大鲵虹彩病毒MCP蛋白在杆状病毒表达系统中的表达与纯化

Expression and purification of major capsid protein of chinese giant salamander (Andrias davidianus) iridovirus in baculovirus expression system

为研制基于杆状病毒表达系统的大鲵虹彩病毒(Chinese giant salamander iridovirus,CGSIV)新型亚单位疫苗,将CGSIV主要衣壳蛋白(major capsid protein,MCP)基因克隆至杆状病毒穿梭载体p Fast Bac1质粒中,构建了重组质粒p Fast Bac-MCP。转化E.coli DH10Bac感受态细胞,经PCR筛选和测序获得了阳性重组杆粒r Bacmid-MCP,在昆虫细胞转染试剂介导下将该重组杆粒转染Sf9细胞,获得重组杆状病毒。重组杆状病毒感染的Sf9昆虫细胞,经超薄切片电镜观察,可见大量重组杆状病毒存在于细胞中。按不同感染复数(MOI=2、5、10)将重组杆状病毒感染Sf9细胞进行CGSIV MCP的表达。SDS-PAGE检测结果表明,在MOI=10时,目的蛋白的表达量最高;间接免疫荧光观察结果显示,目的蛋白在感染细胞中得到表达,且分布在细胞表面。以抗CGSIV MCP单抗为抗体制备的免疫磁珠纯化目的蛋白并利用兔抗CGSIV MCP多抗血清检测目的蛋白的生物学活性。SDS-PAGE和Western blot结果显示,纯化的目的蛋白纯度很高,而且具有抗原活性,能够被兔抗大鲵虹彩病毒MCP多抗血清识别。利用杆状病毒表达系统成功进行了CGSIV MCP的表达,并应用免疫磁珠法进行了目的蛋白的纯化,为CGSIV新型亚单位疫苗的研制奠定了基础。

This work was conducted to provide a useful basis for development of a new subunit vaccine against Chinese giant salamander iridovirus(CGSIV).In order to express major capsid protein(MCP)of CGSIV in baculovirus expression system,the gene of MCP was subcloned into baculovirus transfer vector(pFastBac1).The recombinant plasmid pFastBac-MCP was identified by restriction enzyme digestion and gene sequencing,and then transformed into Escherichia coli DH10Bac competent cells containing baculovirus shuttle vectors.After identification by blue/white selection,PCR analysis,and gene sequencing,a recombinant bacmid(rBacmid-MCP)was obtained.Thereafter,the recombinant bacmid was transfected into Sf9cells with Insect GeneJuice?Transfection Reagent and recombinant baculoviruses were obtained in Sf9cells,which could be observed by eletron microscopy in the ultra-thin sections of infected Sf9cells and were named AcNPV-MCP.The recombinant baculovirus infected Sf9cells with different multiplicities of infection(MOI=2,5,or10)showed the highest production of recombinant protein of MCP.The result of SDS-PAGE analysis showed that the production of expressed MCP could be the highest with MOI=10.By immunofluorescence assay,the presence of MCP was observed on the surface of infected Sf9cells.The recombinant protein of MCP was purified with the prepared immunomagnetic bead with monoclonal antibodies against CGSIV MCP and its bioactivity was verified by western blot assay with the rabbit antisera against CGSIV MCP.The results of SDS-PAGE and western-blotting assays indicated that the recombinant protein was highly purified and had good immunogenicity,which could specifically react with the rabbit antisera.In conclusion,the recombinant protein of CGSIV MCP was successfully expressed in baculovirus expression system and purified with the immunomagnetic bead assay,which laid a solid foundation for the development of a new subunit CGSIV vaccine in the future.