| Avian adenoviruses belong to the genus Adenoviridae,which can be divided into 5 species(A～E)and 12 serotypes(1～7,8a,8b,8～11).Fowl adenovirus serotype 4(FAdV-4),which belongs to the specie of FAdV-C,is a double-stranded DNA virus without envelope.Its nucleocapsid is composed of three structural proteins.The hexon forms the face of icosahedron,and the penton is located at the top.There are two spike proteins on it,Fiber-1 and Fiber-2.The main function of Fiber-1 is bind to cell surface receptors and then invade cells.Hexon and Fiber-2 are the main virulence factors and antigens of FAdV-4,which play an important role in inflammation and virus replication.Since 2015,poultry hepatitishydropericardium syndrome caused by FAdV-4 has broken out in farms in many provinces of China,which has strong transmission ability,short incubation period,rapid onset,high mortality and seriously endangers the development of poultry industry.In order to study the genomic characteristics of FAdV-4 prevalent in China and establish a serological detection method,a strain of FAdV-4 isolated from Liaoning Province was sequenced and Fiber-2 protein of it was cloned and expressed.An indirect ELISA detection method for FAdV-4 was established,which can be used to detect FAdV-4 infection and antibody level after immunization.One strain of FAdV-4 was isolated and identified from the sample which was infected with avian adenovirus in the surrounding area of Shenyang.The complete genome was amplified by PCR and sequenced.Compared with non-pathogenic FAdV-4 strains,the isolated FAdV-4 had multiple amino acid mutations in Hexon,Fiber-1 and Fiber-2,and a deletion of 1966 bp in ORF19 and ORF27,which was consistent with the highly pathogenic FAdV-4 prevalent in China.The Fiber-2 gene of FAdV-4 was amplified by PCR and ligated to the prokaryotic expression plasmid,pET-28a.Then,the plasmid pET-28a-Fiber-2 was obtained.The plasmid,pET-28a-Fiber-2,was transferred into E.coli.BL21(DE3)competent cells,and the recombinant Fiber-2 protein in the form of inclusion body was obtained.The size of the recombinant Fiber-2 protein was about 70 kD.The best induction condition is that the final concentration of IPTG was 0.6 mM,and expression at 30 degrees Celsius for 8 h.After a large amount of expression,the recombinant protein was purified by affinity chromatography.When using imidazole eluent at 200 mM,the purity of the recombinant protein was best.After renaturation,the concentration of recombinant Fiber-2 protein was about 80μg/mL.The indirect ELISA detection method was established by using the purified recombinant Fiber-2 protein.The results showed that the optimum coating concentration of recombinant protein was 0.8 μg/mL,the best dilution of serum was 1:100,and the best dilution of enzyme-labeled antibody was 1:6000.Through the detection of 30 FAdV-4 negative sera,the average OD450 value was 0.145,the standard deviation was 0.080,and the critical value X+3S was 0.385.The established method had good specificity and sensitivity.It was used to detect 377 serum samples from large poultry farms in Liaoning Province.The positive rate of the samples was 86.29%to 97.53%,with an average positive rate of 90.72%.In this study,FAdV-4 strain S Y was isolated,the complete genome sequence and genetic evolution analysis were carried out,the soluble recombinant full-length Fiber-2 protein was prepared and the indirect ELISA detection method of FAdV-4 was established.377 clinical serum samples from some areas of Liaoning were detected,and the positive rate was generally high,with an average positive rate of 90.72%. |
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