The Prokaryotic Expression Of Hexon Of Aviadenovirus

Adenovirus(ADV),found from healthy population of adenoid tissue cell during the culture,it was named so since it tends to infect epithelial cells.Meanwhile,Fowl adenovirus(FAV)was reported as early as in Karachiof Ankara,Pakistan in 1987,therefore,it was also known as Ankara disease.FAV is a double stranded DNA virus without envelope,and it could be divided into 3 subgroups:I,II,III.There are 12 serotypes(FAV1 ~ 12)in group I of FAV,which all possess common antigen and the C type FAV-4 group I FAV is regarded as the main pathogen of Ankara Disease in chicken.Main characteristics of the structure of FAV I are: diameter ranges from 70 nm to 90 nm,unenveloped,spherical,icosahedral symmetry.It has 252 virocapsomeres,of which,the 12 vertex capsomeres are penton while the rest 252 unvertex capsomeres are hexon.Hexon protein is the major structural protein of FAV,andform the shell of FAV with five adjacent body protein and fiber protein.The hexon protein contains the major genera and subgenus-specific antigenic determinants,as well as the secondary species-specific antigenic determinants,which are the targets for neutralizing antibodies and the primary protective antigen gene,which is closely related to pathogenicity and also a promising diagnostic antigen.We conducted to observe a series of sick chicken farms in Hubei Xiantao.In this study,a series of experiments were conducted on sick chicken from farms in Xiantao,Hubei.Through necropsy,pericardial effusion,liver erythrocyte enlargement and other pathological features were observed.Liver tissue steatosis were found by microscopic observation after H.E staining.The PCR results are consistent with the expected size of the amplified fragment.The homology of the sequence was similar to that of I group 4 avian adenovirus,which was 99.2%,which was relatively low compared with other serotypes.Age chicks can also lead to the disease,to determine the incidence of poultry Ankara disease for the avian adenovirus I group 4 type.Hexon gene of I group 4 was isolated by PCR method and cloned into pMD19-T Simple vector.The recombinant pMD19-T Simple-hexon was inserted into p ET-28 a vector after double enzyme digestionand the recombinant plasmid pET-28a-hexon was constructed by hexon.The correct recombinant plasmid was transformed into Escherichia coli BL21 by kanamycin containing LB medium selected positive clones were sequenced.The correct identification of bacteria IPTG expressioncells were broken by SDS-PAGE of soluble expression product expression of target protein was purified by affinity chromatograph.The immunogenicity analysis of the recombinant protein Western-blotwith 3 blind passages containing avian adenovirus inactivated allantoic fluid after immunized with 0.2mL/ only dose by multi point injection mode in three immunized mice after three free blood serum separation and the preparation of mouse polyclonal antibody,the antibody level and the specificity of Western-blot detection of recombinant protein of hexon.Results the expression of hexon protein was successfully expressed.The expression temperature was 37 ?,the concentration of IPTG was 1mM and the induction time was 4h.The results showed that the recombinant hexahedrons were expressed in the form of inclusion bodies and the target protein was purified by affinity chromatography.The target protein was digested in 10 mM,20 mM,50 mM,100 mM,150 mM,250 mM,500 mM Imidazole can be eluted.Westeron-blot analysis showed that the recombinant hexon protein could specifically bind to the positive sera of avian adenovirus and could show specific bands indicating that the recombinant hexon protein had a good reactogenicity.The isolated isolates of mice were identified by Westeron-blot and the murine anti-recombinant hexon protein polyclonal antibody was successfully prepared.The results showed that the recombinant hexon protein was expressed in the form of inclusion bodies and had good antigenicity with had good reactivity with FAV virus 3 mice and laid the foundation for further preparation of new vaccine of avian adenovirus and related research.