pcDNA3.1(-)-Bcr-Abl及pcDNA3.1(-)-Bcr-Abl T3151突变质粒真核表达载体的构建

Construction of the Eukaryotic Expressing Vector of Human Bcr-Abl and Bcr-AblT315I

目的:将Bcr-Abl及Bcr-Abl T3151突变克隆入pcDNA3.1(-)真核表达载体,为研究靶向降解受体型酪氨酸激酶BCR-Abl,抑制肿瘤细胞生长提供研究基础。方法:pcDNA3.1(-)-Bcr-Abl质粒构建:分别设计引物,通过分段PCR将BCR-ABL克隆入pcDNA3.1(-)。首先通过PCR扩增出Bcr-a片段,将其克隆入pcDNA3.1(-)的NheI/XhoI之间;接着将PCR扩增出的Abl-c片段克隆入KpnI/HindIII之间,最后XhoI/KpnI双酶切pGD210,将酶切下片段插入pcDNA3.1(-)的相应位点即可。酶切鉴定及测序正确后,转染293T细胞,Western blot验证质粒的表达。pcDNA 3.1(-)-Bcr-Abl T3151的突变质粒:首先设计引物,第一步以pcDNA3.1(-)-BCR/ABL为模板,以Abl-c-u和ba-M1为引物扩增出A-1:560 bp。第二步,相同模板,以ba-M2和ba-M-down为引物扩增出A-2:870 bp。第三步,以扩增出的A-1和A-2为模板,以Abl-c-u和ba-M-down为引物,扩增出1434 bp的片段A-1+2,以Bcl和Kpn I分别酶切pcDNA3.1(-)-BCR/ABL以及A-1+2,将突变后的A-1+2置换入pcDNA3.1(-)-BCR/ABL。结果:PCR结果显示3.1(-)-Bcr-Abl及3.1(-)-Bcr-Abl T3151突变质粒条带大小符合,重组质粒经酶切鉴定和测序结果正确,转染后可见融合蛋白的表达。结论:成功构建pcDNA3.1(-)-Bcr-Abl及pcDNA 3.1(-)-Bcr-Abl T3151的真核表达载体,并且转染293T细胞后证实其能够正确表达,为后续研究奠定了基础。

Objective： To construct the eukaryotic expressing vector of human Bcr-Abl and Bcr-AblT315I, then test their expression in HEK293T cells. Methods： Two parts of Bcr-Abl （Bcr-a, Abl-c） were amplified from PGD210 by piecewise PCR which were digested by using double restriction enzymes NheI/XhoI and KpnI/HindIII respectively, and inserted into the eukaryotic expression vector pcDNA 3.1 （-）. The part between Bcr-a and Abl-c was digested using double restriction enzymes XhoI/KpnI from PGD210, then subcloned into pcDNA 3.1 （-）. The mutational part was amplified with designed primers by PCR from plasmid pcDNA 3.1 （-）-Bcr-Abl, then replaced the original part in pcDNA 3.1 （-）-Bcr-Abl. Results： PCR showed that the size of the bands are correct, and results of enzyme digestion and sequencing analysis verified the constructed recombination plasmids, the fusion proteins were expressed in HEK293T cell line after transfection. Conclusion： The eukaryotic expressed vector pcDNA3.1 （-）-Bcr-Abland pcDNA 3.1 （-）-Bcr-Abl T3151 were successfully constructed,and were expressed correctly in 293T cell lines after cell transfection, which may be utilized for further study.