CYP4F2和β-actin基因实时荧光定量PCR标准品质粒和标准曲线的构建  

Construction of standard plasmids and standard curves for detecting CYP4F2 and β-actin genes with real-time fluorescence quantitative PCR

作　　者：赵永攀[1,2] 石磊[1] 李晋[1,2] 李晓泽[1,2] 李健[1] 赵树进[1,2] 

机构地区：[1]广州军区广州总医院药剂科广州市老年慢病患者合理用药重点实验室,广东广州510010 [2]华南理工大学生物科学与工程学院,广东广州510640

出　　处：《中国卫生检验杂志》2016年第1期98-100,共3页Chinese Journal of Health Laboratory Technology

基　　金：广州市科技计划项目(201509010012)

摘　　要：目的制备用于CYP4F2和β-actin基因mRNA实时荧光定量PCR检测的标准品质粒与标准曲线。方法通过PCR扩增CYP4F2和β-actin进行双酶切鉴定和测序分析,确保重组质粒完整正确;大量抽提重组质粒,测定其拷贝浓度,10倍稀释成梯度标准品,目的片断纯化后直接连接于p GEM-T easy载体,转化E.coli DH5α宿主菌,获得阳性克隆;通过PCR扩增进行荧光定量PCR检测分析。结果 CYP4F2和β-actin基因目的片段均成功重组,获得的重组质粒保持了目的片段的特异性和序列完整性。梯度浓度标准质粒的实时荧光定量PCR结果显示,循环阈值(Ct)与起始模板量的对数值之间有着良好的线性关系。结论成功构建了CYP4F2和β-actin基因实时荧光定量PCR标准品质粒和标准曲线。Objective To construct the standard plasmids and the standard curves for detecting CYP4F2 gene and β - actin gene by real - time fluorescence quantitative PCR. Methods CYP4F2 and β - actin gene were amplified by PCR for double enzyme digestion and sequencing analysis. The recombinant plasmid was confirmed if it was complete and correct; a large number of recombinant plasmids were extracted, and the copy concentration was determined, diluted 10 times into gradient standard; with the target fragments, after purification, directly connecting to pGEM -T easy vector, and transforming it to E. coli DH5α host bacteria, receiving positive clone; PCR amplification and fluorescence quantitative PCR assay were carried out for analysis. Results Both CYP4F2 and β - actin gene fragments were successfully reorganized, and the recombinant plasmid was obtained to maintain the specificity and sequence integrity of the target fragments. The results of real - time fluorescence quantitative PCR with the series of plasmid standards showed an ideal linear relationship between the logarithmic values of initial template concentration and cycle threshold values. Conclusion A series of recombined plasmid standards for CYP4F2 gene and β- actin gene with real -time fluorescence quantitative PCR analysis have been successfully constructed.

关 键 词：CYP4F2基因 Β-ACTIN基因 实时荧光定量PCR 重组质粒 标准品 

分 类 号：Q78[生物学—分子生物学]