| ChickenShigellawasfirstreportedbyLanjuXuandChuanqingWangat2004. Chicks infected with Chicken Shigella cause a typical clinic sign of bloodydysentery and even worse cause the death of chicks. Researches showed Shigella hadthe transmission possibility between human and avian. O-polysaccharide (O-PS) isthe outermost structure of Shigella’s lipopolysaccharide (LPS) and crucial importantin the process of the evolution of being pathogenic bacteria. O-antigen variation leadto O-antigen gene clusters’ low conservative. Therefore, O-antigen is considered asthe best research material in the study of molecular evolution on the DNA level,especially in the genetic evolutionary relationship of Escherichia coli and Shigella. Inorder to analyze the origination and evolution of the Chicken Shigella on the DNAlevel, we deciphered the gene cluster of O-antigen of Chicken Shigella bogdii. And, tolay a basis for the study of the pathogenic role of the Chicken Shigella bogdii from theaspect of the bacterial endotoxin, we obtained the method of purifying theO-polysaccharide and had a preliminary study of its toxic effect. Specific studies areas follows:Sequencing of O-antigen gene clusters of chicken S. boydii, genetic evolutionanalysis and predicting gene functions. We acquired full-length cluster gene by longPCR using6pairs of chimeric primers, finished gene annotations and predicted genefunctions by means of the bioinformatics methods, analysis galF and gnd gene andconstructed the evolution tree. The result showed that the full-length of O-antigengene clusters is12742bp. It demonstrated high homology in human S. boydii type3and E.coli O167and comparing with chicken S. boydii shared99.9%and99.8%homology at nucleotide level and99.9%and99.5%at amino acid level. It was foundthat the gene cluster encoding to S. boydii type3antigen contained9varieties ofgenes, including UDP-galactopyranose mutase gene (glf,1089bp), O-antigentransferase gene (wzx,1266bp), O-antigen gene (wzy,1251bp), glycosyl transferasegenes (orf3,1203bp; orf4,855bp; orf5,969bp; orf7,1092bp; orf8,639bp; orf9, 633bp). Study indicated that human S. boydii type3evolved from E.coli O167whilechicken S. boydii may evolved from human S. boydii type3.To obtain high purity LPS chicken S. boydii type3, modified hot phenol-watermethod and sephadex gel chromatography were used for obstaining high purity LPS.O-PS was obtained by combinating with acid hydrolysis method and sephadex gelchromatography. That the LPS displayed inhomogeneous ladder. And assaying of theprotein and nucleic acids of the extractive showed that our study achieved a highpurity of the LPS and O-PS, which protein level was only1.83%and0.28%respectively. This indicates that extracted LPS and O-PS could be used for specificpathogenic research and developed effective vaccine candidates.To study the specific pathogenesis of O-PS of chicken S. boydii. We obtainedhigh purity O-PS by using acid hydrolysis method and sephadex gel chromatographyand then observed its toxic effect compared with LPS in vitro. The results showed thatit is a significant difference between the group using O-PS from0.25~250μg/mL todeal with Hela cells and control group. This indicated that O-PS was harm to Helacells and can lead to cytopathic effect while LPS was no harm to the cells. Results ofrabbit ileal loop revealed that O-PS only caused mucosal hemorrhage in vivo.However, LPS could induce congestion, edema and purulent of ileal, causing seriousdamage to intestinal mucosa. Our research demonstrated that LPS subunit O-PS ofchicken S. boydii can cause cytopathic effect is different from LPS. |
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