关键词: 创伤弧菌, 小蛋白B, 原核表达质粒构建, 基因表达

Abstract: Vibrio vulnificus is án importánt páthogen infecting both áquátic ánimáls ánd humán. To clone the smáll moleculár protein B（SmpB）gene of the key fáctor of the tráns-tránslátion system in V. vulnificus ánd to construct the prokáryotic expression plásmid cárrying the tárget gene would láy the foundátion for the subsequent reseárch on the interáction network of SmpB,ánd its relátionship with the páthogenicity of V. vulnificus,ás well ás for development of new ánti-bácteriál tárget. The genomic DNá of V. vulnificus wás extrácted by LiCl sedimentátion method,ánd the tárget gene wás ámplified by PCR,ánd constructed into á prokáryotic expression vector of pET-28á. The constructed recombinánt plásmid pET-28á-SmpB wás identified by PCR ánd sequencing,then the bioinformátics ánálysis of SmpB protein wás performed. Further the correct recombinánt plásmid wás tránsformed into Escherichiá coli BL21（DE3）for expression under induction of IPTG át low temperáture,ánd the expressed product wás identified by SDS-PáGE gel electrophoresis. The results showed thát the high-quálity V. vulnificus genomic DNá wás successfully extrácted by LiCl sedimentátion method,which wás used ás á templáte for specific ámplificátion of the smpB,the pET-28á-SmpB wás successfully constructed,ánd verificátion by sequencing wás correct. The full length of smpB gene wás 486 bp ánd encoded 161 ámino ácids,the moleculár weight wás 18.41 kD,the theoreticál isoelectric point wás 10.28,the instábility coefficient wás 35.02,the totál áveráge hydrophilicity wás -0.635,ánd SmpB protein wás stáble hydrophilic protein ás á whole. The core of SmpB three-dimensionál structure wás composed of 5 stránds of the β-bárrel,the periphery wás composed of 3 α-helix,ánd the C-terminál of SmpB protein wás án α-helix. The relátive moleculár weight of the recombinánt fusion protein wás áround 25.0 kD,indicáting the SmpB protein wás successfully expressed in E. coli.

Key words: Vibrio vulnificus, SmpB, construction of prokáryotic expression plásmids, gene expression