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对金黄地鼠P rP基因序列进行分析,选择其高度保守区域设计引物,对金黄地鼠各组织提取mRNA,经RT-PCR扩增,产物纯化后与pGEM-T-easy连接,转化大肠杆菌DH5α,筛选后得到重组标准品质粒,对重组标准品质粒进行酶切、PCR和测序鉴定,表明P rP基因保守区段已经成功克隆。将107~102拷贝/反应的重组标准品质粒进行荧光定量PCR,10次重复C t值平均分别为17.50±0.5、21.38±0.2、25.29±0.7、29.12±0.7、31.34±0.4、33.89±0.1,系统自动分析软件显示C t值与标准品浓度的对数之间存在良好的线性关系,回归系数为0.998。对动力学曲线分析表明,在该反应体系和反应条件下,该标准曲线的灵敏度为102拷贝/反应。荧光PCR P rP基因表达检测标准品质粒和标准曲线的建立,为检测P rP基因的组织特异性表达和动物传染性海绵状脑病发生的分子机理奠定了基础。
The sequence of P rP gene of golden hamster was analyzed. Primers were designed by selecting their highly conserved regions. MRNA was extracted from each tissue of golden hamster. After RT-PCR amplification, the product was purified and ligated with pGEM-T-easy to transform E. coli DH5α After screening, the recombinant plasmid was obtained. The recombinant plasmid was digested by restriction endonuclease, PCR and sequencing. The result showed that the conserved region of P rP gene was successfully cloned. The 107 ~ 102 copies / reaction of recombinant plasmid for quantitative PCR, 10 times the average value of Ct were 17.50 ± 0.5,21.38 ± 0.2,25.29 ± 0.7,29.12 ± 0.7,31.34 ± 0.4,33.89 ± 0.1, The system automated analysis software showed a good linear relationship between the Ct value and the logarithm of the standard concentration, with a regression coefficient of 0.998. The kinetic curve analysis showed that the sensitivity of the standard curve was 102 copies / reaction under the reaction system and the reaction conditions. Fluorescent PCR P rP gene expression detection standard plasmid and the establishment of standard curve for the detection of P rP gene tissue-specific expression and animal molecular pathogenesis of transmissible spongiform encephalopathies laid the foundation.