was purified by the means of ammonium sulfate precipitation,gel filtration chromatography,ion exchange chromatography and CsCl density gradient centrifugation.The immunoreactivity of purified VLPs were identified by Western blotting,the diameter and morphology of purified VLPs were observed by transmission electron microscopy (TEM).Its components were detected and analyzed by high performance liquid size exclusion chromatography (HPSEC).After purification,the PCV2 rCap protein was detected by SDS-PAGE,the target protein band of 28 ku was detected,and its purity was 97.53% by gray scale scanning.The results of Western blotting showed that the specific protein strips had obvious immunoreactivity.The TEM detected the regular spherical particles with the particle size of 17.35 to 19.24 nm,indicating that the obtained PCV2 rCap protein could express itself in the bacteria.They were assembled into VLPs and deagglomerated without being destroyed during the purification process,and remained in a natural state;In addition,HPSEC detected that the proportion of normally assembled VLPs was 92.67%.The results of this study provided a reference for further study of the spatial structure of PCV2 VLPs and their immune protection mechanisms.