禽流感H7N9亚型病毒样颗粒疫苗的制备与免疫效果评估  

Preparation and immunization effect evaluation of Avian influenza H7N9 subtype virus-like particle vaccine

作　　者：王玉娟 仝晓丹 张盼涛 宋欢欢 田辉 李胜强 谭菲菲 刘武杰 王同燕 田克恭 WANG Yujuan;TONG Xiaodan;ZHANG Pantao;SONG Huanhuan;TIAN Hui;LI Shengqiang;TAN Feifei;LIU Wujie;WANG Tongyan;TIAN Kegong(National Research Center for Veterinary Medicine,Luoyang 471003,China;Pulike Biological Engineering,Inc.,Luoyang 471000,China)

机构地区：[1]国家兽用药品工程技术研究中心,河南洛阳471003 [2]普莱柯生物工程股份有限公司,河南洛阳471000

出　　处：《黑龙江畜牧兽医》2021年第6期113-117,123,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：洛阳市河洛英才计划(新型兽用生物制品研发及产业化)。

摘　　要：为了获得能代替商品化灭活苗的禽流感H7N9亚单位疫苗,试验将H7N9亚型禽流感病毒的HA基因跨膜区进行点突变,构建同时表达HA、NA和M1基因的供体质粒;然后将供体质粒转化至DH10Bac感受态细胞中,获得重组杆粒(Bacmid);将鉴定正确的重组Bacmid转染Sf9细胞,对重组杆状病毒表达的蛋白进行血凝试验、Western-blot、SDS-PAGE、透射电镜、免疫原性等一系列评价。结果表明:重组杆状病毒表达的蛋白液HA效价可达1∶2048;Western-blot显示出现3条特异性条带;透射电镜可见与禽流感病毒结构类似的颗粒物;表达的病毒样颗粒(VLPs)蛋白制备成疫苗,HI效价略高于商品化疫苗。说明3种蛋白能自我组装成VLPs,该VLPs疫苗具有良好的免疫原性。In order to obtain a H7N9 subunit vaccine of Avian influenza that can replace commercial inactivated vaccine,point mutations in the transmembrane region of the HA gene of the H7N9 subtype Avian influenza virus were carried out in this experiment,and a donor plasmid that simultaneously expressed the HA,NA and M1 genes was constructed.The donor plasmid was transformed into DH10Bac competent cells to obtain recombinant Bacmid DNA;the correctly identified recombinant Bacmid was transfected into Sf9 cells,and a series of evaluations including hemagglutination test,Western-blot,SDS-PAGE,transmission electron microscopy,immunogenicity,were performed on the protein expressed by recombinant baculovirus.The results showed that the HA titer of the recombinant baculovirus expressed protein solution reached 1∶2048,and Western-blot showed three specific bands.Transmission electron microscopy showed particles similar in structure to Avian influenza virus.The expressed virus-like particles(VLPs)protein was prepared into a vaccine,and the HI titer was slightly higher than that of the commercial vaccine.The results suggested that the three proteins can self-assemble into VLPs,and the VLPs vaccine had good immunogenicity.

关 键 词：禽流感病毒 H7N9亚型 重组杆状病毒 病毒样颗粒 跨膜区突变 

分 类 号：S855.3[农业科学—临床兽医学] S859.79+7[农业科学—兽医学]