基于Armored RNA的BCR-ABL融合基因质控物研制

Development of quality control substance for BCR-ABL fusion gene based on Armored RNA

目的制备以MS2噬菌体装甲RNA(Armored RNA)为材料的BCR-ABL融合基因质控物。方法获取临床p210型BCR-ABL融合基因骨髓标本cDNA,PCR扩增p210型BCR-ABL融合基因片段和ABL内参基因片段。将MS2噬菌体成熟蛋白基因和衣壳蛋白基因克隆于pACYCDuet-1,制成pACYC-MS2,然后将p210型BCR-ABL融合基因和ABL内参基因分别插入pACYC-MS2载体,经原核表达、分子筛纯化、反转录-PCR鉴定后分别制成包装有p210型BCR-ABL融合基因和ABL内参基因片段的病毒样颗粒(VLPs)。进行稳定性、均一性检测,同时判断质控物的适用性。结果成功制备了包装有p210型BCR-ABL融合基因和ABL内参基因的VLPs质控物。均一性检测表明质控物目的片段拷贝数水平是均一的。稳定性检测显示质控物标本在-20℃可至少稳定保存7个月,4℃稳定保存3个月,室温稳定保存15 d,37℃稳定保存1周。用实时荧光定量PCR试剂盒检测质控物水平,pACYC-MS2-p210 VLPs和pACYC-MS2-ABL VLPs标准曲线的R2分别为0.9940和0.9917,具有很好的适用性。结论将Armored RNA作为材料所制备的BCR-ABL融合基因质控物稳定性、均一性较好,适用于评价BCR-ABL融合基因检测试剂盒的性能,具有广泛的应用前景。

Objective To prepare BCR-ABL fusion gene quality control substance using MS2 phage Armored RNA as material.Methods obtained the cDNA of clinical bone marrow samples of p210 type BCRABL fusion gene,and amplified BCR-ABL fusion gene fragment and ABL reference gene fragment by PCR.The mature protein gene and capsid protein gene of MS2 phage were cloned into pACYCDuet-1 to prepare pACYC-MS2,then,the p210 type BCR-ABL fusion gene and ABL reference gene were inserted into the vector pACYC-MS2 respectively.After prokaryotic expression,molecular sieve purification and reverse transcription-PCR identification,virus-like particles(VLPs)packaged with p210 type BCR-ABL fusion gene and ABL reference gene fragments were prepared.The stability and homogeneity were detected,and the applicability of quality control materials was evaluated.Results The VLPs packaged p210 type BCR-ABL fusion gene and ABL reference gene were successfully prepared.The homogeneity test showed that the copy number level of the target fragment was uniform.The stability test showed that the quality control substance could be stored stably for at least 7 months at-20℃,3 months at 4℃,15 days at room temperature and 1 week at 37℃.Quantitative real-time PCR kit was used to detect the level of the quality control substance,and the R2 of the standard curves of pACYC-MS2-p210 VLPs and pACYC-MS2-ABL VLPs were 0.9940 and 0.9917 respectively,with good applicability.Conclusion The BCR-ABL fusion gene quality control substance prepared by Armored RNA has good stability and homogeneity,which can be used to evaluate the performance of BCR-ABL fusion gene detection kit,and has a wide application prospect.