Study On Prokaryotic Expression Of The Staphylococcus Hyicus ExhB Gene And Its Inoculation In Nude Mice And Balb/c Mice

Staphylococcus hyicus is the causative agent of the Exudative epidermit.With the increase of the intensive extent and the application of the early weaning,the disease will cause more damage on the pig farming.The different strains of the disease can produce differnt exfoliative toxins,designated ExhA,ExhB,ExhC and ExhD.It has been evident that the different toxins play an important role in the disease.The paper is main study the Staphylococcus hyicus which can produce exfoliative toxin B. The pathogenic mechanism of the disease has been the focus of study. However, Staphylococcus hyicus infection is seldomly detected in other mammals rather than swine. So we chose Balb/c mice and nude mice as experimental animals,the mice has some advantage of simple ,economical and easy operate as animal model. The report about the disease study on mice is still uncommon at home and aborad.In this experiment ,we clone and express the staphylococcus hyicus ExhB gene .We also indentify the biological activity of the expression protein. We use the isolation strain infect the mice by muscle injection.In order to find out if nude mice or Balb/C mice are susceptible to Staphylococcus hyicus.Lacking a thymus, nude mouse cannot generate mature T lymphocytes and may be more susceptible to staphylococcus hyicus.A special primer pair P1 and P2 was designed according to the nucleotide sequence of Staphylococcus hyicus ExhB gene(serial number:AF515454) included by the genebank.The Staphylococcus hyicus genomic DNA was PCR ampilified by the primer1 and primer2. A 1280bp fragment was gained.Interest fragment was insert into pMD18-T vector and the recombinant plasmid pMD-ExhB. The result of ELISA and agar diffusion experiments show that the protein could react with the polyclonal serum prepared by the isolation strain.Another special primer P1'and P2' containing Kpnâ… a nd Hindâ…¢was designed. Recombinant plasmid as template, the ExhB gene without N-terminal hydrophobic AA was PCR amplified by P1'and P2'. The PCR product and pET32a vector was digested it with Kpnâ… and Hindâ…¢respectively.The target gene ExhB was subcloned into pET32a vector. Positive clone with interest gene was indentificated by restriction analysis,PCR and DNA sequencing. Then the recombinant was transformed into Escherichia coli BL21(DE3) for ExhB gene expression. The interest gene was induced to express in E.coli with different concentration IPTG.The bacteria containing pET-ExhB was collected and examined by SDS-PAGE.Result showed that the structural protein can expressed in E.coli and melecular weight of the protein was 45 ku.Also, the refolding protein can cause disease in mice.The nude mice and Balb/c mice were injected with the isolation strain,Supernatant and the mixture by intramuscular injection.Trough the observation of eyes,anatomy and do the histopathology biopsy determine the situation of infection.The results show that the nude mice and Balb/c mice are both susceptible to Staphylococcus hyicus. They can generate their respective clinical signs. After the nude mice were injected this bacteria strain 2 or 3 days, we can find a lot of red vesicle in the skin. Some of it would be disappear in 4or 5 days, the other would be form crust. The bacteria strain can cause pyoid secretion in the eyes of some nude mice. The nude mice would be die in 2 weeks. The Staphylococcus hyicus can cause acute clinical signs in Balb/c mice. They will be die in 2 or 3 days. So we can not observe clear clinical signs. Only some of it can generate skin alteration. After anatomy, we found that mice kidneys enlarged significantly and liver congestive obviously. Histological section of skin of mice after infected Staphylococcus hyicus are exfoliation and the hyperplasia of heckle cells, named"acanthosis". This is the characteristics of symptom in pigs. Heart, liver and lung also have different degrees of disease.These results show the Balb/c mice are more susceptible to Staphylococcus hyicus. The nude mice which were infected have visible clinical symptoms,but the Balb/c mice have not .From the study we can know the Staphylococcus hyicus can infect not only swine but also mice. The supernatant and the protein can cause disese in mice which can further make sure the protein is the key factor in the disease.