Development Of An Immune Enhancer For Grass Carp Based On Z-DNA

China is a big country in freshwater fish farming.Although large-scale and high-density farming mode can help increase fishermen’s income and provide people with affordable edible fish,it also has the potential to cause a big outbreak of fish infectious diseases.Most of the existing fish immunization agents only have an effect on some or some kind of pathogen infection,with the mutation of the virus is difficult to produce efficient and positive protective effect on fish.Therefore,the development of a broad spectrum of immune preparation for improving fish immunity and preventing a variety of pathogen infection has a broad market prospect.Grass carp is the main aquaculture species in China.In this paper,grass carp was taken as the experimental object,and an immune enhancer was prepared on pc MV-Flag vector constructed by small molecule Z-DNA and fish-specific protein kinase Z(PKZ).The injection dose of each fish was 1μg /g of fish body weight.After several days of intraperitoneal injection,Ig M antibody levels in grass carp were detected.RNA was extracted from the brain,eyes,intestines,gills,spleen,skin,kidneys,and liver and retrotranscribed into c DNA.q RT-PCR was used to detect the expression of immune effector genes Ci IFN I(Interferon I)、Ci ISG15(Interferon stimulated gene 15)、Ci PKR(Protein kinase R)、Ci IRF3(Interferon regulatory factor3)、Ci IRF7(Interferon regulatory factor 7)、Ci TLR3(Toll like receptors 3)and Ci PKZ(Protein kinase Z)in different tissues of grass carp.Western blot was used to detect the distribution of immune enhancers in different tissues.CAT and SOD activities and MDA content in tissues were detected.At the same time,the virus was extracted from the diseased fish tissues to make GCRV homogenate,and the inactivated vaccine was prepared by heating and inactivating agent treatment.Finally,GCRV challenge was carried out after 15 days of immune protection with the immune enhancer and inactivated grass carp hemorrhagic disease virus vaccine,and the protective effect of the immune agent was detected.The results showed that the expression level of Ig M in serum of grass carp increased gradually after injection of immune enhancer,and reached the highest level on day 16.In cultured grass carp kidney cells,the expression of Ci IFN I was significantly up-regulated by transfection of Z-DNA-Flag and z-DNA-Flag/PKZ-Flag.The expression levels of most genes in various tissues of grass carp reached the highest on day 12 after injection of immune enhancer,which was significantly different from the control group,and the expression levels in spleen,liver,intestine and kidney were higher than those in other tissues.The results of enzyme activity assay showed that SOD,CAT and MDA activities in immune-enhancer injection group reached the highest level from 8d to 12 d,then gradually decreased,and the activity level was significantly higher than that in control group(PBS injection).Western blot results showed that z-DNA-Flag/PKZ-Flag expression was detected in liver,kidney,intestine,spleen,and gills on the 4th day,but not in the 24 th day.The results of grass carp hemorrhagic disease virus challenge showed that the survival rate of grass carp after injection of immune enhancer was 76%,the survival rate of grass carp after injection of inactivated vaccine was 53%,and the survival rate of grass carp without injection of immune enhancer was 21%.The relative protection rate of grass carp after injection of immune enhancer was 70% compared with the group without injection of immune enhancer.The results showed that both immune enhancer and inactivated vaccine could protect grass carp,and the effect of immune enhancer was better than that of inactivated vaccine.