| Fowl Adenovirus(Fowl Adenovirus)is one of the most common avian viruses.Among them,Fowl Adenovirus serotype 4(FAdV-4)infection causes avian pericardial effusion-hepatitis syndrome(Hydropericardium).Hepatitis Syndrome(HHS)is mainly characterized by yellowing and swelling of the liver and the accumulation of a large amount of yellow transparent effusion in the pericardium,which has caused serious harm to my country's poultry breeding industry and serious economic losses.The disease first occurred in the Ankara area of Pakistan in 1987,so the disease is also called Avian Ankara disease.Avian Ankara disease showed a large-scale outbreak in my country in 2015 and began to spread.It is easy to cause infection to chicks aged 3-6 weeks,and the mortality rate can reach 80%.It is very urgent to develop safe and effective drugs and vaccines to prevent the disease.Fiber-2 protein is one of the main structural proteins of FAdV-4 and is considered to be a protective immunogen that can induce the production of neutralizing antibodies.DNA vaccines usually require adjuvants to improve presentation efficiency.In this experiment,Fiber-2 gene was used as the research object and chicken cytokine IL-2 was added as a gene adjuvant to construct pc DNA3.1-Fiber2 recombinant plasmid,pc DNA3.1-IL2 recombinant plasmid,and pc DNA3.1-Fiber2IL2 recombinant plasmid.The three groups of recombinant plasmids were used to immunize chickens to explore its immune effect,and provide a preliminary theoretical basis for the development of a safe and effective vaccine for Ankara disease.The results are as follows:1.Using the newly isolated Xinbin strain liver tissue DNA and chicken spleen c DNA as templates,primers were designed according to the FAdV-4 Fiber2 gene and chicken IL-2 gene sequence on Gen Bank,and the Fiber-2 gene,IL-2 gene and Fiber2-IL2 were fused The gene is amplified by PCR.After amplification,the purified target gene is inserted into the cloning vector,and finally connected to the pc DNA3.1 eukaryotic expression vector to construct a recombinant plasmid.The recombinant plasmid was verified by PCR,Hind? and Xba? double enzyme digestion identification,and sequencing verification.The pc DNA3.1-Fiber2 recombinant plasmid,pc DNA3.1-IL2 recombinant plasmid,and pc DNA3.1-Fiber2IL2 recombinant plasmid were successfully constructed.2.The above constructed recombinant plasmids were immunized in groups of 35 healthy SPF chicks at the age of 14 days.Detect the level of FAdV-4 antibody in the serum of SPF chicks before and after immunization,as well as the content of interleukin 6(IL-6)and interferon gamma(IFN-?)by ELISA method,and then the constructed recombinant plasmid can be used for preliminary immunization effects Evaluation.The test results showed that the recombinant plasmids can induce the body to produce FAdV-4 antibodies and stimulate the increase of IFN-? and IL-6 levels in the body after immunizing the chicks.Cytokine IL-2 as a gene adjuvant can improve the immune effect of pc DNA3.1-Fiber2 recombinant plasmid.Compared with the level of FAdV-4 antibody,there is no significant difference in the immune effect of the pc DNA3.1-Fiber2 recombinant plasmid and pc DNA3.1-IL2 recombinant plasmid and the pc DNA3.1-Fiber2IL2 recombinant fusion plasmid.However,in terms of improving the content of cytokines in the body,as well as the immune dose and cost control,the pc DNA3.1-Fiber2IL2 fusion recombinant plasmid is superior to the pc DNA3.1-Fiber2 and pc DNA3.1-IL2 mixed immune recombinant plasmid.In summary,the recombinant plasmid constructed with FAdV-4 Fiber2 as the target gene can induce the body to produce FAdV-4 antibodies and stimulate the increase of IFN-? and IL-6 levels in the body.Cytokine IL-2 as a gene adjuvant can significantly enhance the immune effect of Fiber2 gene recombinant plasmid. |
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