坎氏弧菌热不稳定溶血素基因的克隆表达、蛋白纯化及其活性  

Expression,purification and characterization of Vibrio campbellii hemolysin (TLH)

作　　者：孙婧[1] 孙铂光[1] 贾爱荣[1] 张晓华[1] 

机构地区：[1]中国海洋大学海洋生命学院,山东青岛266003

出　　处：《中国水产科学》2010年第4期745-752,共8页Journal of Fishery Sciences of China

基　　金：国家自然科学基金委与香港研究资助局(NSFC-RGC)联合科研基金项目(30831160512);教育部科学技术研究重点项目(108082)

摘　　要：坎氏弧菌(Vibrio campbellii)是水产养殖动物的重要致病菌。本研究构建了坎氏弧菌热不稳定溶血素(TLH)基因的重组表达质粒pET26b(+)/tlh,并转入大肠杆菌BL21(DE3)中诱导表达带有6个组氨酸的TLH融合蛋白,然后利用Ni琼脂糖亲和层析柱进行纯化。SDS-PAGE分析显示,该溶血素能够大量表达,分子量约为42kD。纯化的TLH(0.41mg/mL)具有较强的溶血活性(溶血圈直径为16mm)及磷脂酶活性(晕圈直径为15mm)。其溶血活力的最适温度为37℃,在75℃下培养30min即丧失活力;最适pH为6,pH大于或小于6时都会导致其稳定性下降;一价金属离子如Na+、K+对溶血活性几乎没有影响,而部分二价金属离子如Ca2+、Co2+会导致溶血活性降低。本研究结果对阐明坎氏弧菌的致病机理及基因工程疫苗开发有重要意义。Vibrio campbellii,a Gram-negative,facultative anaerobic marine bacteria,which is an important pathogen in aquaculture. V. campbellii can cause the outbreak of the red leg-disease of shrimp（ Penaeus orientalis）,and it can cause abalone suffering from the pyosepticemia when the temperature is higher than 16℃ . However,the pathogenicity mechanism of V. campbellii is not clear. Previous studies showed that V. campbellii VIB285 contained a heat-labile hemolysin（ TLH） gene similar with the VHH hemolysin gene in V. harveyi. The deduced amino acids of TLH shared 85 % sequence identity to the VHH hemolysin of V. harveiy and it had a lipase domain（ Lipase_GDSL） at the 147- 406 amino acid,indicating that the TLH hemolysin may be a kind of phospholipase. In this study,the open reading frame of tlh（ 1254 bp） from V. campbellii VIB285 was amplified by using a specific primer set（ VCAF2 and VCAR2）, The forward primer（ VCAF2：5 ′ -CGGAATTCATGAATAAGACCATTACGTTACTTAGT -3 ′） begins from the initiation codon and adds an EcoRI site at the 5′ end of the gene,and the reverse primer（ VCAR2：5′ -CGCTCGAG GAATGGATGATTCGAAAGTTGGTC-3′） ends before the stop codon,and adds an XhoI site. The PCR product was excised and inserted into the EcoRI/XhoI-cut expression vector pET-26b（ ＋）. The ligated plasmid was transformed into E. coli BL21（ DE3） for expression of the full-length tlh gene. The recombinant TLH hemolysin was successfully expressed in E. coli strain BL21（ DE3） as His-tag fused protein,with the induction of IPTG at a final concentration of 1 mmol·L-1 and the incubation temperature of 37℃ for 3 h. The poly His-tagged TLH hemolysin was purified by Ni-NTA His-Bind Resin according to the manufacturer’s instructions. The molecular weight of the recombinant TLH hemolysin was about 42 kD as assessed by SDS-PAGE. Hemolytic activity against flounder（ Paralichthys olivaceus） erythrocytes was measured in 96-well microtiter plate,and the phospholipase activity was detected on 1%（ v/

关 键 词：坎氏弧菌 溶血素 TLH 表达 蛋白纯化 溶血活性 

分 类 号：S94[农业科学—水产养殖]