Construction Of FAdV-4 PcDNA3.1-Penton Recombinant Plasmid And Preliminary Study On Its Immunogenicity

Hydropericardium-Hepatitis Syndrome(HHS)is caused by ? group of fowl adenovirus serotype 4(FAd V-4),it main characteristics were pericardial effusion and liver tissue injury et al..The disease first erupted in the Ankara area of Pakistan in 1987,so it is also known as the poultry "Ankara disease." The disease is currently prevalent in many countries around the world,causing serious influences.In 2015,the disease was prevalent in many parts of China,resulting in reduced immune function and increased death rate,which brought serious economic losses to the Chinese poultry industry.So far,There is no commercialized vaccine for Ankara disease in China,so it is urgent to develop an effective vaccine to prevent the disease.Penton protein is the main structural protein in FAd V,and with good antigenicity,immunogenicity and high conservativeness.In this study,the Penton gene was used as the research object to construct the pc DNA3.1-Penton recombinant plasmid,and the recombinant plasmid was used to immunize fowls at different doses to explore its immunogenicity,which provided a theoretical basis for the development of effective Ankara vaccine.The test contents and results are as follows:1.The primers were designed according to the FAd V-4 Penton gene sequence on Gen Bank.Using the DNA of liver tissue of FAd V-4 positive material retained in our laboratory as template,the Penton gene was amplified and purified.The purified Penton gene was linked with p MD19-T cloning vector.The recombinant plasmid p MD19-T-Penton was constructed and identified by double enzyme digestion of Hind III and Eco R I.Then the target gene was linked with pc DNA-3.1 vector,then we can obtain the pc DNA3.1-Penton recombinant plasmid.2.90 healthy fowls of 3 days old were selected in this experiment,30 were SPF fowls and 60 were commercial fowls,which were divided into 6 groups,15 in each group.Grouped into: SPF fowls immunization group and negative group;commercial fowls immunization group and negative group with low,high and low dose adjuvant.The immunization mode was intramuscular injection,twice.After the first immunization 7 days,the immunization was strengthened.Blood samples were collected weekly after the first immunization.The levels of FAd V-4 antibody,interleukin-6(IL-6)and interferon-gamma(IFN-?)in serum were detected weekly after immunization by ELISA,and then the immune effect was evaluated.Results: After immunization of fowls with pc DNA3.1-Penton recombinant plasmid,FAd V-4 antibody was produced in the immunized group.The difference of serum FAd V-4 antibody between SPF fowls immunized group and commercial fowls low-dose group was not significant(P>0.05).The difference between the control group and the high-dose group was lower than that of the low-dose adjuvant group(P>0.05).The high-dose and low-dose adjuvant group were significantly different(P<0.05).Compared with the low-dose immunization group,the difference was significant(P<0.05).The difference between the immunized group and the control group was extremely significant(P<0.01).Serum levels of IFN-? and IL-6: At 7?14 and 21 days,there was no significant difference between the SPF fowls immunized group and the low-dose group of commercial fowls(P>0.05),which was significantly different from the control group(P<0.01);the low-dose ? high-dose and low-dose adjuvant groups of commercial fowls were significantly different from the control group(P<0.01);the difference between the low-dose adjuvant group and the low-dose and high-dose groups was significant(P<0.05).There was no significant difference between the low-dose and high-dose groups(P>0.05).After 21 days,there was no significant difference between the immunized group and the control group(P>0.05).The results of this experiment indicated that the constructed pc DNA3.1-Penton recombinant plasmid could produce FAd V-4 specific antibody after immunization of chicks,indicating that the constructed pc DNA3.1-Penton recombinant plasmid has good immunogenicity and Freund's The FAd V-4 specific antibodies produced by the adjuvant immunoassay group and the high dose immunization group were higher than the low dose group.Compared with the blank control group,the serum levels of IFN-? and IL-6 increased in each immunized group,indicating that pc DNA3.After immunization with 1-Penton recombinant plasmid,it can induce the production of related cytokines such as cellular immunity and humoral immunity.