实时定量聚合酶链反应检测心血管活性多肽apelin方法的建立

目的：建立实时定量聚合酶链反应(RQ-PCR)检测心血管活性多肽的方法，为快速、准确地分析心脏组织中apelin基因表达奠定基础。方法：根据心血管活性多肽apelin基因序列，设计并合成引物和荧光标记TaqMan探针。将逆转录.聚合酶链式反应扩增的apeLin基因片段克隆至pGEM-Teasy载体，重组质粒经蓝自筛选、酶切、测序鉴定后，作为阳性克隆标准品，用于标准曲线的制定，由此可定量检测出每一样品模板的起始拷贝数。结果：阳性克隆标准品制作的标准曲线可显示循环阈值与模板浓度具有良好的线性关系(r=0.970)，可用于apelin基因表达的起始拷贝数定量测定。结论：RQ-PCR方法较常规PCR技术更为简便、准确、特异、灵敏，对定量检测及研究心血管活性多肽有积极意义。

The Establishment of Real-time Quantitative Polymerase Chain Reaction Method for Detecting Cardiovascular Bioactive Polypeptide Apelin

Objective:To establish real-time quantitative polymerase chain reaction(RQ-PCR)method for detecting cardiovascular bioactive polypeptide,and to settle the groundwork on analyzing rapidly and accurately the gene expression of apelin in the heart.Method:According to the specific sequence of apelin genes,the primers and TaqMan probe were designed and synthesized.The apelin fragment generated and purified from RT-PCR product as template was cloned into the pGEM-T easy vector.With being identified by blue-white screening,enzyme digestion and gene sequencing,the positive recombinant plasmid would be used as clonal standard template to make the standard curve for sample detection.Results:The standard curve made by the positive clonal standard indicated a good linear relationship between cycle threshold and template concentration(its correlation coeffecient was 0.970)and could be used to detect quantitatively the original copy numbers of apelin gene.Conclusion:Compared with conventional PCR,RQ-PCR with high sensitivity and reliable specificity is a simple,convenient and accurate method, and has a vital significance for detecting quantitatively and studying cardiovascular bioactive polypeptide.