CAJ | 학술논문

为了能够快速、准确定量小鼠 Bax 基因的 mRNA，构建了小鼠 Bax 基因的标准品质粒和标准曲线。根据 GenBank 中小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162 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；将阳性重组质粒10倍递减稀释作为标准模板，通过 SYBR Green Ⅰ实时荧光定量 PCR 方法，建立了 Bax 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＝0．999，表明线性关系好。本试验成功构建了目的基因 Bax 的标准品质粒和标准曲线。
위료능구쾌속、준학정량소서 Bax 기인적 mRNA，구건료소서 Bax 기인적표준품질립화표준곡선。근거 GenBank 중소서기인편마구보수서렬，설계특이성인물，종 N2a 세포중제취총 RNA，이용 RT-PCR 기술확증해기인162 bp 적핵감산편단，경순화、련접화전화후，제취질립병진행매절、측서감정；장양성중조질립10배체감희석작위표준모판，통과 SYBR Green Ⅰ실시형광정량 PCR 방법，건립료 Bax 표준곡선급직선회귀방정。결과표명산물용해곡선봉치단일，인물특이성고；표준곡선상관계수 R2＝0．999，표명선성관계호。본시험성공구건료목적기인 Bax 적표준품질립화표준곡선。
The aim of the present study was to construct the standard plasmid and standard curve of real-time PCR for the quantitation of mRNA expression level of the mouse Bax gene.A pairs of specific primer were designed based on the conserved coding region from GeneBank.Total RNA was extracted from N2A cell and the fragments of target gene about 162bp were amplified by RT-PCR.Plasmid DNA was purified and identified by PCR amplification and sequencing after recycling,purification and transformation.The positive plasmids as standard template were entered into the SYBR Green Ⅰ real-time qPCR steps after 10 times dilution.The standard curve and regressive curve were generated automatically.The results showed that no specific amplicons was found according to the derived melting curve of the real-time PCR products, indicating the specification of the present primers.The correlation coefficients of the standard were 0.999, suggesting the strong linear relationship between the groups.The construction of the standard plasmid and standard curve were established for Bax gene.