Cloning And Biological Function Of Vibrio Alginolyticus HY9901 Type ? Secretion System Effector Protein Va1686

In this study,va1686 gene was cloned from Vibrio alginolyticus HY9901.The total length of its gene is 1164 bp,and it could encode 387 amino acids.The physicochemical properties,protein structure,genetic evolutionary relationship and antigenic characteristics of the effector protein Va1686 of V.alginolyticus HY9901 type ? Secretion System were studied and analyzed by bioinformatics methods and tools.The results showed that Va1686 was a stable hydrophilic and acidic protein without a transmembrane region and a signal peptide,and secondary structure to a-helix.The evolutionary analysis showed that V.alginolyticus HY9901 and Vibrio harveyi were clustered together,which indicated that the genetic relationship between the two species was closest.Va1686 contains a Fic superfamily conserved domain associated with cell division.Bioinformatics analysis showed that the B-cell preponderant epitopes of Va1686 might be localized in the regions of 48?49?82?85?125?126?150?153?185?186?236?237 and so on.The 3D structure model of Va1686 subunit was simulated by SWISS-MODEL software and it was found that the vopS of Vibrioparahaemolyticus were similar and the similarity was 89.46%.In this study,the feasibility of Va1686 as a common antigen of Vibrio was verified from the perspective of bioinformatics,which laid the foundation for the next step in vaccine development.At the same time,Overlap PCR and two homologous recombination techniques were used to knock out T3SS effector gene va1686 from V.alginolyticus HY9901.Using ?2163 and suicide plasmid pLP12,combined with positive and negative screening methods,we succeeded in obtaining the mutant and complement strain.Did a biological phenotype analysis.There was no difference in heritability,growth,biofilm formation,and ECPase activity.In the median lethal dose test of the grouper fish,it was found that the loss of the effector protein Va1686 reduced the virulence of V.alginolyticus HY9901 by about two orders of magnitude.The results showed that the ?va1686 mutant have potential value of an effective live vaccine and be used for resistance against V.alginolyticus in grouper fish farming.It laid the foundation for the future research on the function and pathogenicity of the Va1686 protein.In this study,the grass carp kidney cells(CIK)infected with V.alginolyticus strain HY9901,?va1686 and C-?va1686 at different time points under fluorescence microscopy.The positive control changes similarly,and the cytoskeleton is gradually destroyed and the skeleton collapses until disappears in the field of vision.The caspase-3 enzyme in CIK cells was activated by HY9901 strain,but was not changed significantly in mutant strain,confirming that V.alginolyticus effector protein Va1686 can rapidly induce apoptosis in fish cells.Infection of the HY9901 strain also results in the release of the contents of infected fish cells,such as the release of LDH.In conclusion,these data confirm that V.alginolyticus HY9901 infection of fish cells can result in the abnormal death of fish cells via effector protein Va1686.This study provides important theoretical insights into the mechanisms by which Vibrio cause host cell death.