Eukaryotic Expression Of β-hemolysin Of Staphylococcus Aureus And The Evaluation Of Its Immune Protection Effect

Bovine mastitis is a very common disease that causes mammary gland inflammation. It may cause great economic losses to the dairy industry and even endanger the public food safety.We selected 33 Staphylococcus aureus isolated from bovine mastitis as our study objects. To investigate the hemolysin genotype and phenotype distribution and the corresponding relation between them, hemolysin genes were detected by PCR method, and the hemolysin phenotype was oberserved with the method of bovine blood plate and sheep blood plate.The positive rate of the genes encoding hla,hlb,hld,hlg,hlg-2 were 90.91%,100%,93.94%,78.79% and 66.67% respectively. While the isolates mainly presented β-hemolysis in both bovine and sheep blood agar plates,more isolates expressed hlb phenotypically in bovine blood,which indicated that bovine blood was more sensitive to hlb than sheep blood. There is no corresponding relationship between the hemolysin phenotype and the genetype.hlb gene was inserted into pc DNA3.1 His B Vector,and the recombinant plasmid was renamed as pc DNA3.1 His B-hlb. The recombinant eukaryotic expression vector was transfected into MCF-7 cells by liposome method and the positive cells were obtained by the selection of G418 conditioned culture.Using PCR and Western blot method,we found that vetor pc DNA3.1 His B-hlb had been transfected into MCF-7 successfully and the positive cells could express hlb correctly.The expression products that contained recombinant hlb were purified through a Ni-NTA affinity chromatography column,and then,the hemolysis test and hemolytic activity of purified hlb to bovine erythrocyte and sheep erythrocyte were determinated.SDS-PAGE suggested that the purificated product showed a single band with the molecular weight of about 41.8KDa, and its specific activity were 1658HU/mg,and 829Hu/mg to bovine erythrocyte and sheep sheep erythrocyte respectively.And lastly, Using the mixture of the purified hlb and Freund’s adjuvant,and the mixture of pc DNA3.1 His B-hlb and Freund’s adjuvant, mice were immunized subcutaneously on the back and mucosally in the nasal.Ten days after the third immunity, IL-1、IL-2、IL-4、Ig G、Sig A、IFN-γ and Sig A were determinated by ELISA method and the attack experiment was carried by Staphylococcus aureus.It turned out that the concentration of IL-2 and Ig G between experimental groups and contral group had significant difference,the immune effect was good and these two vaccines both had some immune protection.