含PML/RARα(L型)与ABL双基因质粒标准品的制备及应用

Preparation and application of double plasmid standard containing PML/RARα(L) fusion gene and reference gene ABL

目的建立一个含PML/RARα(L型)与ABL双基因质粒标准品,用于PML/RARαL型)的定量检测。方法从NB4细胞中提取总RNA并逆转录为cDNA,以此为模板,设计PML/RARα(L型)与ABL基因特异性引物进行PCR,PCR产物经琼脂糖电泳、胶回收、PCMV4载体克隆后转化DH5α工程菌,提取质粒进行酶切、测序鉴定,提取质粒用于制作荧光定量PCR标准曲线,同时对质粒的稳定性能进行评价。结果结果NB4细胞提取的总RNA完整性良好,构建的质粒经酶切鉴定后与目的条带一致,测序结果与目的片段几乎完全一致,且质粒的原始浓度为9×1012copies/ml,倍比稀释至9×104copies/ml,均能得到良好的标准曲线(R2≈1),且质粒的稳定性好,提示含PML/RARa(L型)与ABL双基因荧光定量PCR质粒标准品构建成功。结论本研究建立了制备含PML/RARα(L型)与ABL双基因质粒标准品方法,并成功制备出稳定的含PML/RARα(L型)与ABL双基因质粒标准品。

Objective To clone double standard plasmid containing PML/RARa and ABL cDNA for quantitative detection of PML/RARα fusion gene.Methods Total RNA was extracted from NB4 cells and reverse transcribed to cDNA,then amplify PML/RARα and ABL gene using gene specific primer.After agarose gel electrophoresis and gel extraction,the PCR product was ligated with PCMV4 vector and transformed to bacteria DH5α.Plasmids were extracted from positive clones and identified with restriction enzyme,and then were subjected to sequencing.Finally,FQ-PCR standard curve was mede and the plasmid stability was assessed.Results Total RNA extracted from NB4 cells was perfected.The results of restriction enzyme and DNA sequencing were according with aimed fragment.The original concentration of the recombinant plasmid was 9×1012 copies/ml,and then diluted to 9.0×104 copies/ml with multiple proportions.Perfected standard curve were all received（R2≈1） and the plasmid was very stable.The recombined plasmids containing PML/RARa（L） fusion gene and ABL gene were constructed successfully.Conclusions This test set up the method constructing the plasmid containing PML/RARa（L） fusion gene and reference gene ABL,and successfully construct the standard plasmids.