【摘要】 目的研究一种新的人类β3-糖基转移酶β3GnT8/β3GalT7在恶性血液系统肿瘤细胞中mRNA水平的表达情况,并在原核体系中克隆和表达。方法运用半定量RT-PCR方法研究人β3GnT8/β3GalT7在13种恶性血液系统肿瘤细胞中的表达谱,以其中表达该基因最高的THP-1细胞的cDNA为模板,克隆目的基因,构建重组表达载体,并在大肠杆菌中诱导表达。经超声裂菌,尿素溶解包涵体,过Ni-NTA琼脂糖层析柱纯化,透析复性浓缩后进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western blot验证。结果 K562、U937、THP-1和NB4细胞株均较高效表达β3GnT8/β3GalT7,其中THP-1细胞株表达最高;并成功构建原核表达载体pQE30-β3GnT8/β3GalT7,表达出相应的重组蛋白。结论人β3GnT8/β3GalT7在13株细胞中表达有显著差异,并获得大量较纯的目的蛋白,其分子量与预测相符,为单克隆抗体制备及进一步研究该基因的生物学功能奠定了基础。更多还原

【Abstract】 Objective To explore the biological activity of the novel gene in tumor of hematology system,the expression of a novel human β3-glycosyltransferase(β3GnT8/β3GalT7) in 13 human haematopoietic cell lines was examined and the recombinant β3GnT8/β3GalT7 protein was obtained.Method mRNA expression of β3GnT8/β3GalT7 was semi-quantified by RT-PCR and normalized to the expression of the housekeeping gene β2-MG.The full length of β3GnT8/β3GalT7 cDNA except the predicted signal peptide was cloned by RT-PCR and the recombinant prokaryotic expression vector was constructed.After sequencing,the recombinant vector was transformed into E.coli M15 and His-tagged β3GnT8/β3GalT7 was induced by IPTG.The target protein was purified by Ni-NTA affinity chromatography column and characterized by SDS-PAGE and Western blot.Result β3GnT8/β3GalT7 was highly expressed in K562,U937,THP-1 and NB4 cell line,among which THP-1 reached the highest level.The prokaryotic expression vector pQE30-β3GnT8/β3GalT7 was successfully constructed and the purified target protein was obtained.Conclusion The human β3GnT8/β3GalT7 gene is expressed extensively in human haematopoietic cell lines with different expression levels.The recombinant pQE30-β3GnT8/β3GalT7 protein can be highly expressed in E.coli,which provides a basis for the further study on the biologic characteristic and mechanisms of β3GnT8/β3GalT7.更多还原