Establishment Of PCR Assay For Detection Of Fowl Adenovirus Group ? Serotype 4 And Prokaryotic Expression Of Penton Protein

Fowl adenovirus group ? serotype 4 belongs to the genus Aviadenovirus,which can lead latent infection in chickens and cause the incidence of broiler in 3-5 weeks.The disease is characterized by its sudden occurrence with high mortality in broilers and Hydropericardium hepatitis syndrome(HHS).The first outbreak of the disease,called Angara Disease,was occurred at Angara,near Karachi in August,1987 in Pakistan.Since then it has been prevalent in many countries including America,India,Canada,Hungary,Japan,South Korea,and Poland.Since 2012,the occurrence of inclusion body hepatitis(IBH)was reported in some regions in Ourcountry and the death rate of broilers was about 10%to 30%.And the mortality was up to 30%to 90%by 2015.The data shows that the major domestic epidemic strains since 2016 belong to C,D(serotype 4 and serotype 10)and E strains.In this study,the suspected FAdV-4 isolated in this laboratory was used to determine the relevant genetic characteristics of the suspected fowl adenovirus isolate in Shaanxi and to establish a PCR diagnosis method for fowl adenoviruses group ?.This study aimed to clone and prokaryotic express the Penton gene of Fowl adenovirus group ? serotype 4(FAdV-4),to obtain a high degree of accumulation of soluble Penton protein product in the bacterial cell.1.Genetic sequence analysis of suspected Fowl adenovirus serotype 4In this study,the six gene fragments Hexon,Penton,Fiber-1,Fiber-2,33K and pllla of the suspected serotype 4 Fowl adenovirus strain were cloned into prokaryotic expression vector pCold-SUMO.The results of enzyme digestion showed that six recombinant plasmids were successfully constructed.The six cloned gene fragments were sequenced and compared with the corresponding nucleotide sequences and deduced amino acid sequences of 17 different adenoviruses published in the GenBank.The results showed that the cloned six fragments had the highest homology with the fowl adenovirus group ?serotype 4.The results of comparison with type 4 adenovirus are as follows:Hexon gene shared from 99.9%to 100.0%nucleotide identity and 99.9%to 100%amino acid identity with FAdV-I serotype 4.Penton gene shared from 99.9%to 100.0%nucleotide identity and 100%amino acid identity with FAdV-I serotype 4.33K gene shared from 99.3%to 100.0%nucleotide identity and 91.5%to 100%amino acid identity with FAdV-I serotype 4.Fiber-1 gene shared from 98.5%to 100.0%nucleotide identity and 65.9%to 100%amino acid identity with FAdV-I serotype 4.Fiber-2 gene shared from 98.1%to 100.0%nucleotide identity and 99.3%to 100%amino acid identity with FAdV-? serotype 4.p ? a gene shared from 98.6%to 100.0%nucleotide identity and 98.5%to 100%amino acid identity with FAdV-I serotype 4.Phylogenetic analysis revealed that these genes seemed to be closely related to serotype 4.Taken together these data showed that the Shaanxi isolate was identified as a strain of FAdV-I serotype 4.2.Establishment of a PCR assay for diagnosis of Fowl adenovirus group ?.A pair of PCR primers has been designed according to conserved sequences of Hexon protein to detect group ? fowl adenovirus and the reaction conditions were optimized.The PCR amplification was carried out and the ideal annealing temperature was confirmed from 55? to 65?.The best effect of PCR efficiency was confirmed to be at annealing temperature 53.4 ?.According to specificity test results,the PCR amplification was shown to be positive to FAdV-4 and negative to CIAV,EDSV,ILTV and MDV.Then the serotype 4 adenovirus template was diluted to 5 group from 101 to 105 times to perform the sensitivity test.Obvious specific bands can also be detected by electrophoresis when the template concentration is 3.02 × 10-2 ng/?L,the results indicated the sensitivity of the method can be used to detect as low as 3.02 × 10-2ng/?L of viral DNA content.3.Prokaryotic expression and purification of Penton protein and preliminary study on indirect ELISA based on Penton proteinThe Penton gene of SX-17 strain was amplified and cloned into the pCold-sumo vector to construct the plasmid pCold-SUMO-Penton,and then the plasmid was transformed into E.coli DE3 for prokaryotic expression and induced using IPTG.The IPTG concentration was 1 mmol/L and the expression was induced at 15? for 18 h.SDS-PAGE electrophoresis and Western blot results showed that the Penton protein of SX17 strain was expressed as soluble proteins.The expression level and purification concentration of the Penton protein was shown to be with the high qualities,and the protein purity can be up to 80%?90%after separation and the purify concentration is up to 240 ?g/mL.The purified Penton protein had been showed to have the good biological activity and can be used as a good diagnostic antigen to provide a platform for subsequent researches.For this purpose,the purified Penton protein was coated and subjected to an indirect ELISA assay to initially determine the optimal antigen concentration for the ELISA assay and the optimal dilution for the serum,which were 50 ?g/mL and 1:200,respectively.