Preparation And Research Of Ichthyophthirius Multifiliis Nucleic Acid Vaccine

Ichthyophthirius multifiliis(Ich)is a ciliate protozoan that is not strictly hostselective and can infect most freshwater fish and even amphibians,causing a disease in freshwater fish commonly known as cucumber disease or "white spot disease".Due to its high mortality rate and rapid outbreak,it poses a great threat to the freshwater fish farming industry,and there is no effective control method yet.Therefore,we propose to construct a nucleic acid vaccine for Ich to prevent Ich.Ich DNA was sequenced by PCR amplification and compared with the sequence of Ich β-microtubulin gene(accession number: XM＿004037588.1)contained in the NCBI website.The gene was used as the antigen and the p VAX1 plasmid was used as the vector to construct a β-microtubulin nucleic acid vaccine against Ich.Ctenopharyngodon idella used in the experiment was divided into a blank group injected with saline,a blank group injected with empty plasmid,and an immunized group injected with nucleic acid vaccine,and booster immunization was performed at the same dose 4 weeks after the initial immunization.Immunological indexes and survival rates of the immunized,null and blank groups were compared after immunization.The attack experiment was started at week 2 after the second booster immunization with an infection density of 1000 theronts/100 m L.The main results of the study are as follows:Verification of the smooth expression of the antigenic gene Ich β-microtubulin in the immunized fish by examination of m RNA in the blood of Ctenopharyngodon idella in the blank,no-load and immunized groups.Ich nucleic acid vaccine showed better immunity against Ich infection,with a higher survival rate in the immunized group and immune protection up to70.0%compared to the blank and unloaded control groups.Ctenopharyngodon idella sera were tested for antibodies to Ich β-microtubulin by ELISA,the results of which showed that: Only the immune group was positive and persisted until the thirteenth week,while the blank and empty groups were negative,indicating that specific antibodies against Ich β-microtubulin were produced in the experimental fish of the immune group only.The experimental results of serum agglutination potency showed that the sera of all three groups of experimental fish could cause Ich theronts to agglutinate and die.However,the immunized group had the highest serum potency and the longest duration of immunization,the blank group had the lowest duration and potency,and the no-load group was in between.The results of IgM content measurement showed that the content of specific immunoglobulin IgM increased and then decreased after the first and second immunizations.The IgM content increased significantly(P < 0.05)after the second vaccination than after the first vaccination,and reached a peak at the sixth week.The IgM content of the idle group showed a small increase and then decreased,and the IgM content of the idle and blank groups were all lower than that of the immunized group at each week.It can be seen that the specific immune response of Ctenopharyngodon idella was stronger in the immunized group,and more immunoglobulins were produced,which were helpful for the prevention of pathogens including Ich.Moreover,the IgM content of the immunized group increased significantly(P < 0.05)after the second immunization.It indicates that the re-stimulation of antigen activated the immune memory cells,which rapidly produced a large amount of immunoglobulins to help the fish defend against the invasion of Ich.The results of non-specific immune indexes showed that the activities of SOD,MDA,ACP,AKP and the content of cortisol increased after the initial and secondary immunization compared to the blank,empty and immunized groups.However,the peak was higher and and the duration of the increase was longer in the immunized group,indicating that the non-specific immune function of the fish was elevated after vaccination,as was that of the empty plasmid.The quantitative real-time fluorescence expression of four genes,MHII-DAB1,MHCⅠ,IRAK-I,and My D88,was measured in blood.Because these four genes are associated with Ich immunization,the experimental results showed that the gene expressions of these four genes were up-regulated after the first and second immunizations,showing a trend of first increase and then decrease.Only the gene expressions of IRAK-I and My D88 were different from those of the control group,and after the secondary immunization,the up-regulated amounts of all genes were higher and higher than those of the first immunization.It indicated that all immune-related genes were up-regulated when the fish were stimulated by the vaccine,which also supported that the immune response was stronger in the immune group.