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

PrP基因敲除(PrP-/-)小鼠：杂合子(PrP+1-)小鼠交配产生PrP-/-小鼠，得到27只PrP-/-小鼠。抗PrP血清的制备：在大肠杆菌中表达PrP，离子交换层析和凝胶过滤纯化。重组小鼠PrP免疫PrP-/-小鼠后，从PrP-/-小鼠体内获得抗PrP血清，免疫印迹显示PrP信号良好。PrP脂质体的制备：首先，我们阐述了用β-半乳糖苷酶DNA制备脂质体的方法。接种后3d取脑，X-Gal染色。在20只小鼠脑内未检测到X-半乳糖苷酶阳性神经元，这清楚地表明我们所用的方法不能有效地将外源物质引入脑内。虽然用差速离心法从感染C57BT/6的小鼠脑中获得了富含Prion的部分，但我们不能继续进行Prion脂质体的制备和Prion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.