Abd-Bcr／Abl原核表达载体的构建和表达  

Construction and Expression of Abd-Bcr/Abl Plasmid

作　　者：康志杰[1] 李国辉[1] 黄斯勇[1] 梁亮[1] 尹郸丹[1] 何飞[1] 胡兴斌[1] 韩骅[1] 梁英民[1] 

机构地区：[1]第四军医大学,唐都医院血液科,遗传与发育生物学教研室,西安710038

出　　处：《科学技术与工程》2008年第9期2331-2334,共4页Science Technology and Engineering

基　　金：863计划课题编号:2006AA02A111资助

摘　　要：构建Bcr/Abl-Abd原核表达载体,并表达、观察对白血病细胞K562的影响。以慢性白血病PGD210质粒为模板,应用聚合酶链反应(PCR)扩增出Abd编码区序列,克隆入pMD18t载体,经酶切测序鉴定正确。再通过酶切重组克隆入原核表达载体pET32a,构建pET32a-Abd重组表达质粒,经酶切、测序鉴定,序列、读码框均正确。通过转化E.coliBL21(DE3),经IPTG诱导表达,纯化。结果PCR扩增出特异性的501bp的目的片断,以此构建的重组质粒pET32a-Abd能够在上清中表达约36.6kd的目的蛋白,Western blot鉴定为特异表达。证明成功构建了原核表达载体pET32a-Abd,并表达在上清中,表达蛋白约占菌体总蛋白的6%,纯化后达到74.3%,Western blot检测为特异性表达,为进一步研究该蛋白对慢性白血病细胞的作用奠定了分子基础。To construct the expressing plasmid of Abd, and to detect the functions of the expressed protein to cell line k562, the Abd gene was cloned from the plasmid PGD210. In order to obtain expressing recombination gene pET32a-Abd, inserted it into pMD18t vector to construct pMD1St-Abd plasmid, firstly. After sequencing pMD1St-Abd, inserted the Abd into pet32a to construct pET32a-Abd successfully. The recombination plasmid was transformed into E. coli BI21 and the expression of the fusion protein was induced by IPTG. Fusion protein was purified from the supernatant of cell lysate via Ni-NTA affinity chromatography and identified by Western blot. The results showed that the Abd gene has been successfully cloned and expressed in supernatant then identified by anti-his monoelonal antibody with western blot. For the expression of Abd-Bcr/Abl , the molecular basis has been estab- lished to find a new clinical therapeutic medicine or a new target of CML.

关 键 词：慢性髓细胞性白血病 BCR/ABL基因 肌动蛋白结合域(Abd) 

分 类 号：Q813[生物学—生物工程]