| Enterococci are a ubiquitous gram-positive bacteria in nature and also are a major microflora in human and animal intestinal tract . At present,it have achieved 41 species , but E.faecelis and E. faecium is still the main advantage of the existence of the population in various media. Enterococcus infection occupy an important position in hospitals, who can cause peritonitis, endocarditis, and meningitis and urethritis.but in recent years, more and more animals infected by enterococci were reported, not only can cause encephalitis in lamb, sepsis in chicken and duck, may also be caused septicemia, arthritis and meningitis in pig. From the year 2003 to 2007, Forty-two isolates from invasion swine of diseased blood, liver, spleen, kidney, lymph nodes, brain tissue, synovial fluid collected in Luoyang, Jiyuan, Xuchang, Nanyang in Henan province were isolated and identify to the level of species through conventional and molecular biology methods ,including 12 Enterococcus faecalis, 30 Enterococcus faecium. In order to further study pathogenesis of enterococci infection on the pig and establish basis of immunization and serological diagnosis later, we have separated 12 enterococci from fresh pork in the market in Zhengzhou City and 14 strains from pig feces respectively. including 42 strains infected enterococci, phenotype and genotype of virulence factors have been determinated in 68 strains, animal experiments, and cloning and prokaryotic expression of Enterococcus cytolysin cylA .All studies have obtained in the preliminary expected results.cytolysin and Gelatinase of Enterococcus are the two main pathogenic factor and the two main pathogenic characteristics of clinical examination. The two phenotypes , includingβ-hemolysis and gelatin hydrolyze , were performed on agar plate containing 5% rabbit blood and agar plate supplemented with 3% gelatin, respectively in 68 strains swine Enterococci . it was found of high ratio in infected strains, in which Enterococcus faecalisβhemolytic is 91.7%, gelatin dissolved is 100% .cylA, gelE, efaA, esp, AS and the ace is considered the main pathogenic gene of Enterococcus, we designed six pairs of primers and detected the six virulence determinants in 68 isolates by PCR technology. results showed that infected isolates carry a higher percentage of enterococci virulence gene, food isolates and pig fecal isolates carried the relatively lower enterococci virulence gene , and the food isolates carry higher enterococci virulence gene than the fecal isolates; comparing virulence determinants carryed from the two species of enterococci isolated, we found that Enterococcus faecalis carried a higher percentage of virulence determinants in infected isolates, food isolates or pig manure isolates. In the six kinds of virulence determinants, visceral pathogenic isolates cylA (100%), gelE (100%) and efaA (100%) found the highest rate, indicating the three virulence genes may play a major pathogenic role.Establishment of animal model is an effective means to study the pathogenic mechanism of pathogens, clinical treatment, new drug development and evaluation of virulence of pathogenic bacteria . Enterococcus is a opportunistic pathogens, and its pathogenic capability is complexity of the process, including a number of virulence factors. its level of virulence was determined by phenotype measured in clinical treatment, such as the detection of the cytolysin and gelatin, but virulent strains contain two phenotypes are not judged. as a result of weak pathogenicity, the test which infected mice require larger quantities enterococci and are difficult to determine the pathogenicity of different gradient of different strains.20%extracts of rat faeces (SRFE) were added to the Enterococcus faecalis, five Enterococcus faecalis carrying different virulence gene were choiced, by measuring the LD50 of the strain, pathogenic Enterococcus faecalis peritonitis model was successfully constructed.HE1 and HE5 carry all 6 kinds of virulence genes, the LD50 of two Enterococcus faecalis in mice were 106.01CFU and 106.17CFU; HE41 contains only four virulence gene , the lack of AS and esp, the LD50 of HE41 is 107.17CFU; HE9 and HE10 contain five virulence genes, HE9 lacks of ace, HE10 lacks of AS, and their LD50 in mice were 107.01CFU and 106.51CFU.Enterococcus cylA is activated protein gene, according to enterococci cytolysin gene expression primers were designed. cylA protein gene was amplified with XhoI and HindⅢrestriction sites, was subcloned into the prokaryotic expression vector pET32a and constructed pET32a-cylA expression plasmid recombinant. By IPTG induced about 65.9 KD fusion protein cylA-His obtained expression of the success and provide the basis for preparation of monoclonal antibody and development of enterococcus vaccine as well as further studing in pathogenetic mechanisms.
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