Elucidation of the nature of a Creutfeldt-Jakob disease agent using prion protein gene-knockout mice

朊病毒蛋白基因敲除 (PrP-/-) 小鼠：将杂合子 (PrP 1-) 小鼠交配产生 PrP-/- 小鼠，共产生 27 只 PrP-/- 小鼠。 抗 PrP 血清的生产： 在大肠杆菌中表达小鼠 PrP，并使用离子交换色谱和凝胶过滤进行纯化。用重组小鼠 PrP 免疫后，从 PrP-/- 小鼠中获取抗 PrP 血清。这些血清在蛋白质印迹中显示出良好的 PrP 信号。朊病毒脂质体的生成：首先，我们阐明了使用 β-半乳糖苷酶 DNA 制备脂质体的方法。将 β-半乳糖苷酶 DNA 与磷脂酰胆碱和去污剂混合，接种到 C57BL/6 小鼠中脑内。接种后3天取出大脑并用x-gal染色。我们无法在 20 只小鼠的大脑中检测到 xi-半乳糖苷酶阳性神经元，这清楚地表明我们在这里使用的这种方法对于将外源物质引入大脑并不有效。尽管利用差速离心从受感染的C57BT/6小鼠大脑中制备了朊病毒富集级分，但我们无法继续制备朊病毒脂质体和进行朊病毒脂质体的接种实验。

Prion protein gene-knockout (PrP-/-) mice : Heterozygous (PrP+1-) mice were mated to produce PrP-/- mice, which resulted in 27 PrP-/- mice.Production of anti-PrP serum : Mouse PrP was expressed in E.coli and purified using ion-exchange chromatography and gel filtration. Anti-PrP sera were taken form PrP-/- mice after immunization with the recombinant mouse PrP.These sera showed nice signals of PrP on western blotting.Generation of prion liposomes : At first, we elucidated the method for making liposomes using beta-galactosidase DNA.The beta-galactosidase DNA was mixed with phosphatidylcholine and detergent and inoculated into C57BL/6 mice intracerebrally. Their brains were removed 3 days after inoculation and stained by x-gal. We could not detect the xi-galactosidase-positive neurons in brains of 20 mice, indicating clearly that this method we used here was not efficient for introducing of exogenous substances into brain. Although the fraction enriched for prion was prepared from the infected-C57BT/6 mouse brains using differential centrifugation, we could not proceed to make prion liposomes and to inoculation experiment of prion liposomes.