ERAP1基因标准品质粒的构建

Standard Plasmid Construction of Endoplasmic Reticulum Aminopeptidase-1 Gene

目的构建人ERAP1基因的重组质粒。方法以成人皮肤组织中总RNA为模板、Random 6 mers为引物反转录合成cDNA,用该cDNA为模板PCR扩增人ERAP1基因,并克隆到PUC18载体,转化入大肠杆菌DH5α,阳性克隆用酶切和DNA测序鉴定。计算重组质粒原液复制数浓度并制备梯度浓度标准品,real-time PCR构建标准曲线。结果 ERAP1目的片段制备成功,获得稳定的重组质粒,保证了目的片段的特异性和序列完整性,标准曲线参数良好,标准品阈值循环数(Ct)与其相应梯度稀释浓度呈良好线性关系,扩增效率高。结论成功地构建了人ERAP1基因的荧光定量PCR标准品质粒。

Objective To clone human ERAP1 cDNA and use it as the standard for real - time quantifying ERAP1 mRNA. Meth- ods TotaI RNA was extracted from the human skin and reverse transcribed to cDNA using Random 6 mers as primer. Amplified ERAP1 cDNA was ligated with pMD 19 T vector and transferred into the E. Coli for replication. The recombinant plasmid picked out from positive clones was amplified by PCR,and then sequenced. This process was used to calculate the standard concentration of recombinant plasmids from real- time quantitative PCR constructing standard curve. Results The target fragment of ERAP1 gene was successfully prepared and the recombined plasmid contained the target fragment was stable and kept its sequential integrity and specificity. A good linear relation- ship was showed between the Standard threshold cycle number （α） and its gradient dilution concentration, amplification efficiency. Con- clusion The real - time fluorescent quantitative PCR reference standards of human ERAPI gene were successfully constructed.