Identification Of Thermolysin Gene In Flavobacterium Columnare

Columnaris is a world-wide bacterial fish disease, the bacterial pathogen, Flavobacterium columnare belongs to the family Flavobacteriaceae. The infection of the bacterium may cause severe gill-rotting and sometimes skin ulceration. Bacterial gill-rot disease is epidemic in China, and a major disease in several major cultured fish species in China, including the grass carp Ctenopharyngodoe idellus and the mandarin fish Siniperca chuatsi. Proteases of F. columnare, together with its adherence factors and chondroitin AC lyase, have been reported as virulence factors. Outer membrane components of bacteria are considered as an interactional interface between bacteria and its extracellular environment, and outer membrane proteases have been shown bo be important immungens of Gram-negative bacteria in fish. The present study, by using bacteria isolated from outbreaks of columnaris disease in China as the pathogen, aimed to identify genes of possible virulence factors of the bacteria and examin the functions of some of these identified genes.In order to identify genes encoding the outer membrane proteins (OMPs) of F. columnare G4, the expression library of the bacterium was screened by using rabbit antisera developed against its OMPs. Positive colonies of Escherichia coli M15 containing fragments encoding the bacterial OMPs were selected for cloning the relevant genes by genomic walking methods. The full sequence of thermolysin,(TLN) gene was gained from the expression library. Its open reading frame had 2709 nucleotides which encoded a 902 amino acid peptide. The comparison of the deduced amino acid sequence of TLN suggested two conservative motifs of zinc metalloproteinase HExxH and GxxNExxSD. The expression primers were designed to clone the catalytic domain and helix domain of TLN and incorporated additional new restriction sites. The DNA products were inserted into vector pET-32a. After the recombinant plasmids were identified by sequencing, they were transformated in Rosetta-gami (DE3) for chemical inducing with IPTG.. SDS-PAGE analysis suggested the refusion protein molecular weight was about 58 kD in the form of inclusion bodies. Fusion protein was purified by Ni-NTA affinity chromatography, which was injected hypodermically into rabbit to get antisera. Western blotting analyses with anti-6 His monoclonal antibody and rabbit anti-TLN-C sera suggested those antibodies could recognize the refusion protein (58 kD) , Western blotting analyses with rabbit anti-TLN-C sera suggested extracellular proteins recognized a peptide of 42 kD and outer membrance proteins recognized a peptide of 35 kD.