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

    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.

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罗鹏,Hu Chaoqun. Cloning haemolysin gene of Vibrio alginolyticus through reverse PCR and expression of the gene in prokaryotic cell[J]. Journal of Fisheries of China,2009,33(3):410~416

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History
  • Received:March 15,2008
  • Revised:April 13,2008
  • Adopted:September 18,2008
  • Online: May 07,2009
  • Published: