HSPC016的表达、纯化与生物学活性分析  

Expression,purification and bioactivity analysis of HSPC016

作　　者：邹锋[1] 郝飞[1] 易勇[2] 邹全明[2] 宋志强[1] 杨希川[1] 叶庆佾[1] 

机构地区：[1]第三军医大学西南医院皮肤科,重庆400038 [2]第三军医大学检验系临床微生物学及免疫学教研室,重庆400038

出　　处：《中国麻风皮肤病杂志》2007年第9期766-768,共3页China Journal of Leprosy and Skin Diseases

基　　金：国家自然科学基金资助课题(30200249)

摘　　要：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。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 B121 （DE3）. After inducted by IPIG 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.

关 键 词：HSPC016重组蛋白 细胞记数 生物学活性 

分 类 号：R751[医药卫生—皮肤病学与性病学]