| The type III secretion needle-like injectisome from Vibrio alginolyticus or injectisome for short is a complex nanomachine that allows bacteria to deliver protein effectors to eukaryotic cellular membranes. The injectisome from the base located in the bacterial cytosol to the tip of the needle protruding from the host cell surface, which total across three layers of membranes.In this study, two important factors vscP gene and vscX gene of V. alginolyticus from injectisome were cloned.The results showed that vscP gene eneodes a putative protein of 401 amino acids with ORF of 1206 bp.The predicted molecular weight of VscP was 44.4kD with an estimated p I of 5.72. A model of VscP subunits was built and found its three-dimensional structure had a similar configuration with flagellar hook-length control protein FliK. The vscX gene eneoded a putative protein of 125 amino acids with ORF of 378 bp.The predicted molecular weight of VscX was 14.2kD with an estimated pI of 5.75. Searched protein-protein interaction of VscX and other proteins using STRING database and revealed the result of neighborhood relations between vscX and vscY, vopB, sycN.The T3S4 domain(amino acids residues 369 to 389) and Pfam domain(amino acids residues 1 to 125) was an important domain structure to VscP and VscX, respectively.Combining real-time fluorescent quantitative PCR with algorithm of 2- △ △ Ct to investigate the level of the mRNA expression of VscP and VscX respectively from the wild-type of V. alginolyticus strain HY9901, the vscO mutant and the vscO complement in different growth period. Results showed that the mRNA expression of VscP and VscX increased with the growth of V. alginolyticus strain HY9901, and it rase significantly(p < 0.01) in steady phase. The mRNA expression of VscP and VscX as well as increased significant(p < 0.01) in the late growth in vscO gene deletion, suggested that protein VscP and VscX also interacted with vscO.However, the high-level expression of VscP and VscX in the late growth due to the vscO mutant.VscP roles as antigen were used to be subunit vaccine to immunize Sillago sihama Forskál. pGEX-vscP of recombinant plasmid first was constructed and expressed in E.coli BL21(DE).Then the recombinant protein was gained in the optimal induced conditions of 0.4 mM IPTG at an OD600 of 0.4. The culture was incubated at 28℃ for 5 h. Last the recombinant protein was purified by affinity chromatography on glutathione resin.A RPS of 67.7% was in result of that Sillago sihama Forskál were immunized to against V. alginolyticus, while a RPS of 86.6% was challenaged by Vibrio harveyi. These results indicated that the antigen of VscP would be expected to become a good material to be the efficient subunit vaccine with cross protection.In the study, splenic immune protein of Sillago sihama Forskál immunized by the antigen of VscP, which analysed by proteomic.The immune proteins were separated and identified by application of technology of two-dimensional gel electrophoresis and mass spectrometry. Total protein spots were counted on the two-dimensional gel, 1316 spots in immune group, 1149 spots in the blank control group, 1291 spots in the PBS control group and 1316 spots in the PBS with adjuvant control group. The result revealed that 9 different proteins, 5 of which for the raising of protein expression and 4 for new proteins in the immune group compared with the control group.The study laid a foundation for further study on the immune protection mechanism. |
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