Krasemann S, Jürgens T, Bodemer W
Laboratory of Molecular Genetic, Mainz, Germany.
J Biotechnol. 1999 Aug 20;73(2-3):119-29. doi: 10.1016/s0168-1656(99)00115-7.
Prion diseases belong to a group of neurodegenerative disorders affecting humans and animals. The human diseases include kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS) and fatal familial insomnia (FFI). The pathomechanisms of the prion diseases are not yet understood. Therefore, monoclonal antibodies (mAbs) would provide valuable tools in diagnostics as well as in basic research of these diseases. In contrast to conventional strategies we have developed an immunization protocol based on nucleic acid injection into non tolerant PrP0/0-mice. DNA or RNA coding for different human prion proteins including the mutated sequences associated with CJD, GSS and FFI were injected into muscle tissue. The mice were primarily inoculated with DNA-plasmids encoding PRNP and boosted either with DNA, RNA or recombinant Semliki Forest virus (SFV) particles expressing PRNP. After hybridoma preparation, different mAbs against prion proteins were obtained and their binding behaviour was analysed by peptide-ELISA, Western blot, immunofluorescence and immunoprecipitation. Our mAbs are directed against four different linear epitopes and may also recognize discontinuous regions of the native prion protein. It could, therefore, be demonstrated that immunization of non tolerant mice with DNA and live attenuated SF virus is a valuable means to induce a broad immune response leading eventually to the generation of a panel of mAbs for basic science as well as for diagnostics.
朊病毒疾病属于一组影响人类和动物的神经退行性疾病。人类疾病包括库鲁病、克雅氏病（CJD）、格斯特曼-施特劳斯勒-谢inker综合征（GSS）和致死性家族性失眠症（FFI）。朊病毒疾病的发病机制尚未明确。因此，单克隆抗体（mAb）在这些疾病的诊断以及基础研究中都将提供有价值的工具。与传统策略不同，我们开发了一种基于将核酸注射到非耐受性PrP0/0小鼠体内的免疫方案。将编码不同人类朊病毒蛋白（包括与CJD、GSS和FFI相关的突变序列）的DNA或RNA注射到肌肉组织中。小鼠首先接种编码PRNP的DNA质粒，然后用表达PRNP的DNA、RNA或重组塞姆利基森林病毒（SFV）颗粒进行加强免疫。制备杂交瘤后，获得了针对朊病毒蛋白的不同mAb，并通过肽ELISA、蛋白质印迹、免疫荧光和免疫沉淀分析了它们的结合行为。我们的mAb针对四个不同的线性表位，也可能识别天然朊病毒蛋白的不连续区域。因此，可以证明用DNA和减毒活SF病毒对非耐受性小鼠进行免疫是诱导广泛免疫反应的一种有价值的方法，最终可产生一组用于基础科学以及诊断的mAb。
