| Nocardia seriolae, a bacterial fish pathogen of marine fish and freshwater fish, can cause nocardia infection that lead to mass loss of fish farming. It is an opportunistic pathogens.They can also occur when fish with low immunity or fish body damage by feeding the bait or wound infect N. Seriolae and can cause an acute or chronic fester or granulomatous lesions.The challenged fish main symptom is lots of nodules(granuloma) in the liver, kidney and spleen, while occasionally also visible on the heart and swim bladder. Mammalian cell entry protein of Mycobacterium tuberculosis is involved in the uptake of bacteria in non-phagocytic cells and also related to granuloma formation. Mce1 A protein encoding protein involved in cell invasion, decided to mycobacterium tuberculosis invasive phenotype. In this study, mce1 A gene was cloned and the expression of this gene was analyzed to display the pathogenesis of pathogenic mechanism in N. seriolae.Bacterial loads in the tissues of infected tilapia were performed using q PCR assays at different time points after N.seriolae virulent. In the acute stage of infection, N.seriolae exists in all of the tissue. Bacterial tissue tropism was first detected in spleen, a peak in 1d after challenge and the expression quantity have begun to peak off, the results showed that the spleen is the optimal target tissue that the bacterial planting.According to the mce1 A gene sequences of nucleic acids in N. farcinica, N. cyriacigeorgica, M. smegmatis and N. nova design a series of specific primers MCE in conservative area, application of polymerase chain reaction amplification specific fragment, found 24 kinds of mammalian cells invasion factor in providing the 22 strains of N. seriolae exist in cartesian bacteria, show that MCE family protein family in N. seriolae is stable heredity.The full-length(1,254 bp) of mce1 A c DNA in N. Seriolae was cloned in this study. The MCE1 A molecular weight is 43.98 kD, theoretical isoelectric point and theoretical hydrophobic averages are 5.14 and 0.044 respectively. It contains 161 hydrophobic amino acids, as the hydrophobic proteins.The protein mainly consists of random coil, almost has no helix and folding. The MCE domains and the Mtu_fam_mce domain overlap each other and contains the public areas.Speculated that this overlap domain play an important role in functional area, the main role of the domain is to virulence,while 34-160 protein may be involved in the invasion of macrophages and in them survial in them.ABC transporters is composed of The OM_asym_MlaD domains and the MlaD domain, participation in lipid transport.Compared with 12 other species’ same gene sequence, it has the highest homology with chicken(up to 87.1%) and the lowest homology with fishes(down to 11%).It is worth noting that the sequence homology and is only 11.7% between N. seriolae and N. Transvalensis.The study constructed phylogenetic tree based on mce1 A cDNA to showed that the members of N.seriole are closely related to N. nova. This subject aims to construct a recombinant plasmid pET32a-mce1 A and then transferred to E.Coli BL21(DE3) in order to obtain recombinant protein. The Escherichia coli containing recombinant vector expressed protein of 63 kD after induction by IPTG. These results will provide some prerequisites for further researches on pathogenic mechanism.Preparation of pET 32a(+)-mce1 A antigen protein and purified it was emulsion. Trachinotus ovatus were injected intravenously with antigen protein. Results show that the two immune protection effect is not ideal, the highest survival rate of the immunized group was 50%. Objective to develop novel candidate vaccines for nocardia in order to lay a foundation for safe and effective prevention to the nocardia. After 30 d Immune,a challenge trial was conduction by injection of N.seriolae, then fed for 14 d to determine the effect of antigen peotein. The results showed that the immune group experiment fish mortality rates as high as 60% or even 60%. But the survival rate of blank group and PBS group was obviously higher than that of three experimental immune protection.Cord factor separated from N. seriolae was injected into the trachinotus ovatus can induced the granuloma. Purified TDM from N. seriolae was emulsified with Freund’s incomplete adjuvant and Tween 80. Trachinotus ovatus were injected intravenously with various doses of TDM. Results show that the two immune protection effect is not ideal, the highest survival rate of the immunized group was 50%. Objective to develop novel candidate vaccines for nocardia in order to lay a foundation for safe and effective prevention to the nocardia. |
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