| 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. they are worldwide zoonotic pathogens and lead to many different diseases characterized by meningitis, septicemia, arthritis, endocarditis and may cause sudden dealth in pig or human.Fn is a large dimeric glycoprotein composed of aand (3 polypeptide chains, which is generated by several cells.Fn is present in a soluble form in hematoplasma and erevy body fluids and in a fibrillar form in the extracellular matrix of connective tissue. The former is called by hematoplasma Fn, and the latter is called by cell Fn.Fibronectin-binding protein (FNZ) is an important surface protein and virulenceassociated factor of Streptococcus equi subsp.zooepidemicus, which is to mediate substrate adhesion of eukaryotic cells.It is one of the main adhension of SEZ.In the present study,at the molecular levels,we investigated the virulence factors of FNZ in order to get new insights into the pathogenic mechanism and new prevention methods against Streptococcus equi subsp.zooepidemicus.Based on the sequence of FNZ published on the genbank of Streptococcus equi subsp.zooepidemicus,FNZ gene was amplified from genomic DNA of Streptococcus equi subsp.zooepidemicus ATCC35246 strain by polymerase chain reaction(PCR), then the amplified gene was cloned into pMD-19 vector.The recombinant plasmid was verified by restriction endonuclease analysis and nucleotide sequencing.Nucleotide sequencing analysis revealed a 1895bp fragment of FNZ gene of strain ATCC35246,the accession number of this sequence to GenBank is DQ363208.sequence comparison with other published FNZ gene of Streptococci showed that the FNZ gene nucleotide sequence was 89.9% identical to that of Streptococcus equi subsp. zooepidemicus VTU211 strain, and 78 %identical to that of Streptococcus equi subsp. equi, and the FNZ protein sequence was 84.4% identical to that of Streptococcus equi subsp.zooepidemicus VTU211 strain, 44.1 % identical to that of Streptococcus equi subsp. equi. It is low homology with other streptococcus. More mutation exists and 111bp is absent in FNZ gene. Based on the sequence of ATCC35246 FNZ gene, five gene fragments of FNZ gene beween 4-277bp,4-808bp,592-1134bp,895-1615bp and 4-1716bp were amplified from the genomic DNA of SEZ by PCR.Then the amplified fragments were cloned into the plasmid of pET-32a (+),the recombinant plasmids of pFNZ (2-91),pFNZ (2-268),pFNZ (197-378),pFNZ (299-538) and pFNZ were verified by restriction endonuclease analysis and nucleotide sequencing.These recombinant plasmids were trasfomated into its host E.coli strains BL21, recombinant proteins of 31,50,45,25,84kD were highly expressed after induced by IPTG respectively. SDS-PAGE analysis showed that the recombinant proteins had the molecular weight as expected.Western ligand affinity blot assay were used with recombinant fusion protein pFNZ (2-91),pFNZ(2-268),pFNZ(197-378),pFNZ(299-538)and pFNZ,respectively.The results showed that the recombinant fusion protein pFNZ (2-268),pFNZ (197-378),pFNZ (299-538) and pFNZ can bind hFn. Therefore, it is speculated that the epitopes are present in FNZ gene 277-808bp and 895-1134bp, the FNZ protein have linear Fn-binding domain between 197-268 and 299-378 amino acid residue.The BL21 was induced by IPTG to over express, which contains the linear Fn-binding domain recombinant plasmids, SDS-PAGE showed that the fusion protein exist in supernatant. The fusion protein was purified by affinity chromatograph column, and emulsionized by ISA206. The ICR mice were immunized with the purified recombinant protein of pFNZ(2-268),pFNZ(299-538)å’ŒpFNZ (2-268)+pFNZ(299-538) respectively, the 20%,30% and 30% of mice were survival after challenge with Streptococcus equi subsp.zooepidemicus strain ATCC35246. It demonstrated that the recombinant protein was a protective antigen and maybe a critical functional fragment of FNZ. |
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