The Study Of Monoclonal Antibody And Recombinant Protein For Diagnosis And Therapy To Fabry's Disease

Fabry disease is an X-linked inherited alpha-galactosidase A deficient disease. It was reported that alpha-galactosidase A(α-GalA) gene was located in Xq22. Lose of alpha-galactosidase A activity leads to the accumulation of globotriasylceramide in cells, tissues and organs in human body resulting blood circulation problem, and finally the patients will die of cardiovascular, cerebrovascular, renal dysfunction and neural disorders, if the patients can not be treated in time. It is estimated that incidence of Fabry disease was 1/40000 in male and most of patients died of cardiovascular, cerebrovascular, renal dysfunction and neural disorders at about 40 years old. Most female patients showed significant symptoms at 40 to 50 years old. It is of great importance to diagnosis and treat Fabry disease at early stage so as to prevent the progress of disease, improve life quality and extent the lifespan of victims.Determination ofα-Gal A level by monoclonal antibody is a reliable method for Fabry disease diagnosis at early stage. In order to obtain high titer and high specific humanα-Gal A monoclonal antibody, dominant epitopes ofα-Gal A were analyzed. 2 peptides were synthesized and conjugated with KLH. BALB/c mice were immunized with KLH-conjugated peptide. The hybridoma cell lines were selected in CM semisolid culture medium containing HAT. The selected hybridoma cell and its monoclonal antibody were identified by karyotype analysis, enzyme linked immunosorbent assay ( ELISA), SDS-PAGE, as well as Western-Blot. One hybridoma cell line producing high titer humanα-Gal A monoclonal antibody was obtained. The antibody was identified to be IgG1 subtype and showed high affinity and high specificity to humanα-Gal A. Theα-Gal A level in the plasma samples from 12 health donors and 5 Fabry disease patients were detected by the monoclonal antibody. The results showed that theα-Gal A level in patients was lower then that of health people, the mean OD₄₅₀ of patients and health control were 0.159±0.06 (from 0.080 to 0.238,) and 0.481±0.116 (from 0.332 to 0.694 ) .Theα-Gal A activity was also detected and OD₄₀₅ of patient and control was 0156±0.008 (from 0.004 to 0.024) and 0.031±0.006 (from 0.022 to 0.043) respectively. However theα-Gal A level in of one patient was very closed to that of health control. It might be because that mutantα-Gal A without hydrolsis activity was expressed in some patients. It suggested that combination ofα-Gal A level detection withα-Gal A activity determination could increase reliability in Fabry disease diagnosis at early stage.Enzyme replacing therapy by injectingα-Gal A into patient is an effective method in Fabry disease treatment. However, sufficientα-Gal A with high purity and high activity is the perquisite for enzyme replacing therapy. So another purpose of this study is to cloneα-Gal A cDNA and express recombinant humanα-Gal A protein in Pichia pastoris. Humanα-Gal A cDNA was cloned from human liver cancer HepG2 cells by RT-PCR and identified by DNA sequencing. Theα-Gal A cDNA was subcloned into Pichia pastoris expression vector of secretion type pPICZαA with methanol-inducible promoter. The recombinant plasmid was transformed into Pichia pastoris and the transformants were selected by Zeion. After being induced by methanol, the supernatant of transformants was collected and identified by SDS-PAGE, Western blot as well as enzymatic analysis. SDS-PAGE result showed that there was a xx KD protein bind ( same size asα-Gal A protein) in supernatant, which could react with antiα-Gal A antibody in a specific manner. In enzymatic analysis study,α-Gal A activity was detected in the supernatant. Our results indicated that recombinantα-Gal A with hydrolysis activity was obtained. In order to acquire sufficientα-Gal A with high purity and high activity for clinical treatment, expression condition for recombinant protein should be optimal in the future.