Generation Of Antibodies Against Fiber-2 Protein Of Fowl Adenovirus Serotype 4 Strain HN And Its Subunit Vaccine

Fowl adenovirus(FAdV)belongs to the genus Aviadenovirus,family Adenoviridae and has been clustered into five species(A-E)with 12 serotypes.The five species of FAdV include species A(FAdV-1),species B(FAdV-5),species C(FAdV-4 and-10),species D(FAdV-2,-3,-9,and-11)and species E(FAdV-6,-7,-8a,and-8b).Although these species had spread globally prior to 2015,FAdV infections mostly caused subclinical symptoms or were avirulent.However,from the summer of 2015,the outbreaks of high pathogenic serotype 4 fowl adenovirus(FAdV-4)had emerged in many provinces of China.These outbreaks typically occurred in broiler and layer flocks,were associated with inclusion body hepatitis(IBH),hydropericardium syndrome(HPS),gizzard erosion and ulceration.In contrast to a mild disease caused by previous FAdV outbreaks,severe FAdV infections in the past two years have caused a huge economic loss in the poultry industry.The nuclear capsid of fowl adenovirus mainly contains three kinds of structural proteins,penton,hexon and fibers,respectively.Fiber-2 is located on the surface of the fowl adenovirus with a big knob linked to a slim body which makes it look like a match.Fiber proteins are able to identify the specific host cell receptor,which will establish the combination between virus and the surface of host cell.Based on fiber-2 gene sequence data of FAdV-C isolate ON1,which belongs to serotype 4,a pair of primers were designed.A fragment of 1440 bp was amplified by PCR from the DNA of strain HN.The fragment was subcloned into a prokaryotic expression vector,pCold-TF,and was transformed into the competent cells,DH5a.After confirmed by sequencing,named as pCold-Fiber-2,the plasmid was transformed into the E.coli competent cells BL21(DE3).After incubated at 16 ℃,expression of Fiber-2 protein was induced by IPTG.Taken a brief sonication,the samples was collected and analyzed by SDS-PAGE.Another pair of primers was designed to subclone fiber-2 into a eukaryotic expression vector,pcDNA3.The positive plasmid was named pcDNA3-Fiber-2.Then,the plasmid was transfected into 293T cells.After 36 h,the cells were collected and treated with 2×SDS-PAGE sample buffer.The overexpression of Fiber-2 protein was identified by Western blot analysis.pCold-Fiber-2 protein was used as immune antigen to prime and boost immunization in 6-week old Balb/c female mice.After four times immunization,the sera were collected and tested by IFA and WB analysis.The spleen of the most obviously positive mouse was separated after 72 h,and then fused with SP2/0 myeloma cells.The hybridoma cells were selected by HAT selection medium,together with ELISA and IFA analysis.Finally,four hybridoma cell lines secreting monoclonal antibodies(McAbs)against Fiber-2 were developed,named as 1B5,1C6,2A9,and 3H11,respectively.For vaccination,in this study,the expressrd Fiber-2 proteins were used as a subunit vaccine to carry out the protection experiment in chickens.The Fiber-2 immunization groups were divided into a primer immunization group and a boost immunization group.Results showed that Fiber-2 subunit vaccine as well as inactivated whole virus vaccine provided 100%protection.These indicate that Fiber-2 subunit vaccine can protect chickens effectively.In conclusion,the study laid a foundation for further exploration of the immunological function of Fiber-2 and development of the diagnosis tool for fowl adenovirus serotype 4.