Development And Evaluation Of Immune Effects Of DNA Vaccines Against Nocardia Seriolae Based On IVIAT

Fish nocardiosis is an aquatic fish disease caused by Nocardia spp.,which occurs in a variety of economically cultured fish.In recent years,the outbreak of fish nocardiosis has caused serious losses to aquaculture industry.The pathogen of the disease can be infected through the wound or gill of the fish and feed.The hybrid snakehead suffered from fish nocardiosis has no obvious syndromes in the early stage,exhibits long incubation period and slow development.Once infected,it is difficult to treat in the later stage and the mortality is high.Typical disease symptoms include granulomatous nodules in the kidney,spleen,heart,gills and liver with multiple skin ulcers.There are mainly three kinds of pathogenic Nocardia spp.caused fish nocardiosis,among which Nocardia seriolae appeared in the most cases of infection recently.N.seriolae is an opportunistic pathogen,which can infect immunocompromised fish.N.seriolae infection has been reported in more than 40 kinds of fish.Fish nocardiosis caused by N.seriolae is becoming an increasingly epidemic disease,causing more and more serious damage to freshwater and marine aquaculture systems in Asia.In this study,using the whole genome sequence information of 17 strains of N.seriolae,the specific genome fragment ORF3659 was screened,and the specific primers 659-F/R were designed to establish the specific PCR detection method of N.seriolae.At the same time,the in vivo induced gene of N.seriolae was screened and identified by in vivo induced antigen technology(IVIAT)in this study,and the selected in vivo induced genes were used to develop DNA vaccines,and the immune effects of DNA vaccines were evaluated through vaccinating hybrid snakehead(Channa maculate ♀ × Channa argus ♂),which aim to establish the immune prevention of fish nocardiosis.The details of this study are as follows:1.In this study,the published whole genome data of several N.seriolae strains were screened and analyzed by using N.seriolae ZJ0503 strain as a comparable template.It was found that a genome fragment ORF3659 was conserved within species and interspecific variation.A pair of specific primers,659-F/R,were designed.Using this pair of primers,the specific PCR detection method of N.seriolae was established.After the optimization of reaction conditions,the best annealing temperature for PCR reaction was 64℃,the target fragment of ORF3659 could be specifically amplified in N.seriolae,and the hybrid snakehead fry suffering from recessive N.seriolae infection could be detected quickly,accurately and sensitively.The minimum detection limit of nucleic acid concentration of PCR detection was 80 pg/μL,the minimum detection limit of bacterial concentration is 8.6CFU/μL.2.In this study,the in vivo induced genes of N.seriolae were identified by IVIAT: the DNA of N.seriolae was extracted,the genomic expression library of N.seriolae was constructed in Escherichia coli,the serum of hybrid snakehead infected by N.seriolae was collected,then the serum was fully absorbed and treated with the whole cells and ultrasonic lysate of N.seriolae cultured in vitro and the ultrasonic lysate of E.coli without containing genomic library.Six in vivo inducible genes of N.seriolae were screened by immune hybridization: IS701 familytransposase,molybdopterin-dependent oxidoreductase,Yid C,phosphoketolase family protein,Egt A and hypothetical protein 6747.3.In this study,six in vivo induced genes were analyzed by bioinformatics,and six in vivo induced genes were inserted into eukaryotic expression vector pc DNA3.1-myc-his-A and prokaryotic expression vector p ET28 a,respectively.Six eukaryotic expression vectors and six prokaryotic expression vectors were successfully constructed,and six endotoxin free eukaryotic expression recombinant plasmids were extracted to prepare DNA vaccines of fish nocardiosis.4.In this study,six DNA vaccines were used to immunize hybrid snakehead for35 days.Serum,muscle,head,kidney,spleen and liver tissues were collected on day 7,14,21,28 and 35,respectively.On the 7th and 28 th day after immunization,the muscle,liver,spleen and kidney tissues of each experimental group were detected by RT-PCR,and it was found the fragment size of amplified products were consistent with the target bands by electrophoresis,indicating that the six in vivo induced genes were expressed in the hybrid snakehead.Four different serum enzyme activity detection kits were used to detect the nonspecific immune indexes in the serum of hybrid snakehead: LZM lysozyme activity index,SOD superoxide dismutase activity index,ACP acid phosphatase activity index and AKP alkaline phosphatase activity index.The results showed that using pc DNA group and PBS group as negative control,the four non-specific serum detection indexes in each experimental group increased significantly at different vaccine immunization time points,and reached the index detection peak at different time points,indicating that the six DNA vaccines can improve the non-specific immune indexes in the serum of hybrid snakehead.Indirect ELISA was used to detect the serum antibody titers of hybrid snakehead immunized with six DNA vaccines.The test results showed that comparing with the serum antibody titers of pc DNA group and PBS group,the antibody level in the experimental groups all increased significantly after 7 days post immunization,and reached the peak of antibody level detection at different vaccine immunization time points,which indicated that the six vaccines can caused the specific immune response in hybrid snakehead.Real-time fluorescence quantitative PCR(q RT-PCR)was used to detect m RNA expression levels of 6 immune related genes(TNFα、CD4、CD8α、IL-1β、MHCIIα and MHCIα)at 0,7,14,21,28 and 35 days after vaccination.The results showed that the m RNA expression levels of 6 immune related genes in the four tissues of hybrid snakehead increased extreme significantly and reached the peak at different time periods,respectively.The results showed that the relative percent survival(RPS)of the six DNA vaccines was shown as below.IS701 family transposase group: 45.06%;molybdopterin-dependent oxidoreductase group: 61.04%;Yid C group: 65.36%;phosphoketolase family protein group: 53.82%;Egt A group: 61.04%;hypothetical protein 6747 group: 43.73%.