Differentially Expressed Sequences Analysis Of Wild Type Strain And ?tssJ And Preparation Of The Polyclonal Antibody Against Its TssJ Of Vibrio Alginolyticus

Vibrio alginolyticus, one of the most common bacterial causative agents of thediseases in aquatic animals, caused huge economic losses in the world. Recentstudies found that the type VI secretion system, T6SS, had been associated withbiofilm formation, motility, cytotoxicity and survival in phagocytic cells, and itincluded 13 subunits. Among them, TssJ, an outer membrane lipoprotein that wasanchored to the outer membrane through the acylation of its N-terminal cysteineresidue, played an important role in T6SS assembly in Escherichia coli. Up to date,no more information was available about the function of the TssJ in V. algoionlyticus. In this study, the V. algoionlyticus HN08155 (wild strain, WT) was isolated fromdiseased fish in Hainan. RNA-Seq was performed to study the transcriptome of theinitial stage of biofilm formation between wild strain and ?tssJ. After assemblingand annotating, we obtained 3600 Unigenes. Then we compared the gene expressionof these two strains, the results indicated that a total of 180 genes or transcripts weredifferentially expressed (84 up-regulated,96 down-regulated). These differentiallyexpressed genes were closely related to the formation of cell velum, ABC transportprotein, iron carrier conjugated protein, amino acid metabolism, membrane proteinand some hypothetical protein. Among them, the amount of expression of the genesin association with the membrane protein, the flagellin and the glycosyltransferasewere significantly declined in the mutant. Additionally, in this study we alsoanalysised GO, COG and KEGG about the differential expression genes. The resultsindicated that these genes enriched to 26 subclasses in GO, and the genes involved inmolecular function and biological process accounted for high proportion; andenriched 19 classifications in COG, among them, the genes which involved in aminoacid transport and metabolism account for high proportion; and these genes enrichedin 5 pathyway types. To further understand the function of TssJ in V. alginolyticus and the relationshipbetween tssJ and T6SS assembly, tssJ was amplified by PCR from V. alginolyticusHN08155 and cloned into PET-32a(+) vector. The recombinant plasmid PET32a-tssJwas transformed into E. coli BL21(DE3) for expression under induction with IPTG.The optimized induction condition in E. coli was accomplished and the condition wasthat the strain was inducted at 37? for 8h, with lmmol/L IPTG in LB medium. Theexpressed product was analyzed by SDS-PAGE, purified by Ni column. Themolecular weight of the purified protein was identified by SDS-PAGE, indicating that the interest protein with high purity was obtained. The acquired protein was employed as antigen to immunize SPF rats by three separate immunizations and ultimately the polyclonal antibody against TssJ was achieved. The antibody titer was validated by ELISA was 1:32000.