猪瘟病毒重组融合蛋白E-A-E在家蚕杆状病毒表面的展示.doc

猪瘟病毒重组融合蛋白E2-2A-E0在家蚕杆状病毒表面的展示  5 10 15 20 25 30 35 40 45 （1. 浙江理工大学生命科学学院生物化学研究所，杭州 310018； 2. 浙江省家蚕生物反应器和生物医药重点实验室，杭州 310018） 摘要：本研究旨在构建杆状病毒表面展示系统转移载体 pFastBac1-gp64，并将猪瘟病毒重 组融合蛋白展示于细胞和杆状病毒囊膜表面，以用于新型猪瘟疫苗的研制。我们首先利用 SOE-PCR 、PCR、双酶切等相关技术构建出杆状病毒表面展示系统转移载体 pFastBac1-gp64； 然后构建大肠杆菌重组表达质粒 pET-28a-E2-2A-E0，在 E.coli Rosetta 中表达目的融合蛋 白，通过包涵体洗涤及镍离子亲和层析大量纯化目的蛋白，免疫新西兰雄兔制备多克隆抗体， 以备后续实验检测之用，用间接 ELISA 法其效价为 1:51200。同时构建重组转移载体 pFastBac1-gp64-E2-2A-E0，通过 Bac-to-Bac 系统获取重组杆状病毒 Bmgp64-E2-2A-E0，经 SDS、Western blotting 鉴定分析及激光共聚焦显微镜观察，结果表明重组融合蛋白 成功在家蚕 BmN 细胞、家蚕五龄幼虫和家蚕蛹中表达并展示于细胞及杆状病毒囊膜表面，便 于接下来对重组杆状病毒免疫原性的研究及新型猪瘟疫苗的研制。 关键词：生物化学与分子生物学；CSFV；家蚕杆状病毒表面展示系统；猪瘟疫苗 中图分类号：Q78 The surface display of recombinant fusion protein E2-2A-E0 of CSFV on baculovirus envelope Zhang Xuesai1,2, Shan Qianqian1,2, Chen Jianqing1,2, Shu Tejun1,2, Zhang Yaozhou1,2 1. Institute of Biochemistry,College of Life Science,Zhejiang Sci-Tech University, HangZhou 310018; 2. Zhejiang Provencial Key Laboratory of Silkworm Bioreactor and Biomedicine, HangZhou 310018 Abstract: This paper aimed to construct the transfer vector pFastBac1-gp64 of baculovirus surface display system and to be used for develop a new classical swine fever vaccine. This vaccine was a pseudovirion, it was displaying the classical swine fever virus recombinant fusion protein on the surface of BmN cells and baculovirus envelope. We first use SOE-PCR, PCR, restriction enzyme digestion and other related molecular technology to construct a baculovirus surface display system transfer vector pFastBac1-gp64; Then construct recombinant expression plasmid pET-28a-E2-2A-E0, and expressed fusion protein in E.coli Rosetta, and purifying the fusion protein by means of washing inclusion bodies and nickel ion affinity chromatography, after getting enough protein, immunized New Zealand male rabbit to obtain polyclonal antibody for subsequent experimental testing purposes. While we construct recombinant transfer vector pFastBac1-gp64-E2-2A