急性早幼粒细胞白血病PML-RARα融合基因异构体cDNA实时定量PCR标准品及标准曲线的构建

Cloning of human PML-RARα fusion gene and its expression in E.coli DH5a

目的克隆急性早幼粒细胞白血病PML-RARα融合基因异构体L型、S型cDNA作为实时定量聚合酶链反应(FQ-PCR)检测PML-RARα融合基因的标准品,从而构建FQ-PCR检测标准曲线。方法应用反转录-聚合酶链反应(RT-PCR)从急性早幼粒白血病患者骨髓单个核细胞的总RNA中逆转录扩增的PML-RARα融合基因异构体L型、S型cDNA,将纯化的PML-RARα融合基因异构体L型、S型cDNA与pMD18-T载体进行连接,转化宿主菌E.coli DH5a感受态细胞,氨苄青霉素培养基筛选阳性克隆,提取重组质粒cDNA用限制性核酸内切酶HindⅢ、EcoRⅠ进行双酶切鉴定并测序分析,最后进行实时荧光定量PCR检测。通过检测重组质粒260 nm吸光度,确定原液的重组质粒拷贝浓度并以此制备荧光定量PCR标准品。结果酶切鉴定、PCR扩增及测序分析均证实PML-RARα融合基因重组到pMD18-T载体。结论成功克隆急性早幼粒白血病PML-RARα融合基因异构体L型、S型cDNA。

Objective To done the PML-RARα fusion gene and to investigate its expression in E. coli DH5α. Methods The PML-RARα fusion gene was amplified from the total RNA which was isolated from bone marrow sample, then was inserted into TA-vector and cloned into plasmid pMD18. Finally, expression of PML-RARα fusion gene in plasmid pMD18 was detected by PCR amplification, sequence identification, being identified with EcoR Ⅰ and Hind Ⅲ. Results PCR amplification and DNA sequencing confirmed that the isolated PML-RARα fusion gene was identical to the previously reported sequence. The recombinant plasmid pMD18 was constructed and the gene successfully expressed in E. coli DH5α. Conclusion The fusion gene is successfully cloned and expressed in E. coli DH5α.