Isolation And Identification Of Fowl Adenovirus Serotype 4 From Shanxi Province And Expressing Its Fiber-2 Protein In Vitro

Avian adenovirus serotype 4 virus(FAdV-4)can cause diseases characterized by pericardial effusion and hepatitis in chicks,called pericardial effusion-hepatitis syndrome,which is also called Ankara disease because it is found in the Ankara area of Pakistan.In recent years,there have been reports of this disease in different provinces of our country,and the incidence and death are different,which has caused huge economic losses to the poultry industry.In this study,livers of sick chick suspected of being infected with Ankara disease were collected from a chick farm in Shanxi.The hexon fragment of FAdV-4 was amplified using specific primers and identified as FAdV-4 infection.Grind the disease material and inoculate SPF chicken embryos.After three generations of blind transmission,allantoic fluid was harvested and pure-bred virus was obtained.The allantoic fluid DNA was extracted to amplify the Fiber-2 fragment of FAdV-4,followed by homology analyze with other FAdV-4isolates.Fiber-2 protein was successfully expressed using the baculovirus insect cell expression system.It laid the foundation for further preparation of avian adenovirus new vaccine and related research.1.The DNA of the liver of the suspected diseased chick was extracted,and the FAdV-4Hexon fragment was amplified by PCR using specific primers.After agarose gel electrophoresis,the product appeared a target band around 900 bp.PCR products were sequenced and blast in NCBI database.The sequence homology with the published avian adenovirus serogroup 4 Hexon gene sequence reached 99.9%,which was confirmed as FAdV-4 infection.2.The liver of sick chickens determined to be infected with FAdV-4 was taken and inoculated with the yolk sac of 7-day-old SPF chicken embryos after treatment.The chicken embryo appeared obvious lesion after blind transmission for three generations.Extract DNA from chicken embryo allantoic fluid for PCR detection.The product was subjected to agarose gel electrophoresis and the desired band appeared near 900 bp.It indicated that FAdV-4 was successfully isolated and named FAdV4-SX strain.3.Design specific primers to amplify Fiber-2 fragments of FAdV-4.Using DNA of allantoic fluid as a template for PCR detection.The target bands appeared near 1400 bp after agarose gel electrophoresis.After sequencing,blast with domestic and foreign avian adenovirus isolates in NCBI.Results: The Fiber-2 fragment of FAdV4-SX strain has a similarity of 100 % and no variation with domestic isolates in recent years.It can be seen from phylogenetic tree that FAdV4-SX strains are in the same branch with isolates from all provinces in China,and the distance is relatively close.It is not in the same branch with foreign isolates such as Hungary(accession number AF036092.3).4.Recombinant baculovirus shuttle vector containing Fiber-2 gene was constructed by using baculovirus expression system.Then transfected into SF9 cells to obtain recombinant baculovirus.Recombinant Fiber-2 protein was expressed by suspension amplification process.Soluble analysis showed that recombinant Fiber-2 protein was mainly expressed in secretory form.The target protein was isolated and purified by Ni affinity chromatography.The purified Fiber-2 protein can specifically bind to the His-tag antibody,indicating that the recombinant Fiber-2 protein was successfully expressed.