霍乱弧菌TcpA蛋白的表达及其免疫活性分析

Expression and immune characteristics analysis of TcpA protein of Vibrio cholerae

采用PCR方法从鱼源霍乱弧菌Y1株(Vibrio cholerae Y1)扩增毒素共调菌毛蛋白A(toxin-coregulated pilin A,TcpA)基因并克隆至pMD18-T载体。序列分析显示tcpA基因ORF长675 bp,编码224个氨基酸(GenBank登录号EU649677)。同源性比对发现,该基因与GenBank中登录的7个霍乱弧菌参考株相应基因序列的核苷酸同源性和氨基酸同源性均很高,分别为99.6%～99.7%和98.7%～99.1%,表明TcpA蛋白相当保守。将tcpA基因亚克隆到表达载体pGEX-4T-1,并进行IPTG诱导表达,SDS-PAGE分析发现分子量约为47.0 kD的重组TcpA融合蛋白主要以包涵体形式表达。Western blot检测结果显示,重组TcpA融合蛋白可与鼠抗Vc Y1菌株菌毛蛋白抗血清发生特异性结合反应。用纯化的重组TcpA融合蛋白免疫草鱼(Ctenopharyngodon idellus)制备抗血清,双相免疫扩散试验检测抗体效价达1∶16,且该抗血清能够明显抑制Vc Y1菌株对HEp-2细胞的黏附。草鱼免疫后第25天用Vc Y1菌株攻毒,结果相对免疫保护率达到73.33%。研究结果表明,重组TcpA蛋白仍保留着天然菌毛蛋白的免疫原性、免疫反应性和免疫保护性,重组TcpA蛋白可作为霍乱弧菌的候选诊断抗原和疫苗抗原。

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.