Role of Host-coded Protein "PrP"in Scrapie : Preparation of Transformed Cells with Heterologous or Artificially Mutated PrP Gene.

从牛脑cDNA文库中分离出一个牛痒病相关纤维蛋白（PrP）的cDNA克隆，并扩增了6个牛痒病相关纤维蛋白的基因组DNA克隆。这些克隆具有在其他动物PrP基因中观察到的特定特征。然而，根据重复次数，牛PrP可分为两种类型。其中一个具有与其他动物PrP基因相同的4个八肽重复序列，包含256个氨基酸；另一个具有5个八肽重复序列，包含264个氨基酸。原牛PrP的氨基酸序列与羊PrP一致，直到n端第165个氨基酸。牛PrP cDNA导入小鼠L-929细胞后稳定表达。免疫荧光法检测到重组牛PrP在细胞中的表达水平高于原鼠PrP^c。sds -聚丙烯酰胺凝胶电泳结果表明，重组PrP^c与原牛PrP^c共迁移，表明重组产物在L-929细胞中完全糖基化。胞核周围可见明显的中间丝束。减法，核酸部分和PrP部分组成的PrP^<17-25>。PrP^<sc>的核心片段，从小鼠痒病相关原纤维富集部分制备。在核酸酶处理前或处理后，先将核酸部分导入小鼠L-929细胞，然后在2周后制备转染后的细胞裂解液接种于小鼠，或直接接种于有或不含PrP部分的小鼠，以检测其生物活性。将PrP部分单独接种于小鼠。小鼠接种转染的细胞裂解液或单独接种核酸部分，在其一生中没有出现瘙痒症状。单独接种PrP组分的小鼠中有60%出现痒病临床症状，与核酸组分联合接种的小鼠中有67%出现痒病临床症状。

A cDNA clone encoding bovine scrapie-associated fibril protein, PrP, from a bovine brain cDNA library and 6 amplified genomic DNA clones of bovine PrP were characterized. These clones possessed specific characteristics observed in other animal PrP genes. However, the bovine PrP was divided into two types by the number of repeats. One possessed four octapeptide repetitive sequences like other animal PrP genes and consisted of 256 amino acids, the other had five such repetitive sequences and 264 amino acids. The amino acid sequence of the former bovine PrP agreed with that of sheep PrP up to the 165th amino acid from the N-terminal. Bovine PrP cDNA introduced into mouse L-929 cells were stably expressed. The expression level of recombinant bovine PrP in the cells judged by immunofluorescence was higher than that of authentic mouse PrP^c. The reoombinant PrP^c co-migrated with authentic bovine PrP^c in SDS-polyacrylamide gel electrophoresis, suggesting that the recombinant product was fully glycosylated in L-929 cells. Distinct bundles of the intermediate filaments were frequently seen at perinuclear region of the cells.Subtractions, a nucleic acid fraction and a PrP fraction consisting of PrP^<17-25>. a core fragment of PrP^<sc>, were prepared from the mouse scrapie-associated fibril-enriched fraction. To examine the biological activity, the nucleic acid fraction was either first introduced into mouse L-929 cells before or after nuclease treatments, then transfected cell lysates prepared 2 weeks later were inoculated into mice, or directly inoculated into mice with or without the PrP fraction. The PrP fraction alone was also inoculated into mice. Mice inoculated with the transfected cell lysates or with the nucleic acid fraction alone showed no scrapie signs during their lifespan. While 60% of the mice inoculated with the PrP fraction alone and 67% of those inoculated with the fraction together with the nucleic acid fraction showed clinical signs of scrapie.