鸟苷酰环化酶C质粒标准品的构建

目的:制备人鸟苷酰环化酶C(GC-C)质粒标准品,为实时荧光定量PCR检测GC-C含量,探讨大肠癌患者外周血单个核细胞(PBMC)GC-C mRNA表达水平及临床意义奠定基础。方法:在鸟苷酰环化酶C(GC-C)基因高保守区设计特异性的引物和探针,RT-PCR扩增目的片段并连接于pGEM-T载体,以得到GC-C质粒标准品。结果:经过蓝白斑筛选,PCR或EcoR I酶切初步鉴定,阳性克隆测序分析确定GC-C质粒标准品正确性。结论:正确构建GC-C质粒标准品为RFQ-PCR标准曲线的绘制及GC-C的定量检测奠定了基础。

Construction of the plasmid DNA of guanylyl cyclase-C

Objectives:To Construct the plasmid DNA of guanylyl cyclase-C,and make a foundation for measurement of the expression level of guanylyl cyclase-C(GC-C)in the Peripheral blood mononuclear cell(PBMC)and elucidation of the clinical significance of its level in colorectal cancer patients by establishing real-time fluorenscence quantitative polymerase chain reaction(RFQ-PCR).Methods:Specific primers and TaqMan probe were designed,and GC-C fragments were amplified by RT-PCR and connected with pGEM-T to get plasmid DNA of GC-C.Results:positive clone was assayed correctly by detection of the sequence.Conclusions:The plasmid DNA of GC-C constructed correctly made a foundation for the creation of the standard curve and quantitative detection of GC-C.