Detection Of Virulence Related Proteins MRP And EF Of Streptococcus Suis Type 2 And Their Cloning And Expression Of Genome Fragments

Detection of Virulence Related Proteins MRP and EF of Streptococcus suis type 2 and Cloning and Expression of their Genome Fragments Muraniinidase-released protein(MRP) and extracellular factor(EF) of Streptococcus suis type 2, being relative molecular mass 1 36kD and 11 OkD were considered to be virulence-related proteins. Cell envelope proteins and extracellular proteins of Jiangsu isolate HA980 1 of Streptococcus suis type 2 were extracted and separated on a SDS-polyacrylamide gel. The 136kD and I lOkD band were excised from gel and used as antigens for preparation of antibodies.Virulence-related proteins of 19 isolates, including 17 Chinese isolates, 1 German isolate of swine Streptococcus strains and 1 human isolate of Streptococcus suis type 2 were detected with immunoblotting and ELISA using the above antibodies. Bacterium cell envelope proteins and extracellular proteins were analyzed by SDS-PAGE and immunoblotting. The results showed that 11 strains produced MRP and 10 strains possessed EF or EF. They existed four phenotypes: MRP~EF~ (8/19), MRP~EF (1/19), MRP~EF (1/19), MRPEW (10/19) Dot-ELISA and indirect ELISA were established for detection MRP and EF, while reference strain of Streptococcus equi subsp. zooepidemicus ATCC35246 and isolate HA9801 were used as negtive control and positive control respectively. It showd that Dot-ELISA and indirect ELISA appeared correspondent results. Positive rates of MRP and EF both were 61%(11118). Indirect ELISA became deeper background and needed more time for coating antigen,having,but less antisera than that of Dot-ELISA,Comparing with immunoblotting, ELISA was more simple and sensitive, and could be widely applied on epidemiology. Structures of virulence-related proteins of Streptococcus suis Type 2 were predicted by SWISSMODEL, GOLDKEY and DNAstar software according to their amino acid sequences. Amino acid sequences of MRP and EF were compared to the database in Genebank by BLAST software respectively. The results showed MRP had 27梸33% homology with 10 proteins, but had no homology with group A Streptococcal M protein. According to the available messages, structure pattern 108 of MRP was mapped. EF had 48% homology with IgA 1 protease of Sfreptococcus pneumoniae. 304挆 1 168bp sequence of mrp gene and 12O7~?1675bp sequence of epf gene were amplified from genomic DNA of Streptococcus suis type 2 Jiangsu isolate HA9801 by polymerase chain reaction(PCR) tecbnique.PCR product was cloned into the expressing plasmid vector pGEX-6p-l via restriction endonuclease BamH I and Sal I .The recmbinant were verified by restriction endonuelease analysis and nucleotide sequencing. Then they were transformed into its host E.coli strain BL21. After induced by IPTG, the expression of 57kD fusion protein with glutathione S-transferase(GST) and 4lkD fusion protein with GST was confirmed by SDS-PAGE.The expressed fusion protein was recognized by MRP antiserum and showed that it had the expective epitopes. The expressed 4lkD fusion protein could not be recognized by EF antiserum, and there were no EF epitopes in the peptide encoded by 120T?1675bp.