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

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 PCV2 TZ0601株为模板,将PCV2 CAP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PCV2 CAP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25~35 ku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。%The study was aimed to express CAP protein of porcine circovirus type 2 (PCV2 )by eukaryotic expression system.The PCV2 TZ0601 strain was the template,the CAP protein with or without the signal peptide of PCV2 coding sequence were cloned into pOET3 vector.The con-structed plasmid were confirmed by restriction enzyme digestion and DNA sequencing,then sf9 in-sect cells were transfected with recombinant plasmid pOET3-CAP and pOET3-CAP-X.The test was designed to express the CAP protein with or without the signal peptide of PCV2 by flashBAC baculovirus expression system.Expression of PCV2 gene were confirmed by indirect immunofluo-rescent assay (IFA),SDS-PAGE and Western blotting.The results showed that the eukaryotic expression plasmids of pOET3-CAP and pOET3-CAP-X were constructed successfully and the gene was highly expressed in sf9 insect cells.After expression,we could see our target band about 25 to 35 ku with SDS-PAGE and Western blotting.The immuno-reactivity of the protein was con-firmed by antisera against PCV2.This would lay a foundation for a further study of PCV2 subunit vaccine and diagnostic antigen kit.