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

Establishment of a real-time fluorescence quantitative PCR method for detecting the expression of TNF-α mRNA in Bamma minipig inbred line

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 Sits 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 puri- fied 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 HindlIl, 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 10^5 - 10^9 and 10^5 - 10^9 copies, respectively. A linear relationship was found between the threshold cycle number （Ct） and the PCR system （R^2 = 0. 993 and R^2 = 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 gene. This method can serve as a molecular foundation for the study of TNF-α and its role in many clinical diseases.