人干细胞因子基因大肠杆菌表达产物结构分析及活性研究

 目的确证人干细胞因子基因在大肠杆菌表达产物的正确性。方法通过蛋白质杂交，氨基酸末端序列分析，质谱分析及动物体内生物活性的研究，对人干细胞因子基因大肠杆菌表达产物——重组人干细胞因子(rhSCF)分子进行了鉴定。结果大肠杆菌表达产物rhSCF N端15个氨基酸序列及C端2个氨基酸序列与理论推测一致，产物的相对分子质量为18 583.75，质量肽谱与理论序列的重叠率为98.2%，动物试验亦表明当与粒细胞集落刺激因子(G CSF)联用具有显著的外周血干(祖)细胞动员作用。结论人干细胞因子基因大肠杆菌表达产物与理论推测完全一致，为其中试及临床试验奠定了基础。

Structure analysis and bioactivity study of human stem cell factor gene expressed in Escherichia coli

OBJECTIVE To identify the product of recombinant human stem cell factor(rhSCF) gene expressed in Escherichia coli .METHODS The purified product was characterized through Western blot,terminal amino acid sequence analysis,mass spectrometry and in vivo bioactivity assay.RESULTS The molecular weight of rhSCF is 18 583.75.The N -terminal 15 amino acids sequence,C-terminal 2 amino acids sequence and its peptide mass mapping were conformable to the rhSCF gene.The experiments in monkeys also indicated that the rhSCF used with G-CSF had remarkable ability to mobilize the hematopoietic progenitor cell into peripheral blood.CONCLUSION The expression of the rhSCF gene in E.coli is just as expected,which set a solid base for its scale-up and clinical trail.