实时荧光定量PCR构建BIM基因表达标准品质粒和标准曲线  

Construction of the standard plasmid and standard curve of the BIM gene expression using Real-time fluorescene quantitative PCR

作　　者：付文卓[1] 潘博[2,3] 吴英婷[1] 隋雷鸣[1] 王利勇[1] 武文琦[1] 邹林樾[1] 

机构地区：[1]首都医科大学医学实验与测试中心,北京100069 [2]军事医学科学院微生物流行病所国家重点实验室,北京100071 [3]中国农业大学动物医学院国家海绵状脑病实验室,北京100193

出　　处：《黑龙江畜牧兽医》2015年第1期39-42,213,共5页Heilongjiang Animal Science And veterinary Medicine

摘　　要：为了能够快速、准确定量BIM基因的mRNA,试验构建了小鼠BIM基因的标准品质粒和标准曲线,根据Gen Bank中鼠基因编码区保守序列,设计特异性引物,从N2a细胞中提取总RNA,利用RT-PCR技术扩增该基因122 bp的核苷酸片段,经纯化、连接和转化后,提取质粒并进行酶切、测序鉴定,然后将阳性重组质粒10倍递减稀释作为标准模板,通过SYBR GreenⅠ实时荧光定量PCR方法,建立了BIM标准曲线及直线回归方程。结果表明:产物熔解曲线峰值单一,引物特异性高;标准曲线相关系数r2=0.998,线性关系好。说明成功构建了目的基因BIM的标准品质粒和标准曲线。Bcl -2 interacting mediator of cell death（BIM） ; real -time fluorescene quantitative PCR; standard plasmid; standard curve Abstract：To quickly and accurately quantify mRNA of the Bcl - 2 interacting mediator of cell death（BIM） gene, the standard plasmid and standard curve of the mouse BIM gene were constructed. A pairs of specific primer were designed based on the conserved sequence of the coding region of the mouse gene published in GenBank. Total RNA was extracted from N2a cells, and the 122 hp nueleotide fragment of the target gene was amplified by RT - PCR technique. The p lasmids were extracted and used for the identification of enzyme digestion and sequencing after purification, ligation, and transformation. The positive plasmids were used as standard template after ten - fold descending dilution. The standard curve and linear regressive equation of the BIM gene were constructed using SYBR Green I - based Real - time PCR method. The results showed that the peak value of the melting curve of the product was unitary, and the primers had high specificity; the correlation coefficient of the standard was r^2 = 0. 998, suggesting good linear relationship between the groups. The results indicate that the standard plasmid and standard curve of the BIM gene are successfully constructed.

关 键 词：BIM 实时荧光定量PCR 标准品质粒 标准曲线 

分 类 号：S813.3[农业科学—畜牧学] Q754[农业科学—畜牧兽医]