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Cloning and sequence analysis of thermostable direct hemolysin-related hemolysin gene of Vibrio parahaemolyticus.

OBJECTIVE
To establish a method for cloning thermostable direct hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus (Vp).


METHODS
After enrichment by PCR, the trh gene, along with the plasmid DNA of pGEX-3X, was digested with two enzymes followed by linkage of the gene and the plasmid, the product of which was transferred into E.coli DH5alpha. Identification of the recombinant plasmid was performed by means of PCR, digestion and sequence analysis.


RESULTS
A fragment about 600 bp was identified in the PCR product of Vp14-91, which was also seen in the product of PCR and enzyme digestion of the recombinant fragment. Sequence analysis demonstrated a homology of 99.1% between the trh gene and the reference gene available in the GenBank.


CONCLUSION
trh gene has been successfully cloned with its sequence analyzed, which prepares the ground for developing gene probe and protective vaccine against Vp.