Wistar大鼠smad3 cDNA序列测定及其mRNA水平实时荧光定量PCR检测方法的建立  

Cloning and sequence analysis of the partial smad 3 gene of Wistar rat and development of real-time fluorescence polymerase chain reaction based SYBR Green

作　　者：李平[1] 熊仁平[1] 刘苹[1] 赵艳[1] 朱敏[1] 周元国[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所分子生物学中心

出　　处：《感染．炎症．修复》2008年第2期82-86,共5页Infection Inflammation Repair

基　　金：国家自然科学基金资助项目(30400468);973项目(G1999054205;2005CB522602)

摘　　要：目的：克隆和测定Wistar大鼠smad3基因部分序列，构建检测Wistar大鼠smad3基因表达的重组质粒标准品并建立实时荧光定量聚合酶链式反应（PCR）方法。方法：提取并培养Wistar大鼠皮肤成纤维细胞的总RNA，逆转录（RT）-PCR扩增，将扩增产物克隆到PMD-18T载体上，测序分析。用已测定序列的T载体作为标准品建立实时荧光定量PCR方法，并检测转化生长因子-β1（TGF-β1）刺激对培养Wistar大鼠皮肤成纤维细胞smad3 mRNA表达的影响。结果：测定Wistar大鼠smad3基因cDNA序列长415bp，其与人、家鼠、小鼠具有较高的同源性，已被GenBank收录（DQ409172）。建立的实时荧光定量PCR方法在10^3-10^7拷贝数/μl的标准品梯度稀释范围内相关系数为0.98946。25ng/ml TGF-β1刺激后Wistar大鼠皮肤成纤维细胞smad3基因表达升高为PBS刺激对照组的1.5倍。结论：获得Wistar大鼠smad3基因序列并成功建立了smad3实时荧光定量PCR的方法，为Smad3作用的分子机制研究提供了实验基础。Objective： To clone and analyze the partial smad 3 gene of Wistar rat, and to develop real-time quantitative polymerase chain reaction （PCR） assay. Methods： Total RNA was extracted from the cultured skin fibroblasts of the Wistar rat. The partial smad 3 gene was amplified by reverse transcription （RT）-PCR and cloned into eukaryotic expression vector PMD-18 T vector and sequenced. Real-time fluorescence PCR based SYBR Green was then developed with the standard recombinant plasmid gained from cloned PMD-18 T vector and detect expression of smad 3 of the fibroblasts stimulated by TGF-β1 . Results：The partial smad 3 gene of the Wistar rat obtained by RT-PCR and cloned into eukaryotic expression vector PMD-18 T was 415 bp in length,and shared high homology with that of human and mouse, and accepted by GenBank. Then, the quantitative assay was developed, and it showed high coefficient of correlation of the external standard curve. One and half fold up-regulated expression of smad 3 mRNA by TGF-131 on the fibroblasts of Wistar rat was found by the quantitative assay. Conclusion： Cloning and sequencing of the partial smad 3 gene of the Wistar rat are accomplished, and the sequence could be used for the real-time fluorescence PCR based SYBR Green.

关 键 词：SMAD3 基因克隆 序列分析 定量聚合酶链式反应 

分 类 号：R-332[医药卫生] R556.7