| Salmonella enterica serovar Pullorum is the causative agent of pullorum disease in poultry, an acute systemic disease that results in a high mortality rate in young chicks but rarely causes severe clinical disease in adult birds. Salmonella pullorum causes infection by vertical transmission and horizonal transmission. Although pullorum disease has been largely eliminated from many western countries, it still has been happenning in small flocks. The explosion of pullorum disease is popular in china. Searching for virulent genes is very important to understand the pathogenesis of Salmonella pullorum and find therapeutic measures to the disease.With the completion of genomic sequence of microbes, comparative analysis of complete genome provides a powerful tool for studying the molecular pathgenesis of pathegenic mirobes. Although SSH (suppression subtractive hybridization) was first developed for cDNA comparison, it has been applied in genomics study of microbes and screening virulent genes and new genes. In this study, the Salmonella pullorum subtractive library was constructed and analyzed by comparison of genomic differences between Salmonella ullorum 533 (tester) and Salmonella enteritidis 50041 (driver) by SSH, and further ipaJ,mobA gene were cloned and expressed. 1. Construction of suppression subtractive library between Salmonella pullorum and Salmonella enteritidisUsing suppression subtractive hybridization (SSH) technique, the Salmonella pullorum subtractive library was constructed by comparison of genomic differences between Salmonella pullorum strain 533 (tester) and Salmonella enteritidis strain 50041 (driver). Parts of subtracted fragments were sequenced and searched homologically in GenBank after identification by Dot-blot. The results showed four types of fragments were identified: mobile genetic element, type III secretion sequences, sequences for plasmid transfer, sequences with unknown function. The amino acid homology of three subtracted sequences with ipaJ of Shigella flexneri was 50%. The results suggested that the Salmonella pullorum subtracted library was very useful for searching pathogenicity- related sequences and analyzing pathogenesis of Salmonella pullorum.2. Cloning and expression of Salmonella pullorum ipaJ geneThe Salmonella pullorum subtracted sequences PEA2,PE31 and PE44 were blasted on the GenBank. The results of BLASTX showed that the homological ratio of PEA2,PE31 and PE44 was 97%,99% and 100% respectively to ipaJ gene fragment in plasmid psfD10 of Salmonella choleraesuis. The three subtracted sequences were spliced together into the whole sequences of ipaJ in Salmonella pullorum, then a pair of primers were synthesized to amplify ipaJ gene in Salmonella pullorum strain 533 by polymerase chain reaction (PCR). PCR products were cloned into prokaryotic expressive vector pET-30a(+). The recombinant was transformed into the host strain BL21(DE3) and expressed by 0.5mM IPTG induced at 37℃. The expression product was found to be 37KDa by SDS-PAGE. Specific reaction was found between Salmonella pullorum positive serum and expressed protein in vitro by Western-blot assay. It implied that ipaJ was immunogenic and expressed when Salmonella pullorum infected chicks, may be it was related to pathogenicity of Salmonella pullorum. The specificity test proved ipaJ was only amplified from Salmonella pullorum and this gene is carried by all 97 salmonella pullorum isolates tested. The function of ipaJ was remained to be further studied.3. Cloning and expression of Salmonella pullorum mobA geneThe homological ratio of Salmonella pullorum subtracted sequence PE30 was 99% to mobA in Salmonella choleraesuis plasmid psfD10. According to the published sequence of mobA gene in plasmid psfD10 of Salmonella choleraesuis, a pair of primers were designed and synthesized to amplify mobA gene (about 1.6kb) in Salmonella pullorum strain 533 by polymerase chain reaction (PCR). PCR products were cloned into prokaryotic expressive vectors pET-30a(+) and pGEMX6p-1, then the recombinants were transformed into the host strain BL21(DE3) and BL21 respectively. After induction at 37℃with 0.5mM IPTG for four hours, the expression products were found to be 64kDa in BL21(DE3) and 84kDa in BL21 by SDS-PAGE. The His-MobA fusion protein were purified and used to immunize mice. Specific reaction was found between GST-MobA fusion protein expressed in BL21 and positive serum from mice immunized with His-MobA by Western-blot assay. It proved the immunogenicity of His-MobA protein. |
