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/v） egg yolk emulsion plates. The purified TLH（ 0.41 mg/mL） showed strong hemolytic activity against flounder erythrocyte （ the diameter of hemolysis circle on fish blood agar plate was 16 mm） and phospholipase activity（ the diameter of the clear ring on 1% egg yolk emulsion plate was 15 mm）. The optimum temperature for the hemolytic activity of the TLH was 37℃,and the hemolytic activity was destroyed entirely by treatment for 30 min at 75℃,indicating it is a heat-labile hemolysin. The optimum pH for the hemolytic activity of the TLH hemolysin was pH6. The effect of cations on hemolysis was determined by addition of various concentrations of metallic cations（ Na＋,K＋,Mg2＋, Ca2＋,Mn2＋,Co2＋,Ni2＋,Cu2＋,Zn2＋ and Ba2＋） as the chloride forms in 20 mmol/L TBS（ pH 7.5）. It was found that only a few divalent cations can cause the decrease of the hemolytic activity,such as Ca2＋ and Co2＋,but monavalent cations and other divalent cations did not affect the hemolytic activity. To our knowledge,this is the first report on purification and characterization of TLH from V. campbellii. Purified TLH could be useful for vaccine development and a diagnostic tool for vibriosis.
出处
《中国水产科学》
CAS
CSCD
北大核心
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)
关键词
坎氏弧菌
溶血素
TLH
表达
蛋白纯化
溶血活性
Vibrio campbellii
hemolysin
TLH
expression
protein purification
hemolytic activity
