Preparation And Preliminary Identification Of The Trichophyton Mentagrophyte Eno Monoclonal Antibodies

Background:Trichophyton mentagrophytes is an anthropophilic and zoophilic dermatophyte.It is the second most common in the world and more than Trichophyton rubrum in some areas.Trichophyton mentagrophytes usually cause superficial infections limited to the keratinized tissues such as the scalp,smooth skin,nails.But occasionally it can cause deep infections such as kerion,tinea tubercle,granuloma and mycetoma.As more and more people keep pets,the incidence of deep infections caused by Trichophyton mentagrophytes increases year by year.Diagnosis may be difficult in patients in the absence of known risk factors for deep dermatophyte infection.Delays in diagnosing deep dermatophyte infection may lead to scarring,alopecia,and incapacitation secondary to severe pain.Moreover it’s hard to cure,easy to relapse and reinfection and serious to threat human’s health.At present the pathogenesis of deep infection induced by Trichophyton mentagrophyte remains unclear.Owing to the variety of MG’s manifestations,it is necessary to make examinations including direct microscopical,histological examinations and the fungal culture.These traditional diagnosis may be spent a long time so that it is important to clarify the mechanism of deep infection of Trichophyton mentagrophytes and establish a rapid diagnostic method.In previous work,a protease named enolase was found to be the pathogenic factor of the Trichophyton mentagrophytes in deep infection.There is no relevant research on Trichophyton mentagrophytes enolase.But Candida albicans enolase was investigated fully.The reported datas suggest that enolase,also known as 2-phospho-D-glycerate hydrolase,is found extensively in the cytoplasm of fungi and abundant and highly conserved and is an intracellular enzyme necessary for glycolysis and can make the conversion of catalyze-2-Phospho-Glycerate to Phosphoenolpyruvate.Enolase is not expressed when Candida albicans is infected in superficial tissue and it is released only during deep infection.Therefore,we speculate that Trichophyton mentagrophytes enolase may be the key enzyme or an important virulence factor in the deep infection.Candida albicans enolase has a strong antigenicity,can stimulate the body to produce cellular and humoral immune response.So we suppose that the Trichophyton mentagrophytes enlose play the role as an antigen in the deep infection.It stimulated the body to produce anti-enolase antibody.If the above two hypotheses are valid,then enolase as an antigen can be used as an important tool for the early diagnosis of Trichophyton mentagrophytes deep infection.In order to verify the above speculation,the full-length cDNA sequence of Trichophyton mentagrophytes enolase gene has been successfully obtained through a large number of experiments in the early stage,and Trichophyton mentagrophytes enolase recombinant protein antigen was prepared.Objective:To prepare the Trichophyton mentagrophyte Eno monoclonal antibody.Methods:1.The previously prepared bacterial liquid containing pET-16b-ENO was inoculated in an ampicillin-resistant LB medium for expression,purified by Ni-NTA resin affinity chromatography and analyzed by SDS-PAGE electrophoresis.Then,protein was dialyzed overnight in PBS and stored in separate containers.2.BALB/c mice were immuned by the immunogen emulsion made by Freund’s adjuvant and purified Enolase recombinant protein.3.The spleen cells of the immunized mouse with the highest titer were fused with myeloma cells induced by polyethylene glycol in vitro.4.Positive cell line producing monoclonal antibodies was identified by measuring the supernatant titer of the cell with ELISA;5.The screened positive cell lines were subcloned by using limiting dilution method.6.Ascites were prepared by mice injected with positive hybridoma cells.Then the monoclonal antibody was obtained after the ascites antibody protein was purified with affinity chromatography.7.The titer and specificity of monoclonal antibody were detected by the indirect ELISA and Western blot seperetly.Results:1.The expressed and purified protein was identified as the target protein by SAS-PAGE analysis and it had a high purity;2.The titer of the immune mice serum before fusion was 1:32000.After two cell fusions,the resulting cell fusion rates were 63.75%and 55.62%,and the positive rates were 5.22%and 4.87%respectively.3.T wo hybridoma cell lines 4E7 and 3H4 that can stably secrete monoclonal antibodies were obtained.The titres of the two cell lines measured by the indirect ELISA were 1:32000 and 1:64000,respectively and their specificity was confirmed by Western blot.Conclusions:1.A hybridoma cell line that is capable of stably secreting the Eno monoclonal antibody was developed.2.The Trichophyton mentagrophyte Eno monoclonal antibody was prepared and identified.