中国兽医科学2024，Vol.54Issue(3)：357-363,7.DOI:10.16656/j.issn.1673-4696.2024.0046
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
摘要
Abstract
In order to determine the immunological characteristics of the African horse sickness virus(AHSV)VP5 protein,the full length S6 gene sequence of theAHSVVP5 protein was artificially syn-thesized,amplified by PCR,and cloned into the transposable plasmid vector pFastBac 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 pFastBac-AHSVS6 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Key words
African horse sickness virus/VP5 protein/baculovirus expression system/polyclonal antibody/immunofluorescence分类
农业科技引用本文复制引用
范亚亚,王轩莹,户鑫兵,田占成,关贵全,罗建勋,殷宏,郑玉姝,独军政..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[J].中国兽医科学,2024,54(3):357-363,7.基金项目
国家重点研发计划项目(2021YFD1800500) （2021YFD1800500）
中央级公益性科研院所基本科研业务费项目(1610312021014) （1610312021014）
甘肃省基础研究创新群体项目(22JR5RA024) （22JR5RA024）
甘肃省科技厅项目(22CX8NA011) （22CX8NA011）
农业科技创新工程项目(CAAS-ASTIP-2016-LVRI) （CAAS-ASTIP-2016-LVRI）
