中国水产科学2017，Vol.24Issue(6)：1271-1279,9.DOI:10.3724/SP.J.1118.2017.16319
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
摘要
Abstract
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 bacu-lovirus expression system, the gene of MCP was subcloned into baculovirus transfer vector (pFastBac1). The re-combinant 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 identi-fication by blue/white selection, PCR analysis, and gene sequencing, a recombinant bacmid (rBacmid-MCP) was obtained. Thereafter, the recombinant bacmid was transfected into Sf9 cells with Insect GeneJuice? Transfection Reagent and recombinant baculoviruses were obtained in Sf9 cells, which could be observed by eletron micros-copy in the ultra-thin sections of infected Sf9 cells and were named AcNPV-MCP. The recombinant baculovirus infected Sf9 cells with different multiplicities of infection (MOI=2, 5, or 10) 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 sur-face of infected Sf9 cells. 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 re-combinant protein was highly purified and had good immunogenicity, which could specifically react with the rab-bit 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 devel-opment of a new subunit CGSIV vaccine in the future.关键词
大鲵虹彩病毒/主要衣壳蛋白/杆状病毒表达系统/蛋白纯化Key words
Chinese giant salamander iridovirus/major capsid protein/baculovirus expression system/protein purification分类
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张星朗,高志,杨平凹,周小愿,贾秋红,韩亚慧,高宏伟..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[J].中国水产科学,2017,24(6):1271-1279,9.基金项目
陕西省科学技术发展计划项目(2014k01-20-02 （）
2012K01-18). （）
