猪圆环病毒2型CAP蛋白在flashBAC杆状病毒表达系统中的表达与鉴定
发布时间：2018-08-02 13:26
【摘要】：试验旨在通过真核表达系统表达猪圆环病毒2型(porcine circovirus type 2,PCV2)CAP蛋白。以PCV2TZ0601株为模板,将PCV2CAP全基因及CAP去除信号肽的基因编码序列克隆至pOET3载体上,酶切与测序鉴定正确后,将重组质粒pOET3-CAP及pOET3-CAP-X转染sf9昆虫细胞。采用flashBAC杆状病毒表达系统表达PCV2CAP及去除信号肽的CAP蛋白,通过间接免疫荧光法、SDS-PAGE和Western blotting鉴定目的蛋白的表达。结果表明,真核表达质粒pOET3-CAP及pOET3-CAP-X构建成功,目的基因在sf9昆虫细胞中高效表达,得到的蛋白经SDS-PAGE和Western blotting鉴定,在25~35ku处有蛋白条带,表达的蛋白质可被PCV2阳性血清识别。试验结果为进一步制备PCV2亚单位疫苗及诊断抗原试剂盒的研发奠定了基础。
[Abstract]:The aim of this study was to express porcine circovirus type 2 (PCV2) CAP protein by eukaryotic expression system. Using PCV2TZ0601 strain as template, the whole PCV2CAP gene and the gene coding sequence of CAP removing signal peptide were cloned into pOET3 vector. The recombinant plasmids pOET3-CAP and pOET3-CAP-X were transfected into sf9 insect cells after confirmed by restriction enzyme digestion and sequencing. The flashBAC baculovirus expression system was used to express PCV2CAP and CAP protein which removed the signal peptide. The expression of the target protein was identified by indirect immunofluorescence staining and SDS-PAGE and Western blotting. The results showed that the eukaryotic expression plasmids pOET3-CAP and pOET3-CAP-X were successfully constructed. The target gene was highly expressed in sf9 insect cells. The obtained protein was identified by SDS-PAGE and Western blotting, and the expressed protein could be recognized by PCV2 positive serum. The results laid a foundation for the further preparation of PCV2 subunit vaccine and diagnostic antigen kit.
【作者单位】： 天津瑞普生物技术股份有限公司瑞普生物研究院;中国农业大学动物医学院;
【基金】：瑞普生物研究院动物疫病流行病学调查专项基金(RPBVI20130010)
【分类号】：S852.651
，
本文编号：2159576
[Abstract]:The aim of this study was to express porcine circovirus type 2 (PCV2) CAP protein by eukaryotic expression system. Using PCV2TZ0601 strain as template, the whole PCV2CAP gene and the gene coding sequence of CAP removing signal peptide were cloned into pOET3 vector. The recombinant plasmids pOET3-CAP and pOET3-CAP-X were transfected into sf9 insect cells after confirmed by restriction enzyme digestion and sequencing. The flashBAC baculovirus expression system was used to express PCV2CAP and CAP protein which removed the signal peptide. The expression of the target protein was identified by indirect immunofluorescence staining and SDS-PAGE and Western blotting. The results showed that the eukaryotic expression plasmids pOET3-CAP and pOET3-CAP-X were successfully constructed. The target gene was highly expressed in sf9 insect cells. The obtained protein was identified by SDS-PAGE and Western blotting, and the expressed protein could be recognized by PCV2 positive serum. The results laid a foundation for the further preparation of PCV2 subunit vaccine and diagnostic antigen kit.
【作者单位】： 天津瑞普生物技术股份有限公司瑞普生物研究院;中国农业大学动物医学院;
【基金】：瑞普生物研究院动物疫病流行病学调查专项基金(RPBVI20130010)
【分类号】：S852.651
，
本文编号：2159576
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