Immune Effect Of Fowl Adenovirus Type 4 Fiber-2 Protein Expressed By Baculovirus Expression System

Fowl Adenovirus(FAdV)is divided into group I,II and III.The 12 serotypes of group I fowl adenovirus can cause fowl pericardial fluid and inclusion hepatitis hepatitis syndrome(HPS-IBH),among which FAd V-4 is the most common pathogenic pathogen.In recent years,the incidence of HPS-IBH caused by FAd V-4 in China has gradually increased,which has caused great economic losses to the poultry industry.At present,there is no commercial vaccine to control FAd V-4 infection in China.Therefore,it is of great significance to carry out the research on FAd V-4 vaccine.In this study,the Fiber-2 protein of FAd V-4 was expressed in the baculovirus expression system of insects,and the immune effect of Fiber-2protein was studied to provide a basis for the study of subunit vaccine of avian adenovirus genetic engineering.We constructed a donor plasmid containing Fiber-2 gene and transformed it into DH10 Bac strain,Screening Positive Recombinant Bacmid by Blue and White Spot.In order to obtain recombinant Fiber-2protein,we transfected the recombinant Bacmid into sf9 cells to obtain recombinant baculovirus,and the recombinant baculovirus was cultured by suspension amplification technology.The sf9 cell expression supernatant was purified by nickel column and concentrated by ultrafiltration tube to obtain the target protein.Western-Blot results showed that the purified and concentrated Fiber-2 protein was successfully obtained.The results of protein concentration determination showed that the content of purified protein was120 μg / m L,and the content of target protein in concentrated total protein was 114 μg / m L.The purified and concentrated Fiber-2 protein were used to immunize 21 d chickens with 5 μg,1 μg adjuvant and 5 μg non-adjuvant groups,respectively.At the same time,the supernatant of sf9 cells without recombinant baculovirus infection was mixed with adjuvant as the control of purified and concentrated immunization groups.The 7 d,14 d,21 d and 28 d after immunization,the OD450 changes of the antibody were detected using the FAd V-4 antibody ELISA established in the laboratory.The results showed that serum antibodies were detected in the purified and concentrated immunization groups 14 d after immunization,and the antibody levels in each immunization group reached the peak 21 d after immunization.The results of t test showed that there was no significant difference in antibody level between purified and concentrated Fiber-2 protein immunization(P > 0.05),but the antibody level of 5 μg adjuvant group was the highest,followed by 1 μg adjuvant group.At 28 d after immunization,the challenge test was carried out by intramuscular injection and oral administration.The results showed that the purified 5 μg,1 μg and concentrated 5 μg,1 μg adjuvant groups could provide 100 % immune protection,while the concentrated 5 μg without adjuvant group could also provide 100 % protection against the challenge of oral administration of FAd V-4 strain.It suggested that the purified and concentrated Fiber-2 protein could produce antibodies and obtain the protection against virus.The concentrated Fiber-2protein saved the cost of protein purification and was more suitable for clinical application.