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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 prokaryotic express and purify the Bcr / Abl gene OD domain fusion protein of chronic myeloid leukemia (CML). Methods The TAT, OD and HA gene fragments were cloned into pET32a (+) prokaryotic expression vector in order to construct recombinant prokaryotic expression vector pTAT-OD-HA. The correct recombinant plasmid was identified by PCR, double enzyme digestion and sequencing. lDE3) induced by IPTG. The expressed product was analyzed by SDS-PAGE and Western blot and purified by nickel ion affinity chromatography. Results The constructed recombinant plasmid pTAT-OD-HA was identified by PCR, double enzyme digestion and sequencing. The relative molecular mass of the expressed recombinant protein was about 30,000, and the protein expression reached the peak at 6 h, about 10%. Western blot analysis showed that the recombinant plasmid pTAT- The protein can react specifically with mouse anti-HA monoclonal antibody; purity is about 95% after purification. Conclusion The prokaryotic expression and purification of TAT-OD-HA fusion protein has been successfully established, which lays the foundation for further study of its role in CML.