Antibacterial Properties And Preparation Of Polyclonal Antibody Of Nile Tilapia Akirin Genes

The Nile tilapia(Oreochromis niloticus)is one of the most important freshwater fish in the world aquaculture industry and is regarded as one of the main sources of animal protein in the future.Among the many farmed fish in China,tilapia is one of the most suitable fish for industrialized production and international trade,and the breeding prospects are broad.However,bacterial diseases seriously threaten tilapia farming and cause huge economic losses.As the acquired immune system of fish is not complete,when fish are stimulated by a large number of external pathogens,their innate immune system will play a major role.Therefore,to study the molecular mechanism of Nile tilapia against pathogen infection,to further understand the regulatory mechanism of tilapia's innate immune response,and to complement the basic theoretical research on fish immunity,it can provide theoretical basis for improving the disease resistance of fish and developing high-efficiency fish vaccine in the future.Akirin is a multifunctional small molecular nuclear protein found in the defense system of Drosophila.It is highly conserved in evolution,widely exists in invertebrates and vertebrates,and plays an important role in resisting bacterial infections and muscle regeneration,and is involved in Fish's innate immune defense system.But at present,Akirin has done little research on fish.In this study,quantitative Real Time-PCR was used to detect the expression changes of Akirin1,Akirin2-1 and Akirin2-2 genes in different tissues and at different time after laboratory artificial infection of three pathogenic bacteria(Aeromonas hydrophila,Edwardsiella tarda and Staphylococcus epidermidis)in Nile tilapia.In this study,the effects of Akirin protein on 6 immune factors of Nile tilapia and the proliferation of 3 pathogens after infection were further studied at the molecular and individual levels.The mechanism of Akirin protein in Nile tilapia innate immunity was explored,and polyclonal antibodies of Akirin1 and Akirin2-1 with high titers were successfully prepared.The findings are as follows:1.Through qRT-PCR detection,Nile tilapia Akirin1,Akirin2-1,and Akirin2-2 genes were expressed in liver,spleen,and kidney.The results indicate that they play a role in these tissues.The expression levels of Akirin1 and Akirin2-1 genes in the liver,spleen and kidney of Nile tilapia were significantly up-regulated after infection with Aeromonas hydrophila,Edwardsiella tarda and Staphylococcus epidermidis,and gradually returned to normal after up-regulated expression for a period of time.The expression of Akirin2-2 gene in the liver,spleen and kidney of fish infected by Aeromonas hydrophila and Edwardsiella tarda also increased first and then decreased to normal levels,indicating that all three Akirin genes are involved in the process of tilapia's immune defense against pathogen infection,as the infection time prolonged,the pathogen was gradually cleared and the expression of Akirin gradually returned to normal.2.By constructing a recombinant plasmid,prokaryotic expression obtained with 6 * His tag 22 kd,21 kd about size of recombinant protein pEASY Blunt-E2-Akirin1,pEASY Blunt-E2-Akirin2-1,two recombinant proteins are abundantly expressed in inclusion body.After purifying the recombinant protein,the rats were immunized,the polyclonal antibodies of Nile tilapia Akirin1 and Akirin2-1 were obtained.After ELISA test,the antiserum titers were above 1: 10000,and Wertern blot verification was performed to confirm that the antibody specificity was good.3.The expressions of immune factors IL-1,IL-8,IL-12,Caspase-3,TNF,and IFN in the kidneys of Nile tilapia injected with Akirin1 or Akirin2-1 protein were detected by qRT-PCR.The results showed that after injection of Akirin1 protein into Nile tilapia,the expression levels of IL-1,IL-8,IL-12,TNF,and IFN all significantly increased,and the immune factors detected after injection of Akirin2-1 protein were significantly up.It is shown that Akirin1 and Akirin2-1 can activate the expression of cytokines during the immune response,thereby regulating the body's immune function.4.Bacterial counting results showed that the injection of Akirin1 and Akirin2-1 protein in Nile tilapia significantly reduced the growth of Aeromonas hydrophila,Edwardsiella tarda and Staphylococcus epidermidis in the liver,spleen and kidney.It is further verified that Akirin1 and Akirin2-1 play important roles in the defense of Nile tilapia against the pathogens Aeromonas hydrophila,Edwardsiella tarda and Staphylococcus epidermidis.To sum up,the three genes of Nile tilapia Akirin all play an important role in innate immunity against pathogen infection.Akirin protein can activate the expression of a variety of cytokines and inhibit pathogens in Nile tilapia.The research results provide a theoretical basis for improving fish disease resistance and developing a highly efficient fish vaccine,and provide technical support for the practical application of Akirin in the future.