Bcr-Abl酪氨酸蛋白激酶域及其突变体的构建和表达  

Production,expression and purification of Bcr-Abl tyrosine kinase domain and its mutants

作　　者：王淑红[1] 梁英民[2] 邢佩霓[3] 刘海妮[1] 王耀春[4] 杨曦[4] 蒋珊珊[2] 李军林[4] 李军锋[4] 

机构地区：[1]西安交通大学第一医院肿瘤内科,陕西西安710061 [2]第四军医大学唐都医院血液科,陕西西安710038 [3]陕西省人民医院血液科,陕西西安710068 [4]第四军医大学基础部医学遗传学和发育生物学教研室,陕西西安710033

出　　处：《第四军医大学学报》2006年第15期1361-1364,共4页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金(30370598)

摘　　要：目的:制备Bcr-Ab l激酶域及其突变体-H is的重组蛋白.方法:用PCR方法从含有Bcr-Ab l全长的质粒pGD210中扩增出Bcr-Ab l激酶域片段,再通过重组PCR技术获得激酶域的几个主要突变体(T315 I,Y253F,E255K,E255V),测序正确后将其克隆到原核表达载体pET-32 a中,构建表达激酶域片段和H is标签融合表达的载体,IPTG诱导表达,得到的融合蛋白用镍-次氮基三乙酸(N i-NTA)琼脂糖进行亲和层析纯化.结果:成功得到了Bcr-Ab l激酶域及其突变体序列,经序列分析证实后将重组质粒转入BL-21进行表达.结论:通过重组PCR技术获得了野生型激酶域及其突变体蛋白.融合蛋白表达在包涵体,占菌体总蛋白的70%以上.经N i-NTA镍珠纯化后,纯度在95%以上.AIM： To prepare the Bcr-Abl protein tyrosine kinase domain and its mutants, which will lay a foundation for the study on the resistance to imatinib. METHODS： PCR was used to amplify the sequence of Bcr-Abl protein tyrosine kinase domain from plasmid pGD210. Its main mutants （T3151, Y253F, E255K, E255V） were got by recombinant PCR and cloned into proka^otic expression vector pET-32a after sequencing, and the recombinant plasmid was expressed in E. coli strain BL21 induced by IPTG. The fusion protein was purified from the cell lysates by Ni-NTA affinity chromatography and analyzed by SDS-PAGE. RESULTS： The sequence of Bcr-Abl protein tyrosine kinase domain was successfully cloned, and was identical to that in Gen- Bank. Point-mutants were got by recombinant PCR and then the recombinant plasmid was expressed in BL-21. CONCLUSION： The fragments of Bcr-Abl kinase domain and its mutants can be expressed in inclusion bodies, accounting for over 70% of total bacterial proteins. And the fusion protein can be purified to over 95% using Ni-NTA affinity chromatography. It will facilitate the research of chronic myeloid leukemia treatment.

关 键 词：蛋白质酪氨酸激酶 突变 重组 遗传 聚合酶链反应 融合表达 蛋白纯化 

分 类 号：R392.11[医药卫生—免疫学]