携带双串联卵清蛋白T细胞表位的嵌合兔出血症病毒衣壳蛋白自聚能力的研究  

Study on the Self-assemble Ability of Chimeric RHDV VP60 Carrying Double OVA T-cell Epitopes

作　　者：张燕[1,2] 胡波[1] 宋艳华[1] 范志宇[1] 魏后军[1] 姜平[2] 王芳[1] 

机构地区：[1]江苏省农业科学院兽医研究所,农业部兽用生物制品工程技术重点实验室,国家兽用生物制品工程技术研究中心,江苏南京210014 [2]南京农业大学动物医学院,江苏南京210095

出　　处：《华北农学报》2014年第1期55-61,共7页Acta Agriculturae Boreali-Sinica

基　　金：国家自然科学基金项目(31070140);江苏省自主创新项目(CX(12)3079)

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。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 pFastBacTMHT 1 and the recombi-nant 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 RT-PCR. The chimeric protein was correctly expressed in baculovirus system and could self-assemble into VLPs by E-lectron 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.

关 键 词：兔出血症病毒 VP60 双串联OVAT细胞表位 病毒样颗粒 

分 类 号：S858.291.265[农业科学—临床兽医学]