H7N9禽流感病毒HA基因在杆状病毒中的表达及检测  

Expression and detection of hemagglutinin gene of H7N9 avian influenza virus in baculovirus

作　　者：王平[1] 蔡明毅[1] 沈琦[1] 任静[1] 赵文良[1] Wang Ping;Cai Mingyi;Shen Qi;Ren Jing;Zhao Wenliang.(Department of Laboratory, Jingan District Center for Disease Control and Prevention, Shanghai, 20041, China)

机构地区：[1]上海市静安区疾病预防控制中心检验科,200041

出　　处：《国际病毒学杂志》2018年第2期100-103,共4页International Journal of Virology

基　　金：上海市静安区卫生和计划委员会第三批“十百千”项目（JWRC2014G34）

摘　　要：目的利用昆虫杆状病毒表达系统表达H7N9禽流感病毒血凝素（HA）基因，获得重组HA蛋白，并验证其活性。方法全基因合成H7N9禽流感病毒全长HA基因，将HA基因进行扩增并克隆到pFastBacI载体中，构建重组杆状病毒表达载体，经PCR鉴定后将其转染昆虫s丹细胞，再采用SDS—PAGE实验和免疫印迹法检测重组HA蛋白的表达。HA蛋白经纯化后，采用血凝试验鉴定其红细胞凝集活性。结果PCR结果显示重组杆状病毒表达载体的扩增产物大小约为4000bp，SDS—PAGE电泳显示纯化的表达蛋白分子量为63KD，免疫印迹检查结果显示表达蛋白可以与特异性HA抗体进行反应，产生条带与理论大小相当。血凝试验结果显示表达的蛋白能使红细胞凝集，滴度为1：64。结论禽流感H7N9的HA蛋白在重组杆状病毒中获得表达，并具有血凝活性。Objective Baculovirus expression system was used to express the hemagglutinin （HA） protein of H7N9 avian influenza virus and to obtain the protein, so as to verify its activity. Methods Full length HA gene of H7N9 avian influenza was synthesized, the gene was then amplified and cloned into a pFastBacI vector. A recombinant baculovirus expression vector containing the target gene was then constructed by homologous recombination. After identification by PCR, the vector was transfected into Sf9 insect cells. The expression of recombinant HA protein was detected by SDS-PAGE and western blotting. HA protein was purified, and its erythrocyte agglutination activity was identified by hemagglutination test. Results PCR results showed that the size of the amplified product of the recombinant baculovirus expression vector was about 4000 bp. The molecular mass of the purified protein calculated by SDS-PAGE was 63KD. Western blot assay indicated that the expressed protein can react specifically with antibody against HA and the band was in consistent with prediction. The results of hemagglutination test showed that the expressed protein can agglutinate red blood cells, the agglutination activity was 1： 64. Conclusions The HA protein of H7N9 avian influenza virus was correctly expressed in recombinant baculovirus and the expressed protein had hemagglutination activity.

关 键 词：禽流感 病毒 基因表达 HA蛋白 杆状病毒表达系统 

分 类 号：R373[医药卫生—病原生物学]