Expression and immune characteristics analysis of TcpA protein of Vibrio cholerae

Abstract

Vibrio cholerae is a causative agent of mucous-sloughing disease and bacterial enteritis in fish,with high morbidity and mortality.Toxin-coregulated pilus(TCP) is a major virulence factor of V.cholerae.As a major structural protein of TCP,toxin-coregulated pilin A(TcpA) is potential candidate for diagnostic antigen and vac-cine development.In this study,the tcpA gene was amplified by PCR from genomic DNA of the Y1 strain of Vi-brio cholerae isolated from diseased grass carp(Ctenopharyngodon idellus).The tcpA gene was cloned into a pMD18-T vector and sequenced.The tcpA gene fragment containing the open reading frame(ORF) was then sub-cloned into pGEX-4T-1 to construct expression plasmid pGEX-4T-1-tcpA.The recombinant fusion protein(rGST-TcpA) was expressed by IPTG induction in E.coli for subsequent immunological characterization.Se-quence analysis revealed that the ORF of the tcpA gene from the Y1 strain(GenBank accession no.EU649677) is 657 bp and encodes a protein of 224 amino acids.The gene is 99.6%–99.7% identical at the nucleotide level and 98.7%–99.1% identical at the protein level to seven tcpA sequences in GenBank,which suggested that the TcpA protein is considerably conserved.SDS-PAGE analysis showed that the 47.0 kD rGST-TcpA fusion protein was mainly expressed in inclusion bodies.Western blotting demonstrated that rGST-TcpA could react specifically with mouse antisera raised against the pilus protein of strain Y1.The purified rGST-TcpA was used to immunize grass carp(Ctenopharyngodon idellus).The titer of the antisera was 1:16,as determined by a double immunodiffusion test,and it could significantly inhibit adherence of Y1 strain to HEp-2 cells in vitro.A relative percentage survival(RPS) of 73.33% was found in immunized grass carp(Ctenopharyngodon idellus) suffering from Y1 strain chal-lenge at day 25 after immunization.This study indicated that the rTcpA protein possess the same immunogenicity,immunoreactivity,and protective efficacy as the natural pilus protein of V.cholerae.