H5N1亚型禽流感病毒神经氨酸酶蛋白在昆虫细胞中的表达与鉴定  

Expression and Identification of N1 Protein of H5N1 Subtype Avian Influenza Virus in Baculovirus-insect Cell System

作　　者：张洁[1,2] 崔鹏飞 张元成[2] 邢鑫 王丛丛 颜成 王燕 陈源 朱春成 缪葭皓 陈素娟 邓国华[2] 陈化兰[2] ZHANG Jie;CUI Pengfei;ZHANG Yuancheng;XING Xin;WANG Congcong;YAN Cheng;WANG Yan;CHEN Yuan;ZHU Chuncheng;MIAO Jiahao;CHEN Sujuan;DENG Guohua;CHEN Huaan(College of Veterinary Medicine,Yangzhou University,Yangzhou,Jiangsu 225009;National Poultry Laboratory Animal Resource Center,State Key Lab for Animal Disease Control and Prevention,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin,Heilongjiang 150069)

机构地区：[1]扬州大学兽医学院,江苏扬州225009 [2]中国农业科学院哈尔滨兽医研究所,动物疫病防控全国重点实验室,国家禽类实验动物资源库,黑龙江哈尔滨150069

出　　处：《中国家禽》2024年第11期40-44,共5页China Poultry

基　　金：“十四五”重点研发计划项目(2021YFD1800201)。

摘　　要：研究旨在利用昆虫细胞-杆状病毒表达系统制备H5N1亚型禽流感病毒(AIV)的神经氨酸酶(NA)蛋白。试验为获得具有良好反应原性的NA蛋白,将H5N1亚型AIV(DK/YN/S4469/2022)密码子优化后的N1基因插入载体pFastBac1,构建的重组转移载体pFastBac1-N1转化至DH10Bac感受态细胞,经过蓝白斑筛选获得阳性重组杆粒rBacmid-N1,然后将重组杆粒转染至sf9昆虫细胞,获得N1亚型重组杆状病毒。结果显示:N1重组蛋白在sf9昆虫细胞中正确表达,并且该重组蛋白具有良好的反应原性,能够与N1蛋白单克隆抗体3F3发生特异性反应,其仅能识别N1亚型鸡血清,特异性较好。研究成功制备了N1重组蛋白,为NA蛋白表达方法提供参考并为临床血清样品的N1抗体检测方法的建立奠定基础。The aim of this study was to prepare the neuraminidase(NA)protein of H5N1 subtype avian influenza virus(AIV)using an insect cell-baculovirus expression system.To produce a neuraminidase N1 protein with highly reactive,a strain of H5N1 subtype AIV(DK/YN/S4469/2022)was carefully chosen to synthesize N1 gene with optimizing codons and subsequently insected into vector pFastBac1.The recombinant transfer pFastBac1-N1 were transformed DH10Bac competent cells,and positive recombinant clones were identified through blue-white screening,resulting in the isolation of the recombinant bacmid rBacmid-N1.This recombinant bacmid was subsequently transfected into sf9 insect cells to generate N1 subtype recombinant baculovirus.The results showed that the recombinant protein N1 was correctly expressed in sf9 insect cell,and the recombinant protein had good reactivity and could react specifically with the monoclonal antibody 3F3 against N1 protein,which could exclusively identify serum samples of the N1 subtype,indicating excellent specificity.The results indicated that the recombinant N1 protein was successfully prepared in this study,which could provide a reference for the expression method of NA protein and lay a foundation for the detection method of N1 antibody in clinical serum samples.

关 键 词：禽流感病毒 杆状病毒表达系统 N1蛋白 血清抗体 

分 类 号：S852.65[农业科学—基础兽医学]