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 andσC of genotype-Ⅰsubcluster avian reovirus

为获得1株能够融合表达基因Ⅰ亚型禽呼肠孤病毒(avian reovirusⅠ-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ARVGCⅠ-sub的σB和σC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、ARVGCⅠ-subσB和σC蛋白;ⅠFA结果显示,能特异性检测到FAdV-4的Fiber蛋白、ARVGCⅠ-subσB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ARVσB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。

In order to obtain a recombinant fowl adenovirus 4(FAdV-4)strain that could express theσB andσC of avian reovirusⅠ-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 expressingσB andσC proteins was constructed.The successful rescue of recombinant virus was proved by PCR,indirect immunofluorescence assay(IFA),Western-blot,and the kinetics of replication in vitro.The results showed thatσB andσC gene of ARV GCI sub were detected in the LMH cells infected with recombinant virus by the PCR,Fiber protein of FAdV-4,σB andσC protein of ARV GCI sub were detected in the LMH cells infected with recombinant virus by the Western-blot experiment,andσB andσC 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 prevention and control vaccines targetingσB andσC protein of ARV generation at the same time,and lays a foundation for the combined prevention and control of ARV and FAdV-4.