人造血增效因子在大肠杆菌中的表达及其生物学作用

目的研究人造血增效因子(hHSF)在大肠杆菌(E.coli)中的表达及其动员恒河猴外周血造血干/祖细胞的作用.方法应用重叠PCR方法合成编码hHSF的DNA片段,克隆测序证实其序列正确后,重组到表达载体pET30a中,转入E coli BL21(DE3),并用IPTG诱导表达,目的蛋白用凝胶过滤和阳离子交换层析法进行分离纯化和复性.用飞行时间质谱仪和氨基酸测序仪分别测定纯化的目的蛋白相对分子质量、N端氨基酸序列以及氨基酸组成.选用恒河猴8只,随机分为两组,每组4只一组为单次皮下注射重组hSCF 500μg/kg,另一组为皮下注射rhG-CSF 10μg·kg-1·d-1 4 d后,单次注射rhHSF 500μg/kg.观察恒河猴外周血CD34+细胞、CFU-GM数目以及血常规.结果测序证实合成的hHSF序列与设计一致,构建的重组表达载体pET30a-hHSF在E.coli中获高效表达,目的蛋白占菌体总蛋白的30%左右,主要以包涵体形式存在.目的蛋白纯度达95%以上,N端前10个氨基酸序列分析结果与预期一致;相对分子质量为7540;氨基酸组成分析结果与理论值基本一致.恒河猴实验结果表明,单次皮下注射重组hHSF后外周血CD34+细胞、CFU-GM和中性粒细胞数分别在用药后3 h,1 h和45 min达高峰,动员幅度分别为用药前检测值的16.3倍、1.9倍和4.4倍.与G-CSF联用,动员外周血干细胞作用更加明显,CD34+细胞、CFU-GM和中性粒细胞数分别为基值的25.8倍、8.7倍和8.3倍.结论合成的hHSF基因在E.coli中获高效表达,重组hHSF具有动员恒河猴外周血干/祖细胞和中性粒细胞的作用,并与G-CSF有明显协同作用.

Expression of human HSF in E.coli and its effects on mobilization of hematopoietic stem cells in rhesus monkeys

OBJECTIVE: To study the expression of hHSF in E. coli and its effect on the mobilization of hematopoietic stem/progenitor cells. METHODS: The hHSF gene was obtained by overlapping PCR and cloned into the vector pET30a to yield pET30a-hHSF, which was transformed into E. coli BL21(DE3) and expressed with IPTG induction. Subsequently, rhHSF was purified by gel filtration and cation exchange chromatography and subjected to refolding. Molecular weight of hHSF was measured by MALDI-TOF Mass Spectroscopy. The N terminal amino acid sequence rhHSF was determined by protein sequencing. rhHSF was profiled in rhesus monkey for mobilization of peripheral blood stem cells. Eight rhesus monkeys were equally divided into two groups. The first group was administered single subcutaneous injection of 500 microg/kg hHSF, while the other one was administered 10 microg.kg(-1).d(-1) G-CSF for 4 days followed by a single subcutaneous injection of 500 microg/kg rhHSF. RESULTS: The sequence coding hHSF was confirmed by sequencing and the induced-expression level was about 30% of total cell proteins. The purity of target protein was over 95%. The sequence of N terminal 10 amino acids and the amino acid composition were consistent with the theoretical parameters; molecular weight of rhHSF was 7540. The peripheral CD34(+) cells, CFU-GM yields, and neutrophils peaked at 3 h (16.3-folds increase compared with baseline), 1 h (1.9-folds increase) and 45 min (4.4-folds increase) respectively after the single injection of rhHSF. The addition of rhHSF after the last dose of G-CSF boosted these levels to 25.8-folds, 8.7-folds and 8.3-folds respectively. CONCLUSION: hHSF is highly expressed in E. coli and rapidly mobilizes the hematopoietic stem/progenitor cells and neutrophils in rhesus monkeys. hHSF shows distinct synergistic effect with G-CSF.