H5亚型禽流感病毒HA蛋白在杆状病毒-昆虫细胞系统中的表达与纯化  

Expression and Purification of H5 Subtype Avain Influenza Virus HA Protein in Baculovirus-Insect Cell Expression System

作　　者：刘郁夫 陈晓洁 区炳明 容芳 郝文茜 陈瑞爱 LIU Yu-fu;CHEN Xiao-jie;OU Bing-ming;RONG Fang;HAO Wen-qian;CHEN Rui-ai(School of Life Sciences,Zhaoqing University,Zhaoqing,Guangdong 526060,China;Zhaoqing Institute of Biotechnology Co.,Ltd.,Zhaoqing,Guangdong 526238,China;Zhaoqing Branch Center of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology,Zhaoqing,Guangdong 526238,China;College of Veterinary Medicne,South China Agricultural University,GuangZhou,Guangdong 510642,China)

机构地区：[1]肇庆学院生命科学学院,广东肇庆526060 [2]华农(肇庆)生物产业技术研究院有限公司,广东肇庆526238 [3]岭南现代农业科学与技术广东省实验室肇庆分中心,广东肇庆526238 [4]华南农业大学兽医学院,广东广州510642

出　　处：《动物医学进展》2023年第10期19-24,共6页Progress In Veterinary Medicine

基　　金：肇庆市科技项目(2021C001);肇庆学院大学生创新训练项目(S202210580052);肇庆学院校级科研项目(QN202225)。

摘　　要：为表达H5亚型禽流感病毒HA蛋白研制亚单位疫苗,构建了pFastBAC-HA重组质粒,转化至DH10Bac感受态细胞,获得重组HA基因的杆状病毒质粒,再将重组杆状病毒质粒转染至Sf9细胞,拯救重组病毒,通过间接免疫荧光和Western blot试验对Sf9细胞中表达的HA蛋白进行鉴定。结果表明表达的重组HA蛋白均能与抗Strep标签抗体、H5亚型禽流感病毒阳性血清(Re-11株)特异性结合,且HA重组蛋白在昆虫细胞中以细胞分泌上清的形式表达,红细胞凝集效价为1∶64,利用链霉素亲和层析的方法,纯化重组HA蛋白,纯化后HA蛋白浓度为0.364 mg/mL。试验结果可为禽流感病毒亚单位疫苗研制、HA蛋白单克隆抗体制备提供前期基础。To express H5 subtype avian influenza virus HA protein,develop subunit vaccine,in this experiment,pFastBAC-HA recombinant plasmid was constructed and transformed into DH10Bac competent cells to obtain the recombinant baculovirus plasmid containing HA gene,later the recombinant baculovirus plasmid was transfected into Sf9 cells to rescue the recombinant virus,the recombinant HA protein expressed in Sf9 cells was identified by indirect immunofluorescence assay and Western blot.The results showed that the expressed recombinant HA protein could react specifically with anti-Strep tag antibody and H5 subtype AIV positive serum(Re-11 strain),the HA recombinant protein could be expressed in insect cells as the form of cell secreted supernatant,the agglutination titer was 1:64,the HA recombinant protein was purified by streptomycin affinity chromatography,the concentration of purified HA protein was 0.364 mg/mL.This study can provide preliminary foundation for the development of avian influenza virus subunit vaccine and the preparation of monoclonal antibody against HA protein.

关 键 词：H5亚型禽流感病毒 HA蛋白 杆状病毒 蛋白表达 

分 类 号：S852.657[农业科学—基础兽医学]