Generation Of Monoclonal Antibodies Against Hexon Protein Of Fowl Adenovirus Serotype 4

Fowl adenovirus group ?(FAdV-I),a non-enveloped spherical double-stranded DNA virus,belonging to the family Flaviviridea,genus Aviadenovirus,is ubiquitously present in the poultry gastrointestinal and respiratory tract.In most cases,it causes subclinical infection,but leads to high poultry morbidity or mortality if co-infection with other pathogens.According to the restriction enzyme digest patterns and serum cross-neutralization test,FAdV-I is classified into five species(A?E)and 12 serotypes(1?12).Epidemiological studies showed that FAdV-I is widely distributed throughout the world and is one of the most common infectious diseases in poultry,including chicken,ducks,geese and others.All ages of poultry are found to be susceptible to FAdV-I infection.In 2013,an occurrence of one highly pathogenic fowl adenovirus infection in poultry,which was characterized as Inclusion body hepatitis(IBH)and Hydropericardium syndrome(HPS),occurred in China.Since June 2015,the disease had spread across the country with following features:acute onset;infection of 20?30 day-old broilers and meat ducks,etc;disease duration of 8?15 days;death rates peak within 4?8 days after the onset of the disease;mortality rate of 20%?80%.In 2016,we demonstrated that this outbreak was caused by a fowl adenovirus serotype 4(FAdV-4)epidemic strain.In order to facilitate surveillance of clinical infection of the disease,the immunosensing technology based on monoclonal antibody plays an important role in clinical diagnosis.Hexon protein,the main structure protein and the most abundant viral protein of FAdV-4,accounting for 90%of the total viral protein,forms nuclear capsid with the Fiber and Penton proteins,and determines the size of a virus particle.Hexon protein contains the type,group and subgroup specific antigenic determinants as neutralizing antibody targets.It has good application prospect as a diagnostic antigen.Therefore,preparation of monoclonal antibodies against Hexon provides a useful tool for diagnosis and immunological detection of FAdV-4.According to the hexon gene sequence of FAdV-4 JS7 epidemic strain published in GenBank,the N terminal 1?293 amino acids of Hexon was selected as polypeptide epitope candidate region based on prediction and analysis of the Hexon protein antigenicity and hydrophilicity.The targeted fragment of hexon gene was amplified by PCR method,and cloned into the prokaryotic expression pET-30a vector to construct recombinant plasmid pET-30a-hexon.After being induced by IPTG,the recombinant plasmid was expressed in BL21(DE3)E.coli competent cells and analyzed by SDS-PAGE.Balb/c mice were immunized intraperitoneally with the antigen prepared with the prokaryotically expressed 30a-Hexon protein.After the third immunization,the mice anti-sera were collected and exmamined by indirect immunofluorescence assay(IFA),with the antigen prepared with 293T cells trasfected with eukaryotic expression vector pcDNA3-hexon.Spleen of IFA-positive mice were isolated and fused with SP2/0 by PEG4000.Then limited dilution and IFA were performed to screen hybridoma clones secreting monoclonal antibodies against Hexon.In this study,we successfully obtained two positive hybridoma clones producing Hexon antibodies with satisfactory immunogenicity of IFA,ELISA and Western Blot,named as 1B4 and 3G8.The detection results showed the monoclonal antibodies against Hexon with high titers and specificity.The sub-types of these two hybridoma clones were IgGl and ?.The preliminary analysis showed that both 1B4 and 3G8 recognized the same region,181PNPNQGPGRNPLRRVQNANTGVLGRFAKSQYNYAYGAYVK219.The monoclonal antibodies against Hexon will be an important tool to further explore molecular biological characteristics of Fowl adenovirus serotype 4 as well as the structure and function of Hexon.It also provides a technical support for developing diagnostic kits for FAdV-4 infection.