Expression Of β-hemolysin Gene Of Staphylococcus Aureus And Its Hemolytic Activity

According to the published hlb gene sequence of Staphylococcus aureus RF122 from GenBank,using the software of Oligo6.0,a pair of primers were designed and synthesized.The genomic DNA of Staphylococcus aureus Shandong strain zfb were extracted and used as templates to amplify the hlb gene by PCR,then the products were sequenced and analyzed by bioinformatics.Sequence analysis showed that the Staphylococcus aureus Shandong strain zfbβ-hemolysin gene(hlb) was composed of 993 nucleotides encoding a mature polypeptide 330 amino acids.Compared to other Staphylococcus aureusβ-hemolysin gene(hlb) that reported shared the identity of 99.4%.It was showed that our Staphylococcus aureusβ-hemolysin was a Metal-dependent phospholipase by computer analyzing.Computer analyzing showed that our Staphylococcus aureusβ-hemolysin had two conserved domains which were Endonuclease/Exonuclease/phosphatase family and Metal-dependent hydrolase.This proved that theβ-hemolysin which we cloned was a Metal-dependent phospholipase. The phylogenetic tree showed that Staphylococcus aureus Shandong strain zfb had high homology with other Staphylococcus aureus,but we also should recognized there differences.pMD18-T/hlb and pET32a+ were digested by HindⅢand BamHⅠ,then linked,the hlb gene was oriented into pET32a+,and constructed the recombinant expression plasmid pET32a+/hlb,and then transformed into BL21(DE3).Induced the recombinant expression strain by IPTG,the product was fuse expressed as inclusions, SDS-PAGE results showed that recombinant proteins were expressed in inclusion bodies in Escherichia coli with molecular weight of 57 KD and the recombinant proteins accounted for 23.9%of the whole proteins.The expression product was purified by passing the Ni~+ affinity chromatograph Collumn using the recombinant protein with a tag of 6×His,The hemolytic activity was evaluated by 96 well microtiter V-plates using the purified recombination protein against sheep and bovine erythrocytes.And the purified protein hlb has 278 HU/mg to sheep erythrocytes and 9×10~3 HU/mg to bovine erythrocytes.Detected the CAMP reactions between the purified recombination protein and Streptococcus agalactiae, and the purified protein hlb can make obviously CAMP reaction with Streptococcus agalactiaeThe results showed thatβ-hemolysin was successfully cloned and well expressed in Escherichia coli.The purified recombination protein had good hemolytic activity,and its hemolytic activity to bovine erythrocytes is bigger than sheep erythrocytes,suggesting thatβ-hemolysin pathogenic,immune mechanism and mastitis vaccine design might be further researched.Also,a new method of specific diagnosis and discriminating Streptococcus agalactiae from mastitis cases pathogen using the recombinant proteins by CAMP reaction is established.