非洲马瘟病毒VP5蛋白在杆状病毒表达系统中的表达及其抗体制备  

Expression of African horse sickness virus VP5 protein in baculovirus expression system and preparation of its polyclonal antibody

作　　者：范亚亚 王轩莹 户鑫兵 田占成[2] 关贵全[2] 罗建勋[2] 殷宏[2] 郑玉姝[1] 独军政[2,3] FAN Yaya;WANG Xuanying;HU Xinbing;TIAN Zhancheng;GUAN Guiquan;LUO Jianxun;YIN Hong;ZHENG Yushu;DU Junzheng(College of Animal Science and Veterinary Medicine,Henan Institute of Science and Technology,Xinxiang 453003,China;State Key Laboratory for A nimal Disease Control and Prevention/College of Veterinary Medicine of Lanzhou University/Lanzhou Veterinary Research Institute,Chinese Academy of A gricultural Sciences,Lanzhou 730000,China;Gansu Province Research Center for Basic Disciplines of Pathogen Biology,Lanzhou 730046,China.)

机构地区：[1]河南科技学院动物科技学院,河南新乡453003 [2]中国农业科学院,兰州兽医研究所,兰州大学动物医学与生物安全学院,动物疫病防控全国重点实验室,甘肃兰州730000 [3]甘肃省病原生物学基础学科研究中心,甘肃兰州730046

出　　处：《中国兽医科学》2024年第3期357-363,共7页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2021YFD1800500);中央级公益性科研院所基本科研业务费项目(1610312021014);甘肃省基础研究创新群体项目(22JR5RA024);甘肃省科技厅项目(22CX8NA011);农业科技创新工程项目(CAASASTIP-2016-LVRI)。

摘　　要：为了确定非洲马瘟病毒(AHSV)VP5蛋白的免疫学特性,通过人工合成非洲马瘟病毒VP5蛋白的S6全长基因序列,经PCR扩增后克隆到转座质粒载体pFastBac HTb上,将重组质粒转化至DH10Bac感受态细胞,通过蓝白斑试验筛选出重组杆状病毒质粒Bacmid-AHSVS6,转染Sf9昆虫细胞;构建了重组质粒pFastBac-AHSVS6,拯救获得含有AHSV S6基因的重组杆状病毒。SDS-PAGE和Western-blot鉴定表明,在Sf9昆虫细胞中成功表达分子质量大小为60 ku的AHSV VP5重组蛋白。采用组氨酸标签纯化树脂对重组VP5蛋白进行纯化,将其与弗氏佐剂乳化后3次免疫新西兰大白兔收集血清,获得了针对AHSV VP5蛋白的多克隆抗体,并通过Western-blot和间接免疫荧光试验验证了该抗体的特异性。本试验利用杆状病毒表达系统成功表达了AHSV全长VP5重组蛋白,成功制备出特异的VP5蛋白多克隆抗体,这为进一步研究AHSV VP5蛋白的功能及研制亚单位疫苗奠定了基础。In order to determine the immunological characteristics of the African horse sickness virus(AHSV) VP5 protein,the full length S6 gene sequence of the AHSV VP5 protein was artificially synthesized,amplified by PCR,and cloned into the transposable plasmid vector p Fast Bac HTb.The recombinant plasmid was transformed into DH10Bac competent cells and Bacmid-AHSVS6 was screened through blue and white spot test,and then was transfected into Sf9 insect cells,and the recombinant plasmid pFastBacAHSVS6 was constructed successfully,and the recombinant baculovirus containing the AHSV S6 gene was rescued.SDS-PAGE and Western-blot identification indicated that the recombinant AHSV VP5 protein with a molecular weight of 60 ku was successfully expressed in Sf9 insect cells.The recombinant VP5 protein was purified by His-Tag purification resin,emulsified with Freund's adjuvant,and immunized three times to collect serum from New Zealand white rabbits.A polyclonal antibody against AHSV VP5 protein was obtained,and its specificity was verified by Western-blot and indirect immunofluorescence.This study successfully expressed the full length recombinant VP5 protein of AHSV using the baculovirus expression system,and prepared specific polyclonal antibodies against VP5 protein,which laid the foundation for further research on the function of AHSV VP5 protein and the development of subunit vaccines.

关 键 词：非洲马瘟病毒 VP5蛋白 杆状病毒表达系统 多克隆抗体 免疫荧光 

分 类 号：S852.659.4[农业科学—基础兽医学]