Characterization Of Mga0553 Gene Encoding An Unkown Protein From Mycoplasma Gallisepticum

Mycoplasma gallisepticum ( MG ) is a pathogentical organism inducing chronic disease in chickens and infectious sinusitis in turkey/, resulting in considerable economic losses in poultry production. M.galliseptiucum is a prokaryotes between bacteria and virus, presenting the smallest genomes of self-replicating organisms. The total genome of strain Rlow is composed of 996422bp, contains 742 putative coding DNA sequences(CDSs) which include 150 conserved hypothetical protein and 123 hypothetical protein without been identification on function, especially membrane associated proteins and pathogenic factors that play key roles in pathogenesis of M.gallisepticum.In present study, a pair of primers was designed containing the sequence enzymed by BamH I and Sal I in order to clone the mga0553 coding sequence from strain R(F94),S6,F36 by PCR amplification respectively. The products of PCR were ligated to the pGEM-T easy vector , then transformated into E.coli JM109 competence cells for clone and sequence. The hypothetical amino acid sequence was blasted with strain Rlow through DNAStar software. The result indicated that the MGA₀553 was a conserved protein and there were ten mutation sites in strain R(F94),S6,F36, while the distribution ofα-helix domains had not difference with Rlow;The sequence from N-terminal 38 site to 55 was hydrophlicitical domain. The amino acid sequence of MGA₀553 had been analysised that suggested the theoretical pI/MW is 8.95/42kDa, the protein contain 38 negatively charged residues(Asp+Glu) and 44 positively charged residues(Arg+Lys). The DAS-Predication of transmembrane region in prokaryotes using the dense alignment surface method indicated that there was a transmembrane domain, which was a signal peptide analyzed by SingalP3.0 software.The products of mga0553 digested with BamH I and Sal I were ligated to the pET-32a(+) vector, then transformated into E.coli BL21(DE3) competence cells. The gene was expressed in recombinant bacterias induced by IPTG and the target protein was separated by SDS-PAGE, then immunized ICR mice. The result of Western-blot analysis showed that the anti-MGA₀553 sera were successfully acquired.In indirect fluorescence assay (IFA), the result showed that mga0553 was positive on the MG cells. The assay of effection on MG growth suggested that the anti-MGA₀553 sera could inhibited the growth of MG.In conclusion, the mga0553 gene might encode a kind of secreted proteins in MG. The function of MGA₀553 will be opened to us in the future.