溶藻弧菌溶血素基因反向PCR克隆及其原核表达  

Cloning haemolysin gene of Vibrio alginolyticus through reverse PCR and expression of the gene in prokaryotic cell

作　　者：罗鹏[1] 胡超群[1] 

机构地区：[1]中国科学院南海海洋研究所海洋生物资源可持续利用重点实验室,广东省应用海洋生物学重点实验室,广东广州510301

出　　处：《水产学报》2009年第3期410-416,共7页Journal of Fisheries of China

基　　金：国家“九七三”计划项目(2006CB101803);国家自然科学基金(30700016)

摘　　要：溶藻弧菌的胞外产物(ECP)在其致病过程中发挥重要作用,已经观察到溶藻弧菌ECP所致的溶血现象,关于溶藻弧菌溶血素的种类和其对溶藻弧菌的致病性贡献的报道非常稀少。利用已经报道的多种弧菌溶血素基因序列设计通用引物,检测溶血素基因在溶藻弧菌中的分布,发现96个菌株中有74株扩增出溶血素基因(vah)片段。对致病株ZJ0451的vah片段测序。根据已测得的片段序列设计引物,通过反向PCR扩增及后续克隆测序获得全长vah基因及部分侧翼序列。经比对证实溶藻弧菌vah与多种弧菌的TLH类溶血素高度相似,且其肽链N端有信号肽。利用pET32a载体构建了两种包含不同长度融合标签的vah表达载体,并均在大肠杆菌BL21(DE3)中获得可溶性表达。在26℃的诱导温度下,9 h时vah表达量达到相对最大。在原核表达系统中,vah蛋白信号肽可以被切除。Extracellular products （ECP） play an important role in the pathogenicity of Vibrio alginolyticus. In some studies, it has been observed that ECP of V. alginolyticus could cause haemolysis. However, there are few reports regarding the types of haemolysins from V. alginolyticus and the contribution of the haemolysins to the pathogenicity of V. alginolyticus. In the present study, based on reported haemolysin gene sequences from Vibrio, universal primers were designed and used for detecting the distribution of haemolysin gene among V. alginolyticus strains. 74 out of 96 V. alginolyticus strains produced amplification segments of haemolysin gene （vah）. vah segment from pathogenic strain ZJ0451 was sequenced and the primers for reverse PCR were designed based on obtained sequence. Through reverse PCR, and the following gene cloning and sequencing, complete vah gene and flanking sequences were obtained. By blast, it was confirmed that the vah from V. alginolyticus has high similarity with TLH haemolysin gene from various vibrios. The predicted peptide strand has a signal peptide at its Nend. With pET32a vector, vah expression vectors were constructed, containing fusion tags with different lengths. They were successfully expressed in Escherichia coli Bl21 （DE3）. At 26 ℃, induced proteins reached the relatively highest amount. vah expressed protein has a signal peptide and it can be cut out in the prokaryotic expression system.

关 键 词：溶藻弧菌 溶血素 反向PCR 表达 

分 类 号：Q786[生物学—分子生物学] S917[农业科学—水产科学]