Vh-TAA Gene Cloning, Expression Analysis Andadhesion Active Research In Vibrio Harveyi

Vibrio harveyi belongs to Gram-negative bacterium, which is widely exist inmarine environment and is considered as a severe opportunistic pathogen that can infecta wide range of marine species, including both vertebrates and invertebrates. That canresult in heavy economic losses. Adhesion of bacteria to cell surfaces is the first step ininfections, in which adhesins play an important role at same time adhesins are alsotreated as a vital virulence factor of pathogens. A new kind of adhesins-trimetricautotransporter adhesins (TAAs) which derived from Gram-negative bacteria are found.These proteins have a characteristic arrangement of functional domains, including anN-terminal signal peptide, an internal passenger domain, and a C-terminal translocatordomain. The C-terminal translocator domain is the only domain that is homologousthroughout TAAs and, as such, defines this family. TAAs as an important virnlencefactor of gram-negative bacterium to the host cell interaction have become the focus ofresearch pathogen pathogenic mechanism.At present there is no reports about TAA in vibrio harvey, through the homologousalignmen we get a636bp ORF in vibrio harveyi by Gene walking, this ORF codes212amino acids. SMART software analysis found that the amino sequence has theremarkable structure characteristics of TAA, signal peptide analysis found that theamino sequence has a1-22aa signal peptide, Multiple-sequence alignment of theC-terminal translocation units found they all contain4β-barrel and a Coiled coil, finally,through daTAAanalysis found that the sequence of we get in vibrio harveyi is a TAAs.In order to understand the biological functions of the protein, through the rearchwe know that the passenger domain is the functional region of TAA,so we prokaryoticexpression the passenger domain which is predicitived in this study. We constructed aprokaryotic recombinant plasmid pRSET A-vhp and observed its expression in bacterialstrain BL21(DE3)plysS by IPTG induce, we got a22KDa soluble recombinant proteinand confirmed by Western Blot. Finally, We purified the recombinant protein useing the Ni-agarose gel,the polyclonal antibody of the protein was achieved after the injectioninto mice,after that determinedvthe antibodytiter is1:16000by indirect ELISA. Theadhesion activity experiment of the recombinant protein was performed byimmunofluorenscence double-staining, and the recombinant protein showed itsadhesivity to epithelial cells of Carp. The adhesion inhibit experiments on bothrecombinant protein and its antibody could restrain the vibro harveyi adhere to epithelialcells of Carp. Finally,we concluded that the Vh-TAA of Vibro harveyi has a celladhesion activity.For study of vh-taa gene expression of vibrio harveyi follow by environmentalfactors change, Apair of primers are designed by targeting the vh-taa gene,16SrDNAasan internal standard gene, using real-time quantitative PCR (Q-PCR) detect vh-taa geneexpression under different conditions. The results showed that gene vh-taa expressiondifference under different condition, Temperature change on the influence of vh-taagene’s expression is no significant difference (P﹥0.05); salinity change on theinfluence of vh-taa gene expression is significant (P﹥0.05), in the concentration ofNacl for40‰vh-taa gene’s expression is highest; iron ion concentration change on theinfluence of vh-taa gene’s expression is significant (P﹤0.05), in the iron ionconcentration for1umol/L vh-taa gene’s expression is the highest.A one step loop-mediated isothermal amplification (LAMP)assay was developedfor detection of vibrio harveyi. A set of four LAMP primers were designed by targetingthe rpoA gene and optimized the reaction, finally determinded the reaction at65°C for55min. The detection limit of V. carchariae culture by LAMP was2.4×102cfu/mL andthe resulrs showed that the LAMP was more sensitivity than PCR. Futhermore, thisLAMP method’s operation was simple and the result was discernible by eye. If joinfluorochrome SRYB Gold, positive reaction for green, negative control for shalloworange, the result was more convenient for observation. So this assay was expected tobecome a valuable tool for rapid detection and identification of vibrio harveyi.