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

从牛脑cDNA文库中克隆了一个编码牛瘙痒症相关原纤维蛋白（PrP）的cDNA克隆，并对6个扩增的牛PrP基因组DNA克隆进行了表征。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，牛PrP被分为两种类型的重复序列的数量。其中一个基因与其他动物PrP基因一样，具有4个八肽重复序列，由256个氨基酸组成;另一个基因具有5个八肽重复序列，由264个氨基酸组成。原牛PrP的氨基酸序列与绵羊PrP的一致，从N-末端开始的第165个氨基酸。将牛PrP cDNA导入小鼠L-929细胞中，获得了稳定表达。免疫荧光检测结果表明，重组牛PrP在细胞中的表达量高于小鼠PrP^c。SDS-聚丙烯酰胺凝胶电泳显示重组PrP^c与真牛PrP^c共迁移，表明重组产物在L-929细胞中完全糖基化。在细胞核周区常可见明显的中间纤维束<17-25>。从<sc>小鼠瘙痒症相关原纤维富集级分制备PrP β的核心片段。为了检查生物活性，在核酸酶处理之前或之后首先将核酸级分引入小鼠L-929细胞中，然后将2周后制备的转染细胞裂解物接种到小鼠中，或直接接种到有或没有PrP级分的小鼠中。也将单独的PrP组分接种到小鼠中。用转染的细胞裂解物或单独用核酸级分接种的小鼠在其寿命期间未显示出瘙痒症体征。而60%的单独用PrP组分接种的小鼠和67%的用该组分与核酸组分一起接种的小鼠显示出瘙痒症的临床体征。

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.