慢性粒细胞白血病Bcr/Abl基因OD域融合蛋白的原核表达及纯化  

Prokaryotic Expression and Purification of OD Domain of Chronic Myeloid Leukemia Bcr / Abl Fusion Gene

作　　者：季茂胜[1] 黄峥兰[1] 黄世峰[1] 曾建明[1] 刘钉宾[1] 罗红伟[1] 曹唯希[1] 冯文莉[1] 

机构地区：[1]重庆医科大学临床血液学教研室临床检验诊断学教育部重点实验室,重庆400016

出　　处：《中国生物制品学杂志》2010年第4期354-357,共4页Chinese Journal of Biologicals

基　　金：国家自然科学基金(30670901).

摘　　要：目的原核表达并纯化慢性粒细胞白血病(CML)Bcr/Abl基因OD域融合蛋白。方法将TAT、OD和HA基因片段顺次克隆入pET32a(+)原核表达载体,构建重组原核表达质粒pTAT-OD-HA,经PCR、双酶切和测序鉴定正确的重组质粒转化E.coliBL2(lDE3),IPTG诱导表达,表达产物经SDS-PAGE及Westernblot分析后,用镍离子亲和层析柱纯化。结果所构建的重组质粒pTAT-OD-HA经PCR、双酶切及测序鉴定正确;表达的重组蛋白相对分子质量约为30000,诱导6h蛋白表达量达最高,约为10%;Westernblot分析显示,该蛋白可与鼠抗HA单克隆抗体发生特异性反应;纯化后纯度约为95%。结论已成功原核表达并纯化了TAT-OD-HA融合蛋白,为进一步研究其在CML中的作用奠定了基础。Objective To express the OD domain of chronic myeloid leukemia（CML）Bcr / Ab1 fusion gene in prokaryotic cells and purify the expressed product. Methods TAT, OD and HA gene fragments were cloned into prokaryotic expression vector pET32a（＋）in turn, and the constructed recombinant plasmid pTAT-OD-HA was identified by PCR, restriction analysis and sequencing, then transformed to E. coli BL21（DE3）for expression under induction of IPTG. The expressed product was identified by SDS-PAGE and Western blot, then purified by nickel ion affinity chromatography. Results PCR, restriction analysis and sequencing proved that recombinant plasmid pTAT-OD-HA was constructed correctly. The relative molecular mass of expressed protein was about 30000. The expression level of recombinant protein reached a peak value 6 h after induction, which accounted for about 10% of total somatic protein. The expressed protein showed specific reaction with mouse anti-HA monoclonal antibody, and reached a purity of about 95% after purification. Conclusion TAT-OD-HA fusion protein was successfully expressed in prokaryotic cells and purified, which laid a foundation of further study on its role in CML.

关 键 词：慢性粒细胞白血病 BCR/ABL基因 OD域 原核表达 纯化 

分 类 号：R733[医药卫生—肿瘤]