Purification, Chemical Modification, Gene Detection And Sequence Analysis Of Hemolysin Of Streptococcus Suis Type 2 Strain Isolated In Jiangsu Province

Streptcoccus suis(S.suis) type 2 is an important swine pathogen and produces varieties of virulence factors.Hemolysin of S.suis type 2 had been identified as a major virulence factor and a good protective antigen. In the present paper,purification,chemical modification of hemolysin and the gene encoding hemolysin was detected,sequenced and analysed.The hemolysin of Streptococcus suis type 2 strain HA9801 isolated in Jiangsu province was purified from the culture supernatant,by using different methods,including selective (NH4)SO4 precipitation,desalting,anion-exchange chromatography,and sephaceryl chromatography,and showed the activity of 128,2048,4096,1024 hemolytic units respectively.The specific activity of the purified hemolysin was enhanced 200 folds.The purified hemolysin appeared a single band with molecular mass of 54,OOODa by SDS-PAGE.It sugguested the purified hemolysin from Jiangsu isolate was analogue of suilysin purified from U.S.A. isolate previously reported.Effects of protein modification reagents on the Streptococcus suis type 2 hemoysin activity were detected. The hemolysin was not affected by PCMB, succinic anhydride( SA), EDC, and N-AI modification, and it indicated that sulfhudryl groups, Amino groups, Carboxyl groups and tyrosine residues were not essential to hemolysin activity. The hemolysin activity was significantly decreased after NBS, DEPC, 2,3-Diacetyl, H2O2 modification and was greatly increased after DTT modification.The results indicated that tryptophane residues, histidine residues, arginine residues and disulfides groups seemed to be essential to the hemolysin activity.A hemolysin gene from six Streptcoccus suis type 2 strains isolated in Jiangsu was amplified by PCR using a pair of specific primers.The PCR product from strain HA9801 was sequenced and nucleotide sequence analysis revealed the hemolysin gene from strain HA9801 had a high homology to strains 1933(99%).