Adhesion Function Of Streptococcus Equi Subsp.zooepidemicus M-like Pretein To Cell

Streptococcus equi subsp. zooepidemicus made substantial economic losses for pig industry and became a substantial threat to human health especially those involved in the pig industry .M-like protein is a major surface protein and virulent factor of Streptococcus equi subsp. zooepidemicus. In the present study, at the molecular levels we investigated the adhensive machanism of M-like protein in order to get new insight into the pathogenic mechanism of the virulent factors and prevention methods against these streptococci.The M-like protein gene without signal peptide was amplified from genomicus DNA of Streptococcus equi subsp. zooepidemicus ATCC35246 strain by polymerase chain rection (PCR),then the amplified fragment was cloned in the proper orientation into the site between EcoR I and Xho I of pET-32a (+) via restriction endonuclease EcoR I and Xho I . The recombinant plasmid was verified by nucleotide sequencing and transformed into E. coli BL21 .An fusion protein was expressed in BL21 affer induced by IPTG, SDS-PAGE and Western bloting analysis showed that the recombinant protein had a molecular weight of 60 kD, and a positive reaction with the antiserum against ATCC35246.In this study, hybridoma cell lives secreting McAbs against M-like protein of Streptococcus equi subsp. zooepidemicus were generated, these McAbs would be for the resesarch of pathogenetic mechanism of streptococcus to prepare the McAbs against M-like protein 6~8weeks old BABL/c mice were immuniszed endermicly with purified recombinant M-like protein. Splenocytes from the immuniszed mice were fused with SP2/0 and indirect ELISA was used to secreen hybridoma cells. 12 hybridoma cell lines specific antibodies against the M-like protein of streptococcus were established. Indirect ELISA confirmed that these McAbs only reacted with M-like protein, but not reacted with streptococcus. The indirect ELISA titers of these 12 ascites McAbs were about 1:10000.HEp-2 cells, a kind of typical cell model, was used in the adhesion and adhesive inhibition experiments to evaluate the adhesion of M-like protein from Streptococcus equi subsp. zooepidemicus strain ATCC35246. The adhesion results showed that ATCC35246...