EML4-ALK融合基因变体1和变体3质控品的建立

The establishment of control materials for variant 1 and variant 3 of human EML4-ALK fusion gene

目的:建立EML4-ALK融合基因质控品,用于评价人类EML4-ALK融合基因突变检测试剂盒的性能。方法:根据人类EML4-ALK融合基因序列,合成EML4-ALK融合基因变体1(V1)和变体3(V3)的基因序列。采用限制性内切酶KpnⅠ和HindⅢ对带有目的基因的质粒和表达载体分别进行酶切,将目的基因与载体连接,转化DH5α感受态细胞,筛选阳性菌落进行测序。将测序结果正确的单个菌落进行超声诱导,制备假病毒溶液,并用荧光定量逆转录-聚合酶链反应(RT-PCR)法对假病毒溶液提取的RNA进行检测。结果:对人类EML4-ALK融合基因V1和V3质控品进行序列测定和荧光定量RT-PCR检测,结果表明成功建立了人类EML4-ALK融合基因V1和V3假病毒质控品。结论:本研究建立了人类EML4-ALK融合基因变体假病毒质控品的方法。可为药物靶向治疗基因检测试剂质量控制提供参考。

Objective： To establish the control materials of human EML4- ALK fusion gene for evaluating detection kits of human EML4- ALK fusion gene. Methods： According to the EML4- ALK fusion gene sequence,the gene sequences of selected variant 1（ V1） and variant 3（ V3） were synthesized. The target gene and expression vector were respectively digested with the restriction enzyme of Kpn Ⅰ and Hind Ⅲ. Then the target gene was ligated into the expression vector,transformed into DH5α,and the positive clones were screened for sequencing. The correct clones were induced by ultrasound. The pseudotyped virus solution was prepared to test the extraction of RNA by fluorescence quantitative reverse transcription- polymerase chain reaction（ RT- PCR）. Results： The control materials for V1 and V3 were successfully established by sequencing and fluorescence quantitative RT- PCR. Conclusion： The method established control materials of human EML4- ALK fusion gene. And it can provide a reference for the quality control of the gene detection reagent for drug targeted therapy.