摘要： 以兔出血症病毒(RHDV)衣壳蛋白VP60为载体,构建VP60携带双串联OVA T细胞表位(Ovalbumin,卵清蛋白第257~264位氨基酸)的嵌合蛋白,研究外源基因对VP60蛋白表达及病毒样颗粒(VLPs)装配的影响,分析VP60作为载体对外源基因长度的耐受性。运用基因克隆和重组技术,通过2次SOE法(Splicing by overlap extension,重叠延伸剪接术),将双串联OVA T细胞表位(257~264位氨基酸)的基因序列取代VP60第302~309位氨基酸的基因序列,得到嵌合VP60基因。利用杆状病毒表达系统表达嵌合蛋白,命名为DC。经IFA、SDS-PAGE、Western Blot、RT-PCR方法鉴定嵌合蛋白的表达,并通过电镜观察嵌合蛋白自聚为病毒样颗粒的能力。嵌合VP60蛋白在杆状病毒表达系统中得到高效表达,且可自聚形成病毒样颗粒。携带双串联外源表位的嵌合VP60蛋白得到有效表达,并可形成VLPs。该研究为嵌合VP60 VLPs的形成、结构功能的研究奠定基础,同时扩展了VP60作为载体可携带的外源基因耐受性的研究资料。

关键词: 兔出血症病毒, VP60, 双串联OVA T细胞表位, 病毒样颗粒

Abstract: The aim of present study was to investigate whether the amino acid replacement of VP60 could affect the expression of VP60 and the capable of self-assembling into VLPs. In order to obtain the chimeric protein of VP60 carrying double OVA T-cell epitopes,the sequences of VP60 302-309aa were replaced by the genes of double OVA T-cell epitopes by SOE. The fused genes were cloned into the donor vector pFastBac^TMHT 1 and the recombinant baculoviruse(rAc-V-302-309) was constructed using Bac-to-Bac baculovirus expression system. The chimeric protein named DC was expressed effectively in insect cells and confirmed by IFA,SDS-PAGE,Western Blot and RTPCR. The chimeric protein was correctly expressed in baculovirus system and could self-assemble into VLPs by Electron microscopy analysis. This manuscript should be very valuable for enhancing the understanding of using RHDV-VLPs as a carrier for foreign genes. Meanwhile,the length of the foreign genes which RHDV-VLPs carried was extended.

Key words: RHDV, VP60, Double OVA T-cell epitopes, VLPs

中图分类号: 

• S858.2912.65