| Streptococcus equi subsp.zooepidemicus and Streptococcus suis type 2 are worldwide zoonotic phathogens of many different diseases such as meningitis, septicemia, arthritis, endocarditis and may cause sudden death in pig or human, which made substantial economic losses for pig industry and became a substantial threat to human health especially those involved in the pig industry. The M-like protein of Streptococcus equi subsp.zooepidemicus and muramidase released protein (MRP) of Streptococcus suis type 2 are important virulence factors and protective antigens. In the present study, at the molecular levels we investigated the virulence factors in order to get new insight into the pathogenic mechanism of the virulence factors and prevention methods against these streptococci.1 Cloning and sequence analysis of M-like gene of Streptococcus equi subsp.zooepidemicus ATCC35246 strain and the detection of M-like gene of streptococci from pigFirst of all, the M-like gene was amplified from genomic DNA of S. zooepidemicus ATCC35246 strain by polymerase chain reaction (PCR). Then the amplified fragment was cloned in the proper orientation into the site between BamH I and Sac I of pET32-a(+) via restriction endonuclease BamH I and Sac I . The recombinant plasmid was verified by restriction endonuclease analysis and nucleotide sequencing. Nucleotide sequencing analysis revealed that there is one open reading frame of 1137 bp in M-like gene of strain ATCC35246, which encodes a protein of 379 amino acids. The accession number of this sequence in GenBank is AY263781. Compared with the other two sequence published in GenBank, it is showed that the homology of nucleotide acids between the M-like gene andthe other two was29.4% - 86.9%,and that of predicted amino acids was 23.4% -84.3%. In the other hand, the predicted amino acid structure of M-like of strain ATCC35246 showed significant homology with the N-terminal signal peptide and the carboxy terminal of group A and C M-like protein .The M-like protein, although showing an extensive region of alpha helix, lacks the A ,B and C repeats found in group A M protein. A proline-rich region upstream from the LPSTGE motif contains 11 repeats of the tetrapeptide PEPK. The presence of these repeats may account for the slow migration of the M-like protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The M-like gene was detected by polymerase chain reaction in 34 strains streptococci of pig origin ,and it is found that the M-like gene could be amplified in all group C streptococci of pig origin , but not in Streptococcus suis type 1 and 2, group B and C streptococci.2 Sequence analysis of the M-like protein gene of 9 isolates of S.zooepidemicus from pig9 isolates of streptococci from Chinese pigg were examinated for the features of their M-like gene. The M-like protein gene of each isolate was cloned, sequenced and analyzed . Nucleotide sequence analysis revealed one open reading frame in M-like protein gene of all isolates, with different length of 1125 bp in CP-0104 isolate, 1143 bp in CG-74-63 isolate, and 1137 bp in the others . The accession number of ATCC35246, CC, CXY012, SH0016, CY in GenBank were AY263781, AY263781, AY263781, AY263781 and AY265990 respectively.The nucleotide identities of M-like genes among 9 isolates except CP-0104 isolate were higher than 95.4%, while the identities between isolate CP-0104 and the remaining 8 strains were 87.1%-88.3%. W60 strain , an equine origin isolate .shared identities of 81.6%-87.0% with these 9 isolates from pig, and 25064 strain ,a human origin isolate , shared identities of 81.7%-91.8%with these 9 isolates from pig .In the deduced amino acid sequences, the 9 isolates except CP-0104 shared similarities up to 92.1%-100%, while those between CP-0104 isolate and the remaining 8 isolates were 82.4%-83.5%. All the 9 isolates shared the common membrane anchor region, and except CP-0104 isolate, the others shared common N-terminal signal peptide.In the hypervariable region (HV) of M-like pr...
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