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Chinese Veterinary Science
2024,54
(9)
1175-1181
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
作 者：
许壮壮;郭茹;王素艳;高立;陈运通;张涛;高宁玉;吴龙波;祁小乐;张艳萍;崔红玉;刘永振;段雨路;王牟平;高玉龙
单 位：
黑龙江八一农垦大学动物科技学院;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
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
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.
译 名：
Rescue and identification of recombinant fowl adenovirus 4 expressingσB and σC of genotype-Ⅰ subcluster avian reovirus
关键词：
avian reovirus Ⅰ-sub%fowl adenovirus 4%σ B%σ C%recombinant virus
摘 要：
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.
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