表达重症急性呼吸综合征病毒刺突蛋白重组新城疫病毒的构建及其免疫原性研究  

Construction and immunogenicity evaluation of a recombinant Newcastle disease virus expressing SARS-CoV spike protein

作　　者：汤筱艳 刘任强[1] 山丹 潘丹 王喜军[1] 葛金英[1] 温志远[1] 步志高[1] TANG Xiao-yan;LIU Ren-qiang;SHAN Dan;PAN Dan;WANG Xi-jun;GE Jin-ying;WEN Zhi-yuan;BU Zhi-gao(State Key Laboratory of Veterinary Biotechnology,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兽医生物技术国家重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2020年第11期1145-1151,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划课题“边境地区外来动物疫病阻断及防控体系研究”(2017YFD0501804)。

摘　　要：重症急性呼吸综合征(SARS)是一种急性、发热并伴有呼吸系统甚至多脏器感染的人兽共患病。虽然自2004年以来未见SARS感染病例相关报道,但是2019年12月我国湖北省武汉市暴发的新型冠状病毒肺炎(COVID-19)疫情表明SARS冠状病毒(SARS-CoV)或者类SARS冠状病毒(SARSL-CoV)在人群中再次大面积流行的可能性极高。本研究首先构建了表达SARS-CoV刺突蛋白(S蛋白)的全长cDNA克隆pBRN-FL-SARS-CoV-S,并利用新城疫病毒(NDV)LaSota疫苗株反向遗传操作系统,拯救出表达SARS-CoV S蛋白的重组病毒rLa-SARS-CoV-S并鉴定。将rLa-SARS-CoV-S以MOI 0.01的剂量感染BHK-21细胞36 h后分别通过western blot和激光共聚焦试验对SARS-CoV S蛋白进行检测,结果显示S蛋白在感染的细胞中能正确表达并准确定位于细胞膜上。将rLa-SARS-CoV-S以1×104 EID50的剂量接种10日龄的SPF鸡胚后在不同时间点收集尿囊液并测定EID50,病毒的生长动力学曲线显示rLa-SARSCoV-S能够在鸡胚中高滴度生长,与亲本病毒一致。将rLa-SARS-CoV-S和亲本病毒10倍倍比稀释后接种10日龄的SPF鸡胚并记录每个鸡胚的死亡时间,按病毒最高稀释度引起鸡胚的死亡时间计算鸡胚平均致死时间(MDT),结果显示rLa-SARS-CoV-S的MDT为112.8 h,NDV LaSota的MDT为96 h,说明该重组病毒仍然保持NDV LaSota疫苗株的低致病力特性。将rLa-SARS-CoV-S和NDV LaSota分别以5×106 EID50的剂量通过肌肉注射接种6周龄的BALB/c小鼠并在第21 d加强免疫,同时设置PBS对照组。小鼠安全性试验结果显示接种rLa-SARS-CoV-S的小鼠全部存活并且无任何临床症状,体质量增长情况与NDV LaSota组及对照组保持一致;免疫后21 d和42 d对小鼠采血制备血清,通过ELISA检测小鼠体内针对SARS-CoV S蛋白的IgG抗体水平,结果显示在初免和加强免疫后该重组蛋白均可以诱导小鼠产生较高水平的特异性IgG抗体,并且抗体水平可以维持较长一段时间。本研究结�Severe acute respiratory syndrome(SARS)is a zoonotic disease whose symptom is acute,febrile,accompanied by respiratory infections and even multiple organ infections.Although there is no more SARS case reported in China since 2004,a novel SARS-like coronavirus(SARS-CoV-2)caused COVID-19 epidemic which broke out in December 2019 in Hubei province,indicating that the high possibility of SARS-CoV or SARS Like-CoV reemerges in a large population.In this study,a full-length cDNA clone of pBRN-FL-SARS-CoV-S expressing SARS-CoV spike protein(S)was first constructed,and the recombinant virus rLa-SARS-CoV-S expressing SARS-CoV S protein was rescued and identified by using the reverse genetic system of Newcastle disease virus(NDV)LaSota vaccine strain.rLa-SARS-CoV-S was identified and its immunogenicity was analyzed in BALB/c mice.BHK-21 cells were infected with rLa-SARS-CoV-S at MOI of 0.01,Western blot and IFA were used to detect the expression of S protein at 36 h post-infection,and the results showed that the S protein was correctly expressed and positioned on the cell membrane of the infected cells.10-day old SPF chicken embryo was inoculated with rLa-SARS-CoV-S at 1×104 EID50,then allantoic fluid was collected at different time points for viral titration,and the results showed that rLa-SARS-CoV-S strain grew well in chicken embryo with high viral titers.10-day old SPF chicken embryo was inoculated with a series of 10 times dilution of rLa-SARS-CoV-S and NDV LaSota,death time of each chicken embryo was recorded.The mean death time(MDT)of chicken embryos was calculated according to the death time of chicken embryos caused by the highest dilution of the virus.The results showed that MDT of rLa-SARS-CoV-S was 112.8 h,and MDT of NDV LaSota was 96 h,indicating that the recombinant virus still maintained the low pathogenic characteristics of NDV LaSota vaccine strain.6-week-old BALB/c mice were inoculated with rLa-SARS-CoV-S at 5×106 EID50 by intramuscular injection and PBS control group was set at the same time.The immuni

关 键 词：重症急性呼吸综合征病毒刺突蛋白 重组新城疫病毒 免疫原性 

分 类 号：S852.65[农业科学—基础兽医学]