版纳微型猪近交系TNFα基因mRNA实时荧光定量PCR检测方法的建立.pdfVIP

版纳微型猪近交系TNF-α基因mRNA实时荧光定量 PCR检 测方法的建立 霍金龙;李颖;赵跃;潘伟荣;刘丽仙;霍海龙;曾养志 【摘要】目的 快速、灵敏、可靠的检测与临床多种疾病密切相关的重要基因 TNF-α 的表达情况,构建 TNF-α 基因及内参 GAPDH 基因荧光定量 PCR 质粒标准 品.方法 利用版纳微型猪近交系 4 ～6 月龄猪建立动物模型,提取皮肤创面总 RNA, 设计特异引物.进行 RT-PCR 扩增.纯化目的片段与 pMD18-T 载体连接,转化宿主菌 DH5α,提取重组质粒 DNA,并经酶切、PCR 和测序鉴定,计算重组质粒原液拷贝数 浓度并制备梯度浓度标准品,进行实时荧光定量 PCR,生成标准曲线.结果 建立的 TNF-α 基因和 GAPDH 内参基因 mRNA 表达实时荧光定量 PCR 检测方法灵敏度 分别可达 103和 105拷贝,线性范围分别为 103 ～109和 105 ～ 109拷贝,阈值循环数(Ct)与 PCR 体系中起始模板量的对数值之间存在的线性关 系 R2分别为 0.993 和 0.999,扩增效率 E 分别为 111.073%和 95.948%.结论 成功的构建了版纳微型猪近交系 TNF-α 基因质粒标准品和标准曲线,并用内参基因 GAPDH 进行校正,此方法可为探讨 TNF-α 基因在临床多种疾病中所发挥的分子机 理奠定基础.%Objective In order to develop a real-time fluorescence quantitative method of fast, sensitive and reliable detection system, this study build a standard plasmid of house keeping gene and TNF-α gene that was closely associated with clinic diseases. Methods Bamma mini inbred Sus scrofa, aged 4 to 6 months, were used to construct an animal model. The total RNA from the skin tissue was extracted, designed specific primers, and then amplified by RT-PCR. The purified products of PCR were conjugated with a pMD18-T vector and transferred into the bacteria DH5α for replication. The recombinant plasmid picked out from positive clones was amplified by PCR, digested with EcoRI and HindⅢ, and then sequenced. This process was used to calculate the standard concentration of recombinant plasmids from real-time quantitative PCR. Results The sensitivity of this method for creating TNF-α and GAPDH by expressing mRNA gene was 105 ～ 109 and 105 ～ 109 copies, respectively. A linear relationship was found between the threshold cycle number (Ct) and the PCR system (R2 =0.993 and R2 = 0.999), and amplification efficiency was determined to be 111. 073％ and 95. 948％ , respectively. Conclusions A standard of plasmid used in inbred minipig and the standard curves are successfully established and rectified by using the GAPDH internal control