| Transmissible spongiform encephalopathies (TSE), also known as Prion diseases are neurodegenerative fetal disorders induced by conformational changes of host-encoded PrPc. The disease-related isoform, PrPSc, has biological and physicochemical characteristics that differ significantly from those of other microorganisms including viruses, it is composed principally or entirely of a host-encoded glycoprotein. PrPSc is partially resistant to proteolytic digestion, its accumulation in the central nervous system(CNS )gives rise to neuronal vacuolation and a series of clinical symptoms such as ataxia, dementia etc. It is believed that the conversion ( or missfolding ) of the normal cellular prion protein (PrPc) to the denatured, malignant prion protein (PrPSc) is the cause of this mysterious illness. The conformational changes lead to missfolding from PrPc α -helices into β -sheet has been speculated as the structural basis by which PrPc acquires pathogenicity and causes TSEs. Human TSE include Creutzfeldt-Jakob disease(CJD), new variant CJD(nvCJD), Gerstamann Straussler syndrome(GSS) and Fetal familial insomnia(FFI). In animals, the recently emerged bovine spongiform encephalopathy (BSE), scrapie in sheep and goats, a deadly disease described 200 years ago, belong to this special group of diseases. TSE can transmit through many pathways, such as: oral (gastro-intestinal) route, neurosurgery, organ transplantation and most probably through other iatrogenic transmission like blood transfusion. TSEs are characterised by a long and silent incubation period lasting 5 (in animal) to 15 (in humans) years without apparent clinical symptoms, once started, however, the infected individuals die quickly within months. At present, the pathogenetic mechanisms of TSE are still unknown. None of the prion diseaseshave effective treatment, the specific diagnosis is barely based on identification of PrPSc in brain tissues by immunological methods, such as irnmunohistochemistry, Western Blot and ELISA.In order to study the pathogenesis of TSEs, develop specific and sensitive methods, genomic DNAs were isolated from peripheral blood lymphocytes, prion(PrPc) genes of bovine(Bo) PrP(25242), hamster(Ha) PrP(23231), and human(Hu) PrP(23231) were amplified by PCR and cloned into pGEM-T plasmid, respectively. Their sequences were analysed. Recombinant BoPrP(25242)-GST, HaPrP(23231) -GST and HuPrP(23231) -GST fusion proteins were expressed with pGEMCS vector in Kcoli Ml5 and identified by Western blot. After amino acid sequence aligning, bovine PrP peptide 2948(BoPi) n 89108(BoP2) ^ 106125(BoP3^ PrP209228 (BoP4) were selected as haptens, which were coupled to keyhole limpt hemocyanin by glutaraldehyde or/and m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) respectively. BALB/c mice were immunized three times with interval of 2 weeks, serum antibody titer was monitored by ELISA, at the same time, antibody titers of two coupling methods were compared. The hybridoma cell lines secreting monoclonal antibodies against these peptides were generated by cell fusion , ELISA detection of the supernants. Cell clonings were performed by minimum terminal dilution. The subtypes of these McAbs were screened and identified. Large quantities of McAbs were produced from ascites fluid , purified by caprylic acid and ammonium sulfate pricipitation. The reactivity and validity of McAbs to the recombinant BoPrP(25242), HaPrP(23231), HuPrP (23231) proteins as well as to PrPc of normal bovine brain were verified one by one by means of Western blot. The specificity of McAbs for the immunohistochemical detection of PrPc in normal bovine brain and PrPSc accumulated in the medulla oblongata of bovine spongiform encephalopathy (BSE) was stringently determined.The results showed that the cloned BoPrP(25242), HaPrP(23231) and HuPrP(23231) genes sequences were identical to the sequences published. The fusion proteins of the BoPrP(25242)-GST, HaPrP(23231)-GST and HuPrP (23-231)-GST expressed with pGEMCS vectors in Ml5 E. Coli were identifiedby Western blot, respectively. Mice immunized with 4 peptides had a specific immune response to the respective antigens, generating antibodies in their sera. There seemed no differences observed between the two methods. Through cell fusions, ELISA detections and 2 to 3 rounds of cell screening and clonings, eight hybridoma cell lines, named as Dn, Dg, D4, Eio, F3, B4, Bg and Fg, were finally established, all them showed secreting McAbs against B0P1, BoP2, B0P3 and B0P4 respectively, Through classifying and sub-typing of the monoclonal antibodies obtained, they were sub-typed as followings: Dn^ D4> Eio> F3 belong to IgGj, and Dg^ B4> Bg> Fg belong to IgG2b- Western blot analysis demonstrated that all the McAbs reacted with recombinant BoPrP (25-242 ), HuPrP (23-231) proteins, but failed to react with the recombinant HaPrP (23-231) protein, whereas, the reactivity of Bg and Fg was more intense, the band could distinctly visualized by DAB. The Bg and Fg did recognize the PrPc in bovine normal medulla oblongata, but the Dn, D8, D4, Eio> F3 and B4 did not. In immunohistochemical analysis, Dg, Eio,B4, Bg, Fg showed a diffuse reaction within the cytoplasm of neurons in normal cattle while very strong for Bg,. Fg, some non-specific reactions were observed in Dg. Dn, D4, F3 showed no reaction. In BSE sections the F3, B4, Bg, Fg detected granular deposits of PrP80 associated with neurons and neuropiles. Bg, Fg also reacted strongly with PrPSc, but Di 1, Dg, D4, Eio showed no activity.In conclusion, the genes encoding bovine, hamster and human PrPc were cloned and expressed, a series of monoclonal antibodies were successfully generated. This work has laid an important base for the development of measures detecting prion diseases in animal and humans. It should be also significant for studying the pathogenetic mechanisms of TSEs. To our knowledge, various works have been done to prepare and identify antibodies against cellular PrP in many mammalian species the data have shown that all the species investigated share a great deal ( about 90%) of homology in the amino acid sequence. Therefore, the generation of antibodies to PrP has been generally hampered by the effects of immunotolerance. BALB/c mice whose PrP genes were ablated (prpm ) were used in most of the research. In this study, we have thoughtfully designed four PrP peptides possessing immunogenicity enhanced by coupling with KLH, when...
|
PDF Full Text Request