Establishment of Prion Protein Immunoassay for Creutzfeldt-Jakob Disease.

首先，我们试图阐明克雅氏病(CJD)中人和小鼠对蛋白酶抗性的Prion蛋白的器官分布，并利用半定量Western印迹分析来检测异常Prion蛋白(PrP)的浓度。人PrP局限于中枢神经系统，而小鼠PrP在CJD末期存在于中枢神经系统和淋巴网状系统。PRP在中枢神经系统中的浓度与人类几乎相同。CJD感染小鼠脑、脾、脊髓、淋巴结、胸腺和肠道的最小湿重分别为0.3 mg、1~3 mg、3 mg、3 mg、10 mg和10~30 mg。CJD感染小鼠的300 mg肝、肺、肾和人的脾、淋巴结、肝或周围神经系统的300 mg PrP组分均未见免疫反应。在我们的方法范围内，小鼠…的分布更多的PrP与人类PrP不同。虽然PrP的免疫学检测确实有一定的敏感性，但PrP浓度确实与感染瘙痒病或CJD的小鼠的传染性滴度有关。其次，我们检测了FUKUKA-1和FUKUKA-2小鼠适应的CJD株。感染福冈-2病毒的小鼠比感染福冈-1病毒的小鼠有更高的库鲁斑发生率、更高的PrP浓度和更高的传染性滴度。第三，为了证明PrP是库鲁菌斑核心的一种成分，我们对库鲁菌斑核心衍生的多肽进行了分离和测序，然后用Achromobacter lyticus蛋白酶I消化。我们用反相高效液相色谱鉴定了3种PrP衍生的多肽，并发现了PrP(102Leu)错义变体的一段酶切片段。在Gerstmann-Straussler综合征患者中也存在PrP变异体。从氨基酸序列数据来看，由于N-末端的异质性，我们无法鉴定N-末端的序列。为了阐明PrP的N-末端序列是否与体内淀粉样蛋白的形成有关，我们制备了人工合成的N-末端多肽。抗N端抗体免疫标记库鲁斑阳性。较少

At first, we attempt to clarify the organ distribution of human and murine protease-resistant prion protein in Creutzfeldt-Jakob Disease (CJD), and to measure the concentration of abnormal Prion Protein (PrP) using semi-quantitative Western blot analysis. Human PrP was restricted to the central nervous system, whereas murine PrP was present in the central nervous system and in the lymphoreticular system at the end stage of CJD. PrP concentration in the central nervous system was almost identical to that of humans. The minimum wet weight of an organ with a positive reaction was 0.3 mg for brain, 1 to 3 mg for spleen, 3 mg for spinal cord, 3 mg for lymph node, 10 mg for thymus and 10 to 30 mg for intestine of the CJD-infected mice. There were no immunoreactions in purified PrP fractions form 300 mg of liver, lung or kidney of CJD-infected mice, nor from 300 mg of spleen, lymph node, liver or peripheral nervous system of humans. Within the limits of our method, the distribution of murine … More PrP differed from that of human PrP. Although the immunological detection of PrP does have limits of sensitivity, PrP concentration did correlate with infectivity titers in scrapie infected or CJD-infected mice.Second, we examined Fukuoka-1 and Fukuoka-2 mouse-adapted CJD strains. Mice infected with Fukuoka-2 strain have a higher incidence of kuru plaques, a higher concentration of PrP, and a higher infectivity titer than do mice with the Fukuoka-1 strain. Thus, it must be kept in mind that there is a difference in the strain of the infectious agent in murine CJD.Third, to demonstrate that PrP is a component of kuru plaque cores, we fractionated and sequenced kuru plaques core derived peptides, following digestion with Achromobacter lyticus protease I. We identified 3 PrP-derived peptides by reverse-phase high performance liquid chromatography and found a fragment of digests derived from a missense variant of PrP (102 Leu). Variant PrP was also present in the prion rod fraction in patients with Gerstmann-Straussler syndrome. From the data of amino acid sequence, we could not identify the N-terminal sequence because of heterogeneous N-termini. To elucidate whether N-terminal sequence of PrP is related to amyloid formation in vivo, we prepared synthetic N-terminal peptide. Anti-N-terminal antibody immunolabeled kuru plaques positively. Less