表达基因Ⅰ亚型禽呼肠孤病毒σB和σC的重组4型禽腺病毒的拯救及鉴定
Rescue and identification of recombinant fowl adenovirus 4 expressingσB and σC of genotype-Ⅰ subcluster avian reovirus
许壮壮 1郭茹 2王素艳 2高立 2陈运通 2张涛 2高宁玉 2吴龙波 2祁小乐 2张艳萍 2崔红玉 2刘永振 2段雨路 2王牟平 2高玉龙2
作者信息
- 1. 黑龙江八一农垦大学动物科技学院,黑龙江大庆 163000;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禽免疫抑制病创新团队,黑龙江哈尔滨 150069
- 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禽免疫抑制病创新团队,黑龙江哈尔滨 150069
- 折叠
摘要
为获得1株能够融合表达基因I亚型禽呼肠孤病毒(avian reovirus Ⅰ-sub,ARV GCI-sub)σB和σC的重组4型禽腺病毒(fowl adenovirus 4,FAdV-4)株,以禽腺病毒rHN20为病毒载体,构建表达σB和σC蛋白的重组黏粒rHN20-r1966-ARV GCI-sub,使用新型Fosmid黏粒拯救系统进行病毒拯救.通过PCR、间接免疫荧光试验(IFA)、Western-blot以及体外复制动力学曲线鉴定重组毒株.结果显示,在感染的LMH细胞中成功检测到ARV GCI-sub的σB和σ-C基因;Western-blot结果显示,在感染重组病毒的LMH细胞中成功检测到FAdV-4的Fiber蛋白、ARV GCI-sub σB和σC蛋白;IFA结果显示,能特异性检测到FAdV-4的Fiber蛋白、ARV GCI-sub σ-B和σC蛋白;重组毒rHN20-r1966-ARV GCI-sub与亲本毒rHN20的复制能力无明显差异;表明重组病毒rHN20-r1966-ARV GCI-sub拯救成功.重组病毒rHN20-r1966-ARV GCI-sub的成功构建为同时靶向ARV σB和σC蛋白的ARV防控疫苗的研制提供了技术支撑,为ARV与FAdV-4的防控奠定了基础.
Abstract
Inorder to obtain a recombinant fowl adenovirus 4(FAdV-4)strain that could express the σB and σC of avian reovirus Ⅰ-sub(ARV GCI-sub),a novel Fosmid clay rescue system was used to rescue the virus using the fowl adenovirus rHN20 as the vector and the recombinant virus rHN20-r1966-ARV GCI-sub ex-pressing σB and σC proteins was constructed.The successful rescue of recombinant virus was proved by PCR,indirect immunofluorescence assay(IFA),Wes tern-blot,and the kinetics of replication in vi tro.The results showed that σB and σC gene of ARV GCI sub were detected in the LMH cells infected with recombi-nant virus by the PCR,Fiber protein of FAdV-4,σB and σC protein of ARV GCI sub were detected in the LMH cells infected with recombinant virus by the Western-blot experiment,and σB and σC protein of ARV GCI sub were specifically detected in the LMH cells infected with recombinant virus by the IFA experiment.The recombinant virus showed no significant difference in replication ability between the recombinant virus and the parent virus,indicating successful rescue of recombinant virus rHN20-r1966-ARV GCI-sub.The construction of recombinant virus rHN20-r1966-ARV GCI-sub provides technical support for ARV pre-vention and control vaccines targeting σB and σC protein of ARV generation at the same time,and lays a foundation for the combined prevention and control of ARV and FAdV-4.
关键词
基因Ⅰ亚型禽呼肠孤病毒/4型禽腺病毒/σB/σ-C/重组病毒Key words
avian reovirus Ⅰ-sub/fowl adenovirus 4/σ B/σ C/recombinant virus引用本文复制引用
基金项目
国家重点研发计划项目(2022YFF0710500)
国家肉鸡产业技术体系项目(CARS-41)
出版年
2024
国家科技期刊平台
NSTL
万方数据