Diferential Transcriptome Analysis Of Intestinal Tissues Of Grass Carps Before And After Immunization With Vaccine And Construction And Expression Of The Recombinant Plasmid Carrying Key Gene

Vibrio mimicus(V.mimicus)is a common pathogen causing serious ascites disease in fish,which lead to large economic losses to aquaculture industry.In order to effectively prevent and control ascites disease in fish,a double targeted nucleic acid vaccine of V.mimicus was constructed in our laboratory,and confirmed that the vaccine had a good enhancement efifect of intestinal mucosal immune by animal immune test.However,the exact molecular regulatory mechanism is still unclear and needs further study.High-throughput RNA sequence technology is the most mature and widely used transcriptome research method.This technology has the advantages of high throughput and accuracy.It can directly conduct comprehensive transeriptome analysis of species with or without genome or gene information,and detect rare transcripts with low abundance.It also provides an effective method for the study of mucosal immune regulation mechanism in fish.In this regard,based on the preparation of double targeted nucleic acid vaccine of V.mimicus,the grass carps were immunized via oral gavage,and differential transcriptomics analysis of intestine tissues from grass carps before and after immunization was carried out by using RNA-seq technique in order to identify the key immune-related differentially expression genes(DEGs)and their involved pathways and construct their expression vector.The primary research work and results were summarized as follows:1)Preparation and Immunization of double targeted nucleic acid vaccine of V.mimicus,and sample collection.In this dissertation,double targeted nucleic acid vaccine of V.mimicus was prepared according to the method established by Ji Cao in our laboratory.First,endogenous targeted nucleic acid vaccine of V.mimicus(pcDNA3.1-Iclp-Ovepis)was extracted in large quantities.Then,the lyophilized DH5a-BG was prepared.Finally,vaccine plasmid pcDNA3.1-Iclp-ovepis was loaded into lyophilized DH5a-BG to form double targeted nucleic acid vaccine of V.mimicus.On this basis,the experimental grass carp was immunized twice via oral gavage.Nine fishes were randomly selected from the vaccine-immunized group and PBS control group respectively on the 21st day after immunization.The posterior intestinal segments were collected and three fish intestinal segments were mixed into one sample.Three biological repeats were obtained in each experimental group.2)RNA-seq of intestinal tissues of grass carps before and after immunization and their bioinformatics analysis.Total RNA of intestinal tissues was extracted used by TRNzol-A+reagent,the cDNA library was constructed.The cDNA library was sequenced with pair-end strategy.After filtering the raw reads,the expression levels of genes and transcripts were calculated by RSEM software.Differentially expression genes were screened by Fold Change>2.0、False Discovery Rate ≤0.001 and p<0.05 as standard.The result showed that 5 334 DEGs were identified,of which 1 275 were up-regulated,4 059 were down-regulated and 1 471 were immune-related DEGs.The bioinformatics of DEGs was analyzed by GO,KEGG and STRING databases,DEGs were involved in 17 cell components,14 molecular functions and 27 biological processes.Among 5 334 DEGs,3 479 DEGs were significantly enriched in 53 KEGG Pathways,including 10 immune-related pathways.TABl、TAK1、p38MAPK、NF-κB、BCR、CTS、Iclp、Hsp70、MR、Vtype ATPase and TGFβR genes were central node genes in the interaction network of immune-related DEGs.3)Verification of differentially expressed genes by qRT-PCR.30 immune-related DEGs were randomly selected for qRT-PCR validation.On the basis of establishing melting curve,amplification curve and standard curve of each target gene,qRT-PCR was used to detect the relative expression of each gene with appropriate diluted sample as initial template concentration,the relative expression of each gene was analyzed by 2-ΔΔCt method.The results showed that the relative expression levels of the differentially expressed genes were significantly different between the vaccine-immunized group and the PBS control group.The trend of qRT-PCR results was consistent with that of transcriptome sequencing.The results showed that the data of transcriptome sequencing were accurate.4)Construction and expression of the vector carrying DEGs TAB1.A pair of specific primers were designed to amplify gcTAB1 gene by PCR using the intestinal tissue cDNA after vaccination.The correctly sequenced gcTAB1 gene was subcloned into the prokaryotic expression plasmid pGEX-6p-1,and then transformed into BL21(DE3)for induced expression.The results showed that the recombinant plasmid pGEX-6p-1-gcTABl was successfully constructed.After induced expression,rgcTAB1 protein was mainly expressed in the form of inclusion bodies.In conclusion,5 334 DEGs,including 1 471 immune-related DEGs,were identified from the intestinal tissues of grass carp post-immunization.932 out of immune-related DEGs were significantly enriched to 10 immune-related pathways,such as phagosome,antigen processing and presentation and B-cell receptor signaling pathway etc.Meanwhile,the expression vector of the key immune-related gene TAB1 was successfully constructed.