Cloning, Expression And Characterization Analysis Of The Arginine Deiminase Of Streptococcus Suis Type 2

PCR analysis demonstrated the presence of the arginine deiminase gene (ad) in all 29 Streptococcus suis (S. suis, SS) strains tested, but none of the seven S. equi subsp. zooepidemicus strains. The ad of HA9801, ZY05719 and T15 were sequenced and analysised by BLAST and DNAStar. It revealed that the ad of the above three strains were almost the same, and they had 99% and 89% identiy with ad of SS2 I9841/1 and sagp of S. pyogenes, respectively. The ad was ubiquitous in Streptococcus suis.The fragment of ad amplified from virulent isolate HA9801 was later cloned into pBAD/Myc-HisC vector via restriction endonuclease and then transformed into host strain TOP10. A recombinant protein of 47000 was highly expressed after induced by L-ararose and purified by Ni-nitrilotriacetic acid affinity chromatography. Western blotting demonstrated that the recombinant protein can react to the polyclonal antibody raised against whole-cell protein of SS2-HA9801, which suggested that it possessed some immunogenicity and may be important for further research. Enzymatic assay revealed that the optimum temperature for its activity is 37℃and pH is 6.5. Studies with class-specific inhibitors supported the assignment of a Sulfhydryl enzyme with some metallo class characteristics.Bacteria has to display rapid responses to survive when encountering an array of adverse environmental conditions such as changes of temperature, oxygen pressure, and pH, as well as limitations in nutrients or iron. Presumably, these responses involve the expression of proteins induced when the respective signals are sensed. It is quite likely that this capacity is a virulence-associated feature. Based on this assumption we attempted to identify proteins of SS2-HA9801 which were expressed or up-regulated in response to a shift in temperature, a stress signal to which the pathogen was exposed when entering deeper tissues and the bloodstream. The membrane protein expressed at 37℃and 42℃were prepared and were separated by two dimensional gel electrophoresis, respectively. Our findings presented here demonstrated that S. suis responds to changes in growth temperature from 37℃to 42℃with a change in the expression of several proteins among which one was AD. Western blot showed that it was up-regulated when temperature rose, which was coincident with other references. These data would further facilitate the research of SS2 escaping from the immune system of the host.