【摘要】 目的:构建实时荧光定量PCR(FQ-PCR)标准以定量检测人肝细胞生长因子mRNA的表达。方法:采用RT-PCR法利用肝组织提取的总RNA制备肝细胞生长因子cDNA目的片段。与pGEM-T Easy Vector连接成重组质粒并转化E.coli DH5α。联合用直接PCR、α互补和氨苄青霉素筛选法、EcoRⅠ限制性酶切和序列分析法鉴定其特异性。测定纯化的重组质粒A260,确定浓度并以此制备FQ-PCR梯度浓度参考标准。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FQ-PCR定量参考标准。更多还原

【Abstract】 Objective: To construct serial standards for detecting human hepatocyte growth factor mRNA expressionabsolutely with real-time fluorescence quantitative polymerase chain reaction(FQ-PCR) .Methods: The target fragment of HGF cDNA was constructed with total RNA isolated from liver tissue and amplified by conventional RT-PCR and then was Jinked with pGEM-T Easy Vector to construct recombined plasmid and to transformate to E. Coli DH 5α. Its specificity was testified by direct PCR,α-complementation and Ampicillin screening, EcoR Ⅰ single restriction enzyme cleavage and sequencing method. The concentration of purified recombined plasmid was verified by detecting absorbance in λ260nm and men was diluted to serial standard concentrations 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 specificity and sequence completion. Conclusion: Reference standards for real-time FQ-PCR are constructed successfully.更多还原