摘要
目的:构建实时荧光定量PCR标准品以定量检测人肝细胞生长因子mRNA的表达。方法:以Trizol裂解抽提法提取新鲜肝组织总RNA。以RT-PCR法制备HGF cDNA目的片段并进行扩增;以A—T载体克隆法将纯化的目的片段与pGEM—T Easy Vector连接成重组质粒并转化E. coli DH5_α。采用碱裂解法提取重组质粒,联合用直接PCR、α互补法和氨苄青霉素筛选法、EcoR Ⅰ限制性酶切和序列分析法鉴定其特异性。用聚乙二醇沉淀法纯化质粒并检测λ260nm吸光度,确定原液的重组质粒拷贝浓度并以此制备FQ—PCR梯度浓度标准品。结果:HGF cDNA目的片段成功制备并获得稳定的重组质粒,保持了目的片段的特异性和序列完整性。结论:成功构建了实时荧光PCR定量标准。
objective:Objective To construct a series of standards for detecting human hepatocyte growth factor mRNA expression absolutely with real-time fluorescence quantitative polymerase chain reaction(FQ -PCR). Methods :Total RNA was isolated form fresh live tissue sample using Trizol lysis extration method. The target fragment of HGF cDNA was constructed and amplified with the conventional RT-PCR by using Applied Biosystems 7700 Sequence Detector and then was linked with pGEM-T easy vector to construct recombined plasmid with A-T clone method. Recombined plasmids transformated to E. Colt DH 5, were extracted with alkaline lysis method. The specificy of the recombined plasmid was testified by direct PCR,α-complementation and Ampicillin sereenning,EcoR I single restriction enzyme cleavage and sequence analysis method. The concentration of DNA template purified by PEG precipitation was detected by analysing absorbance in λ260nm and then was diluted to series standard concentrations of HGF recombined plasmid from 10~6 to 0 eopies/ml for FQ-PCR. Results: The target fragment of HGF cDNA was constructed successfully. The recombined plasmid contained the target fragment is stable and keeps its specificy and sequence completion. Conelutions: A series of standards for real-time FQ-PCR analysis have been constructed successfully.
