Expression Purification And Application Of Penton Protein Of Flow Adenovirus

As an infectious disease pathogen that seriously threatens the development of poultry industry,Fowl adenovirus has been widespread in China since 2015.It mainly causes diseases characterized by fowl hepatitis and pericardial effusion.The vertical transmission and recessive infection characteristics of fowl adenovirus,combining with epidemiological investigation and analysis,make the fowl adenovirus infection existing in the poultry industry in my country and should not be underestimated.This study had an in-depth understanding of the genetic evolutionary relationship between different serotypes of fowl adenovirus strains,and established a monitoring method for fowl adenovirus antibodies that could cover all serotypes and could be used for the detection of foreign viruses in fowl-derived live vaccines(chicken inspection method)and clinical sample testing.In this study,the Hexon gene and Penton gene of 12 serotypes fowl adenovirus representative strains stored in the laboratory were cloned,sequence and analyzed,and the sequencing results were compared with the standard sequences published in NCBI.Nucleotide sequence analysis showed that the homology of Hexon gene with the standard strain was 98.7%,except for E7,while the homology with other serotype strains was higher than 99%,among which homologous with C10,D2,D3,E8a,E8b strains was 100%,and the homology between different serotypes of the same species was80.4%-99.1%.The homology of Penton gene D9,A1 and B5 strains with the standard strain was 97.5%,99%,and 99.2%respectively while the remaining 9 serotype strains were all 100%.The homology among different serotypes with the same species was 95.5%-99.6%.The experimental results showed that the laboratory strains were highly consistent with the international standard strains,and the Penton gene was more conservative than the Hexon gene.The ELISA method is widely used in virus antibody monitoring due to its advantages such as simple operation,short time-consuming,and high sensitivity.The establishment of the ELISA method requires a clear background quality control serum.Therefore,in this study,five representative strains A,B,C,D,and E were prepared by methods such as fowl adenovirus culture and SPF chicken immunization.The more conservative Penton gene was selected for codon optimization with Escherichia coli as the host bacteria,and the prokaryotic expression system was used to express the Penton protein,and a large amount of Penton protein was successfully expressed and purified.Thus,a competitive ELISA method with 4?g/m L Penton protein for antigen coating,1:40 dilution of the serum to be tested,and 1:6000 dilution of the fowl adenovirus Penton monoclonal antibody was established.This method could effectively detect the positive sera of the five genotypes of fowl adenovirus A,B,C,D,and E,and there was no cross-reaction phenomenon with other common fowl virus diseases.At the same time,in order to study whether there was an immune response between the fowl adenovirus Penton monoclonal antibody and other species of the fowl adenovirus genus,this article successfully constructed a eukaryotic expression system combined with an IFA test epitope identification method.This method was simple to operate and took a short time.And it could identify conformational epitopes.Through this method,the epitope identification of the fowl adenovirus Penton monoclonal antibody was preliminarily carried out.The test results showed that the epitope of the fowl adenovirus Penton monoclonal antibody was ¹¹⁵GLQRVLITDDQRRP¹²⁸.The sequence alignment and BLAST found that this sequence was relatively conservative.