表达2.3.4.4b分支H5亚型禽流感病毒HA基因重组鸭瘟病毒的构建及其免疫保护效力的研究  

Construction of recombinant duck plague virus expressing the HA gene of calde 2.3.4.4b H5 avian influenza virus and evaluation of its efficiency

作　　者：张小雨 刘静 赵玉博 焦晨晨 麻琦 陈普泽 谢艳 陈普成[1] 姜永萍[1] 陈化兰[1] 柳金雄[1] ZHANG Xiao-yu;LIU Jing;ZHAO Yu-bo;JIAO Chen-chen;MA Qi;CHEN Pu-ze;Xie Yan;CHEN Pu-cheng;JIANG Yong-ping;CHEN Hua-lan;LIU Jin-xiong(National Avian Influenza Reference Laboratory,State Key Laboratory for Animal Disease Control and Prevention,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]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/国家禽流感参考实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2024年第10期1049-1056,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金面上项目(32072878);黑龙江省重点研发计划项目(2022ZX02B11)。

摘　　要：近年来,2.3.4.4b分支H5亚型禽流感病毒(AIV)引起的禽流感疫情在全球多国暴发,切断水禽作为AIV传播媒介的途径对于禽流感的防控意义重大。为研制针对当前H5亚型AIV流行株和鸭瘟病毒(DEV)感染均具有免疫保护作用的疫苗,本研究经PCR扩增2.3.4.4b分支H5亚型AIV HA基因,按照文献方法构建以DEV疫苗株为载体,表达2.3.4.4b分支H5亚型AIV代表株A/whooper swan/Shanxi/4-1/2020(H5N8)HA基因的重组DEV,命名为rDEV-14株。将rDEV-14株在CEF中连续传代后,经PCR、间接免疫荧光试验(IFA)和western blot检测,并检测各培养时间rDEV-14株的TCID_(50),分析其生长特性。结果显示,在10、15代rDEV-14的PCR产物中检测到HA基因,IFA和western blot检测结果显示HA基因可在rDEV-14株各代中稳定遗传和表达。生长曲线测定结果显示rDEV-14株在CEF中的生长曲线与亲本病毒DEV疫苗株趋势一致。以不同剂量rDEV-14株免疫4月龄SPF鸭14 d后,采用DEV强毒AV1221株攻毒,结果显示,各剂量rDEV-14株均可诱导SPF鸭对DEV感染的完全免疫保护。以不同剂量rDEV-14株免疫2周龄SPF鸭后,分别利用同源和2021年~2022年分离的H5亚型异源高致病性AIV(HPAIV)(H5N6及两株H5N1 AIV)攻毒后记录各组鸭发病和死亡情况、检测排毒情况和HI抗体,结果显示,10^(4)TCID_(50)和10~5TCID_(50)免疫组鸭在攻毒后两周内无发病、无死亡、不排毒,并可诱导鸭产生良好且持久的HI抗体。上述结果表明本研究制备的重组病毒r DEV-14可对DEV强毒、同源H5亚型AIV强毒以及2021年~2022年分离的H5N1和H5N6异源HPAIV攻毒鸭产生完全的免疫保护力。本研究为我国水禽禽流感和鸭瘟的防控提供了新的候选疫苗株。In recent years,avian influenza outbreaks caused by clade 2.3.4.4b H5 subtype avian influenza viruses(AIVs)have occurred in many countries around the world,and blocking the pathway of waterfowl as a vector of AIV is of great significance for the prevention and control of avian influenza.In order to develop a vaccine with protective effect of immunity against both of the current epidemic strains of H5 avian influenza virus and duck plague virus infection,this study amplified the HA gene of clade 2.3.4.4b H5 subtype AIV,in accordance with the literature to constructed a recombinant duck plague virus rDEV-14 strain(a duck plague virus vaccine strain)as a vector expressing HA gene of the representative strain of the clade 2.3.4.4b H5 subtype avian influenza virus,A/whooper swan/Shanxi/4-1/2020(H5N8)strain.The recombinant virus rDEV-14 strain was able to pass continuously on CEF,after that we performed PCR,indirect immunofluorescence assay(IFA)and western blot analysis,and detected the TCID_(50) of the virus at different time to analyze its growth kinetics.The results showed that HA gene was detected in PCR products of the 10th and 15th generation of rDEV-14,and the results of IFA and western blot assay showed that HA gene could be stably inherited and expressed in all generations of rDEV-14 strain.The growth curve showed the growth trend of rDEV-14 strain in CEF is consistent with the parent DEV vacclne strain.After vaccinated 4-month-old SPF ducks with different doses of recombinant virus rDEV-14 strain,and then challenged those ducks with DEV AV1221 strain,the results showed that all doses of recombinant virus rDEV-14 strains induced complete protection of SPF ducks against DEV infection.After vaccinated 2-week-old SPF ducks with different doses of recombinant virus rDEV-14 strain,and recorded the morbidity,mortality,detoxification of every group after challenged respectively with homologous and 2021-2022 isolated H5 subtype heterologous highly pathogenic AIV and detected HI antibody,the results showed that ducks o

关 键 词：鸭瘟 H5亚型禽流感病毒 重组鸭瘟病毒 活载体疫苗 

分 类 号：S852.65[农业科学—基础兽医学]