HSPC016的表达、纯化与生物学活性分析

目的:测定大肠杆菌中造血干细胞(hematopoietic stem/progenitor cells,HSPC)016融合蛋白的表达,纯化及其生物学活性。方法:采用PCR技术从pET-28a( )/HSPC016中扩增出目的基因HSPC016重组到质粒pET-22b( )中,转化BL21(DE3),IPTG诱导表达,镍离子亲和层析和分子筛法纯化;用胰蛋白酶消化对数期生长的第3代和第9代DPC,细胞记数法记录生长曲线。结果:原核表达载体pET-22b( )/HSPC016在大肠杆菌中高效表达,目的蛋白表达率约28%,纯化后纯度达95%以上;经重组蛋白处理的DPC增殖活跃;细胞记数结果初步证实HSPC016重组蛋白能促进第9代DPC的增殖及其DNA合成,对第3代DPC无明显作用。结论:HSPC016基因在大肠杆菌中得到了高效表达;HSPC016重组蛋白具有促进高传代DPC增殖的作用。

Expression, purification and bioactivity analysis of HSPC016

Objective:To determine the expression,purification and bioactivity of HSPC016 fusion protein.Methods:The HSPC016 gene was amplified from the plasmid of pET-28a( )/HSPC016 by PCR,then cloned into the plasmid(pET-22b)( ).The recombinant pasmid pET-22b( )/HSPC016 was transformed into E.coli Bl21(DE3).After inducted by IPTG and purified by affinity chromatography and gel filtration,the HSPC016 protein was added to the passage 3 and 9 DPCs in log growth phase which was digested with trypsin.Cell count was used to detect it proliferation and cellular activity.Results:The HSPC016 protein was highly expressed in E.coli,was accounting for 28% of total expression products and the purity was above 95% after purification.The DPCs treated with HSPC016 protein proliferated more actively than control.The result of cell count showed that HSPC016 protein promoted the proliferation and DNA synthesis of passage 9 DPCs.However,HSPC016 protein had no effect on passage 3 DPCs.Conclusion:Recombinant HSPC016 protein had the function of promoting the proliferation of high-passage DPCs.