A transfectant RK13 cell line permissive to classical caprine scrapie prion propagation

To assess scrapie infectivity associated with caprine-origin tissues, bioassay can be performed using kids, lambs or transgenic mice expressing caprine or ovine prion (PRNP) alleles, but the incubation periods are fairly long. Although several classical ovine scrapie prion permissive cell lines with the ability to detect brain-derived scrapie prion have been available, no classical caprine scrapie permissive cell line is currently available. Therefore, the aims of this study were to generate a rabbit kidney epithelial cell line (RK13) stably expressing caprine wild-type PRNP (cpRK13) and then to assess permissiveness of cpRK13 cells to classical caprine scrapie prion propagation. The cpRK13 and plasmid control RK13 (pcRK13) cells were incubated with brain-derived classical caprine scrapie inocula prepared from goats or ovinized transgenic mice (Tg338, express ovine VRQ allele) infected with caprine scrapie. Significant PrPSc accumulation, which is indicative of scrapie prion propagation, was detected by TSE ELISA and immunohistochemistry in cpRK13 cells inoculated with classical caprine scrapie inocula. Western blot analysis revealed the typical proteinase K-resistant 3 PrPres isoforms in the caprine scrapie prion inoculated cpRK13 cell lysate. Importantly, PrPSc accumulation was not detected in similarly inoculated pcRK13 cells, whether by TSE ELISA, immunohistochemistry, or western blot. These findings suggest that caprine scrapie prions can be propagated in cpRK13 cells, thus this cell line may be a useful tool for the assessment of classical caprine prions in the brain tissues of goats.为评估与山羊源组织相关的羊瘙痒病（scrapie）传染性，可使用山羊羔、绵羊羔或表达山羊或绵羊朊蛋白（PRNP）等位基因的转基因小鼠进行生物测定（bioassay），但该方法的潜伏期普遍较长。尽管目前已有多款可检测脑源性羊瘙痒病朊病毒的经典绵羊瘙痒病朊病毒易感细胞系，但尚未有针对经典山羊瘙痒病的易感细胞系问世。因此，本研究的目标为构建稳定表达山羊野生型PRNP的兔肾上皮细胞系（RK13），命名为cpRK13，并评估cpRK13细胞对经典山羊瘙痒病朊病毒增殖的易感性。将cpRK13细胞与质粒对照RK13细胞（pcRK13）分别与经感染山羊瘙痒病的山羊或转绵羊VRQ等位基因的转基因小鼠（Tg338）制备的脑源性经典山羊瘙痒病接种物共孵育。通过传染性海绵状脑病ELISA（TSE ELISA）与免疫组织化学（immunohistochemistry）检测发现，接种经典山羊瘙痒病接种物的cpRK13细胞中出现了显著的PrPSc积累，该现象提示羊瘙痒病朊病毒已完成增殖。蛋白质免疫印迹（Western blot）分析显示，接种山羊瘙痒病朊病毒的cpRK13细胞裂解液中存在3种典型的耐蛋白酶K的PrPres亚型。值得注意的是，无论是通过TSE ELISA、免疫组织化学还是蛋白质免疫印迹检测，均未在同等条件下接种的pcRK13细胞中检测到PrPSc积累。上述结果表明，山羊瘙痒病朊病毒可在cpRK13细胞中增殖，因此该细胞系可作为评估山羊脑组织中经典山羊朊病毒的有效工具。