摘要
采用杆状病毒表达系统表达并纯化裂谷热病毒(RVFV)Gn蛋白,并对其反应原性进行鉴定。根据GenBank公布的Gn蛋白的序列,选取其主要抗原区Gn1设计引物进行PCR扩增,将扩增产物克隆至pFastBac HTB载体,构建重组转移载体,并将其转化DH10Bac感受态细胞进行蓝白斑筛选。将鉴定正确的重组杆粒转染Sf9细胞获得重组杆状病毒,经SDS-PAGE和Western blot鉴定蛋白正确表达后,将重组杆状病毒感染High5细胞,大量表达重组蛋白,并用镍柱亲和层析法进行纯化,最终获得纯度较高的Gn1重组蛋白。本研究为RVF新型疫苗研发及诊断试剂的研发提供了重要材料。
To produce Gn protein of Rift Valley fever virus(RVFV)using Baculovirus expression system and study its reactivity,the main antigen region Gn1 was selected to design primers for PCR amplification according to the Gn sequence published by GenBank.The amplified products were cloned into pFastBac HTB vectors to construct recombinant transfer vectors and then transformed into DH10Bac receptive cells for blue-white screening.The recombinant rods were transfected into Sf9 cells to obtain recombinant baculoviruses.The recombinant baculoviruses were confirmed in SDS-PAGE and Western blot and used to infect High5 cells.The recombinant Gn1 protein was produced in large quantity and purified by nickel column affinity chromatography.This study provided important materials for the development of RVF vaccines and diagnostic reagents.
作者
孙潇
张艳芳
叶静
曹胜波
陈政
SUN Xiao;ZHANG Yanfang;YE Jing;CAO Shengbo;CHEN Zheng(College of Veterinary Medicine,Huazhong Agricultural University,Wuhan 430070,China;State Key Laboratory of Agricultural Microbiology,Huazhong Agricultural University,Wuhan 430070,China)
出处
《中国动物传染病学报》
CAS
北大核心
2022年第2期126-133,共8页
Chinese Journal of Animal Infectious Diseases
基金
“十三五”国家重点研发计划(2017YFD0501803,2016YFD0501102)。
关键词
裂谷热病毒
Gn1蛋白
重组杆状病毒
蛋白纯化
Rift valley fever virus
Gn1 protein
recombinant baculovirus
protein purification
