Kang Sang-Gyun, Lee Deog-Yong, Kang Mi Lan, Yoo Han Sang
Department of Infectious Diseases, KRF Zoonotic Disease Priority Research Institute and BK21 Program for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea.
J Vet Sci. 2007 Jun;8(2):131-7. doi: 10.4142/jvs.2007.8.2.131.
A normal prion protein (PrPc) is converted to a proteaseresistant isoform by an apparent self-propagating activity in transmissible spongiform encephalopathy, a neurodegenerative disease. The cDNA encoding open reading frame (ORF) of the bovine prion protein gene (Prnp) was cloned from Korean cattle by PCR, and was transfected into Chinese hamster ovary (CHO-K1) cells using lipofectamine. The gene expression of the cloned cDNA was confirmed by RT-PCR and Western blotting with the monoclonal antibody, 6H4. Cellular changes in the transfected CHO-K1 cells were investigated using parameters such as MTT, lactate dehydrogenase (LDH), and superoxide dismutase (SOD) activities, as well as nitric oxide (NO) production, and an apoptosis assay. In the MTT and LDH assays, the bovine PrnP-transfectant showed a lower proliferation rate than the wild-type (p < 0.05). Production of NO, after LPS or ConA stimulation, was not detected in either transfectants or CHO-K1 cells. In SOD assay under ConA stimulation, the SOD activity of transfectants was 10 times higher than that of CHO-K1 cells at 6 h after treatment (p < 0.05). The genomic DNA of both the transfectants and control cells began to be fragmented at 6 h after treatment with cyclohexamide. Caspase-3 activity was reduced by transfection with the bovine Prnp (p < 0.05). Conclusively, the viability of transfectants expressing exogenous bovine Prnp was decreased while the capacities for cellular protection against antioxidative stress and apoptosis were increased.
在一种神经退行性疾病——传染性海绵状脑病中，正常的朊病毒蛋白（PrPc）通过一种明显的自我传播活性转化为一种蛋白酶抗性异构体。通过聚合酶链反应（PCR）从韩国牛中克隆了编码牛朊病毒蛋白基因（Prnp）开放阅读框（ORF）的cDNA，并使用脂质体转染试剂将其转染到中国仓鼠卵巢（CHO-K1）细胞中。通过逆转录聚合酶链反应（RT-PCR）和使用单克隆抗体6H4的蛋白质免疫印迹法确认了克隆的cDNA的基因表达。使用诸如MTT、乳酸脱氢酶（LDH）和超氧化物歧化酶（SOD）活性等参数，以及一氧化氮（NO）产生和凋亡检测，研究了转染的CHO-K1细胞中的细胞变化。在MTT和LDH检测中，牛PrnP转染细胞的增殖率低于野生型（p<0.05）。在转染细胞或CHO-K1细胞中均未检测到脂多糖（LPS）或刀豆蛋白A（ConA）刺激后NO的产生。在ConA刺激下的SOD检测中，处理后6小时转染细胞的SOD活性比CHO-K1细胞高10倍（p<0.05）。在用环己酰胺处理后6小时，转染细胞和对照细胞的基因组DNA均开始片段化。转染牛Prnp后，半胱天冬酶-3活性降低（p<0.05）。总之，表达外源性牛Prnp的转染细胞的活力降低，而细胞对抗氧化应激和凋亡的保护能力增加。
