EML4-ALK-G1202R点突变质粒及其体外稳转细胞系构建  

Construction and expression of EML4-ALK-G1202R point-mutated plasmid in vitro

作　　者：宋惠玲 勾文峰 马玉花 翟鑫[2] 吴英良[1] 左代英[1] SONG Huiling;GOU Wenfeng;MA Yuhua;ZHAI Xin;WU Yingliang;ZUO Daiying(School of Life Science and Biopharmaceutical,Shenyang Pharmaceutical University,Shenyang 110016,China;School of Pharmaceutical Engineering,Shenyang Pharmaceutical University,Shenyang 110016,China)

机构地区：[1]沈阳药科大学生命科学与生物制药学院,辽宁沈阳110016 [2]沈阳药科大学制药工程学院,辽宁沈阳110016

出　　处：《沈阳药科大学学报》2018年第9期776-780,共5页Journal of Shenyang Pharmaceutical University

基　　金：国家自然科学基金资助项目(81673308);沈阳药科大学中青年教师事业发展支持计划资助项目(ZQN2015003);沈阳药科大学大学生创新创业训练计划资助项目(201710163000221)

摘　　要：目的融合PCR法构建棘皮动物微管相关蛋白样4-间变性淋巴瘤激酶(echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase,EM L4-ALK)G1202R点突变载体,并构建体外克唑替尼耐药细胞模型。方法利用RT-PCR从H2228细胞中获得EML4-ALK融合基因,随后利用融合PCR技术构建EML4-ALK-G1202R点突变融合基因,酶切后定向连入真核表达质粒pEGFP-N1中,构建pEGFP-N1-EM L4-ALK-G1202R重组质粒。脂质体法将重组质粒转入A549细胞中,G418抗生素筛选构建EML4-ALK-G1202R稳定高表达细胞系。MTT法验证细胞模型对克唑替尼的耐受性。结果测序结果表明EML4-ALK-G1202R点突变正确,Real time-PCR和Western blot检测结果表明稳转细胞系构建成功,M TT结果表明克唑替尼耐药模型构建成功。结论成功构建了pEGFP-N1-EML4-ALK-G1202R真核表达载体,转染A549细胞后,可稳定表达并发挥克唑替尼耐药性,为进一步研究EML4-ALK点突变导致的细胞耐药机制奠定了基础。Objective To construct the recombinant plasmid carrying EML4-ALK-G1202R fusion gene and obtain crizotinib drug resistant cell model. Methods EML4-ALK fusion gene was obtained from H2228 ceils by RT-PCR and the G1202R point mutation was constructed by fusion PCR. Then EML4-ALK fusion gene was inserted into vector pEGFP-N1 to build the pEGFP-N1-EML4-ALK-G1202R recombinant plasmid, which was transfected into A549 cells and the cell lines which stably expressed the EML4-ALK-G1202R fusion gene were screened by G418. The tolerance of cell model to crizotinib was detected by MTT. Results The results showed that the EML4-ALK-G1202R point mutation was correct, and the stable transformation cell line was verified by the real time-PCR or western blot. MTI＂ results showed that the crizotinib drug resistant model was successfully constructed. Conclusion pEGFP-N1-EML4-ALK-G1202R eukaryotic expression vector was successfully constructed, and EMIA-ALK-G1202R can be stably expressed after being transfected to A549 cell, and could develop the drug resistance of crizotinib. Our experiment laid a foundation for further mechanism study on EML4-ALK mutations-leaded cell resistance.

关 键 词：EML4-ALK-G1202R 非小细胞肺癌 融合PCR 

分 类 号：R96[医药卫生—药理学]