Screening Of Specific Detection Antigens Of Sporothrix And Construction Of Sterilization Method

Sporotrichosis is caused by the dimorphic fungus Schenckii,which is currently distributed all over the world,especially in tropical and subtropical regions.In Chin a,spherical sporotrichomonas is mainly distributed.The disease is usually confined to the skin,subcutaneous cells and adjacent lymphatic vessels,and can spread to other organs.In rare cases,inhalation of conidia can lead to systemic disease,and in severe cases can be life-threatening.At present the study of spore silk mainly concentrated in genotyping,epidemiology and drug sensitivity,such as clinical diagnosis relies mainly on the fungal culture and pathological tissue staining,strict operation and therefore need a high detection sensitivity,simple and rapid diagnostic reagents,this research mainly to the development of a kind that can detect spore silk patients serum antibody antigen.In the early stage of the study,the gene encoding the specific antigen gp70 of Sporothrix schenckii,which has the clearest research and the most extensive pathogenicity in the world,was synthesized,and pET-28a(+)and pET-NTMST were constructed.Kan.Two prokaryotic expression systems were transformed into expression strains BL21 and Rosetta,respectively.The protein concentration of BL21 was higher than that of Rosetta by Bradford method.The expression of gp70-His and gp70-TEV-NusA-His antigens was gp70-His is easier to isolate than gp70-TEV-NusA-His after TEV digestion,and the specificity of expression of the antigen is detected by indirect ELISA and Western Blotting,and the specificity ispoor,considering the spatial structure and glycosylation site of the expressed antigen.Differences affect its antigenic determinants,and subsequent work will construct eukaryotic expression systems.Subsequently,we synthesized the gene encoding the enzymatic cleavage site of Sporothrix schenckii gp70 and successfully constructed the recombinant plasmid of gp70-TEV-His-pPICZalpha B,which was successfully transformed into Pichia pastoris X-33.Glycoprotein antigens still have poor specificity after serological verification.In order to solve the problem of poor antigenic recognition,we will extract active natural antigens.Because the cell wall structure of the yeast phase of the spores is dense and thick,we finally use physical methods combined with enzymatic hydrolysis to release the glycoprotein embedded in the cell wall.It was also found that the natural antigen and the serum antibody of had a binding band of 80 KD,and the antigen-antibody cross-reaction was excluded by the serum of the systemic fungus patient and normal human.The three-dimensional structure of this protein was slightly different from gp70 by bioinformatics analysis.In order to achieve the purpose of preventing and controlling the spread of the disease source,an effective sterilization system is required to effectively kill the bacteria.Traditional UV lamps contain a large amount of mercury,and the environmental pollution is irreversible.We develop a mercury-free light source through an electronic excitation beam and use it for surface sterilization.Through the investigation of the published sterilization literature,the new sterilization system developed has a significantly bactericidal effect using a lower irradiation dose,which is lower than Mohr,H.and Gravemann,U.Using 300 mJ/cm~2(3.0×10~3 mW·s/cm~2)Irradiation measures to kill 10 species of platelet concentrate,and at the same time we have further studied the sterilization mechanism.In summary,the main pathogens of sporotrichosis in the three northeastern provinces of China are spherical bacteria.The 80KD purified antigen of the clinical isolate extracted by the physical method combined with the enzymatic hydrolysis technique was compared with the gp70 protein,and the three-dimensional structure was different.The 80KD protein shows high affinity and good specificity for the patient's serum antibody,which lays a foundation for the development of the next clinical sporozoite detection reagent.In addition,we also explored the killing effect of deep ultraviolet on surface spores,and provided reference for the prevention of sporotrichosis.