重组欧洲禽源H1N1亚型猪流感病毒疫苗株的构建及免疫保护效力  

Construction of a reassortant European avian-like swine H1N1 subtype influenza virus vaccine and its protective efficacy

作　　者：阮宝阳 宫晓倩[2] 刘晓敏[2] 汪琪[2] 张鹏[2] 汪秀会 王振勇[1] 李泽君[2] 王林[1] 童光志[2] 于海[2] RUAN Bao-yang GONG Xiao-qian LIU Xiao-min WANG Qi ZHANG Peng WANG Xiu- hui WANG Zhen-yong LI Ze-jun WANG Lin TONG Guang-zhi YU Hai(College of Veterinary Medicine, Shandong Agricultural University, Taitan , Shandong 271000, China Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241 ,China)

机构地区：[1]山东农业大学动物科技学院,山东泰安271000 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国兽医学报》2016年第12期2106-2112,2118,共8页Chinese Journal of Veterinary Science

基　　金：国家青年自然科学基金资助项目(31201916);上海市自然科学基金青年资助项目(12ZR1453500);中央级公益性科研院所基本科研业务费资助项目(2015JB07);中国农业科学院创新工程科研团队“动物流感病毒病原生态学”资助项目

摘　　要：以国内欧洲禽源H1N1亚型猪流感病毒流行株A/swine/Shanghai/1/2014(H1N1)(SH1)为材料,RT-PCR扩增HA和NA基因并将其克隆至PBD载体中,构建HA和NA基因的重组质粒。将HA和NA基因重组质粒及来源于流感病毒高产株PR8的6个内部基因(PB2、PB1、PA、NP、M和NS)重组质粒共转染293T细胞,从而成功构建了重组欧洲禽源H1N1亚型猪流感病毒疫苗株SH/PR8。以相同病毒剂量接种MDCK细胞,检测不同时间点的血凝效价,绘制病毒生长曲线,结果表明,重组病毒SH/PR8相对于原始野生株SH1在MDCK细胞上具有更高的病毒滴度。重组病毒SH/PR8制备的灭活疫苗经过一免和二免小鼠后,检测血清中血凝抑制抗体、中和抗体和IgG抗体,结果显示首免后抗体效价持续升高,4周后抗体效价达到峰值。小鼠攻毒试验结果表明,免疫组体质量下降不明显且未出现死亡,而非免疫组小鼠体质量持续下降并且于攻毒后6d小鼠出现100%死亡。肺脏组织病毒含量滴定以及组织病理学观察结果显示,免疫组能够有效抑制SH1病毒在肺脏中的复制,减轻肺脏病理变化。总之,本研究表明重组SH/PR8疫苗候选株相对于原始毒株SH1在MDCK细胞上具有更高的复制能力,制备的重组灭活疫苗能够诱导机体产生高水平的抗体,能够针对欧洲禽源H1N1亚型猪流感病毒的攻击提供很好的免疫保护。The material of this study was an European avian-like swine HIN1 subtype influenza virus A/swine/Shanghai/I/2014 （H1N1） （abbreviated as SH1） which was isolated in China. The HA and NA segments of SH1 swine influenza virus were amplified by RT-PCR and inserted into the PBD vector respectively,the HA and NA recombinant plasmids were constructed. In this stud- y,the 6 internal segments （PB2,PB1,PA,NP,M,NS） of A/Puerto Rico/8/34 （H1N1） （abbrevia- ted as PR8） which could efficiently replicate were selected, as the backbone of the influenza reverse genetic system. The six plasmids from PR8 and HA,NA recombinant plasmids from SH1 were co- transfected into 293T cells,and the reassortant European avian-like H1N1 subtype influenza virus vaccine strain（abbreviated as SH/PR8） was rescued successfully. The HA titers were detected at different hours and growth curve of the two viruses were pictured. The result showed that the re- assortant virus SH/PR8 had higher viral titers in MDCK cells than wild type virus SH1.The reassortant virus SH/PR8 was served as the seed virus for the preparation of inactivated swine influenza vaccine and mice were immunized twice. The titer of HI antibodies, IgG antibodies and neutralization antibodies was detected in all mice serum of each group. The antibody titers in- creased continuously at first immunization and reached the highest titer at 4th week. The challeng- ing experiments showed the vaccinated group mice did not lose weight obviously, however, the group mice without being vaccinated lost weight drastically and all died at 6th day. The lungs were used for virus titers detection and histopathological observation. The result showed that in vacci- nated group mice the viral replication abilities in lungs was limited effectively and attenuated his- topathological damages. Based on our research, we concluded that reassortant virus SH/PR8 serving as seed virus had higher replication ability than the wild type virus SH1 in MDCK cells. Pre- pared inactivated reassortant

关 键 词：H1N1亚型 猪流感病毒 反向遗传 抗体效价 免疫保护 

分 类 号：S852.65[农业科学—基础兽医学] S855.99[农业科学—兽医学]