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期刊名称:药物分析杂志
主管单位:中国科学技术协会
主办单位:中国药学会
承办:中国食品药品检定研究院
主编:金少鸿
地址:北京天坛西里2号
邮政编码:100050
电话:010-67012819,67058427
电子邮箱:ywfx@nicpbp.org.cn
国际标准刊号:ISSN 0254-1793
国内统一刊号:CN 11-2224/R
邮发代号:2-237
 

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重组人粒细胞刺激因子的N端氨基酸序列异质性分析

Analysis of N-terminal amino acid sequence heterogeneity of recombinant human granulocyte colony-stimulating factor

作者(英文):
分类号:R917
出版年·卷·期(页码):2018,38 (11):1887-1892
DOI: 10.16155/j.0254-1793.2017.01.01
-----摘要:-------------------------------------------------------------------------------------------

目的:分析重组人粒细胞刺激因子(rhG-CSF)的N端异质性。方法:采用反相超高效液相色谱法分离rhG-CSF中N端不一致的各组分;色谱条件:采用Waters BEH 300 C4(2.1 mm×100 mm,1.7 μm)色谱柱,以0.1%三氟乙酸水溶液(A)-0.1%三氟乙酸乙腈溶液(B)为流动相,梯度洗脱,流速0.2 mL·min-1。分别收集各组分并冷冻干燥,采用N端测序仪测定各组分的N端氨基酸序列;液质联用测定rhG-CSF的相对分子质量,对N端测序结果进行验证质谱仪采用Waters公司Xevo G2-S质谱仪,正离子电喷雾离子源,毛细管电压3.0 kV,锥孔电压40 V,去溶剂气体温度350℃,去溶剂气体流速800 L·h-1,扫描范围(m/z)500~3 000。结果:色谱分离得到3个组分;N端氨基酸序列测定结果显示,3个组分分别为rhG-CSF主成分及2个N端异质性相关蛋白;质谱相对分子质量测定结果与N端测序结果一致。结论:应用色谱法及质谱法可对rhG-CSF中的N端异质性相关蛋白进行分析检测,检测结果可为国产rhG-CSF制品的质量控制和标准提升提供参考和数据支持。

-----英文摘要:---------------------------------------------------------------------------------------

Objective: To analyze the N-terminal heterogeneity of recombinant human granulocyte colony-stimulating factor (rhG-CSF). Methods: The N-terminal inconsistent components in rhG-CSF were separated by reversed phase ultra high performance liquid chromatography. The chromatographic separation was carried out on a Waters BEH 300 C4 column(2.1 mm×100 mm,1.7 μm) with the mobile phase consisting of 0.1% trifluoroacetic acid aqueous solution (A)-0.1% trifluoroacetic acid acetonitrile solution (B) in a gradient mode at the flow rate of 0.2 mL·min-1. Each component was collected and freeze-dried,and the N-terminal amino acid sequences of them were determined by N-terminal sequencer. The relative molecular mass of rhG-CSF was measured by liquid chromatography mass spectrometry so as to verify the results of N-terminal sequencing. Waters Xevo G2-S mass spectrometer with electrospray ionization source in positive mode was used,the capillary voltage was 3 kV,Cone voltage was 40 V,gas temperature was 350,gas flow was 800 L·h-1,mass scan range was m/z 500-3 000. Results: Three components were separated by chromatography,and the results of N-terminal amino acid sequence analysis showed that the three components were rhG-CSF principal components and two N-terminal heterogeneity related proteins,respectively. The results of relative molecular weight determination were consistent with N-terminal sequencing. Conclusion: N terminal heterogeneity of rhG-CSF can be analyzed and detected by chromatography and mass spectrometry,which could provide reference and data support for quality control and standard improvement of domestic rhG-CSF products.

-----参考文献:---------------------------------------------------------------------------------------
[1] ASANO S.Function,molecular structure and gene expression of granulocyte colony-stimulating factor[J].Nihon Rinsho,1992,50(8):1854
[2] MORSTYN G,FOOTE M,PERKINS D,et al.The clinical utility of granulocyte colony-stimulating factor:early achievements and future promise[J].Stem Cells,1994,12(Suppl 1):213
[3] BABAEIPOUR V,KHANCHEZAR S,MOFID MR,et al.Efficient process development of recombinant human granulocyte colony-stimulating factor (rh-GCSF) production in Escherichia coli[J].Iran Biomed J,2015,19(2):102
[4] 梁逊,郭小丽.多肽、蛋白质固相分析[J].生物化学与生物物理学进展,1988,15(2):105 LIANG X,GUO XL.Solid phase analysis of polypeptide and protein[J].Prog Biochem Biophys,1988,15(2):105
[5] 史新昌,杨靖清,韩春梅,等.N-末端氨基酸测序数据估计N-末端不均一的单链重组蛋白制品主肽链比例的方法[J].中国生物制品学杂志,2016,29(10):1073 SHI XC,YANG JQ,HAN CM,et al.Estimate most-peptide-chain-component-ratio of heterogeneous N-terminal single subunit recombinant protein biological products using N-terminal sequencer data[J].Chin J Biol,2016,29(10):1073
[6] 毕华,陶磊,韩春梅,等.重组人血管内皮生长因子抑制剂理化对照品质控方法及质量标准的建立[J].中国生物制品学杂志,2017,30(8):833 BI H,TAO L,HAN CM,et al.Establishment of methods and standard for quality control of physicochemical reference substance for recombinant human vascular endothelial growth factor inhibitor[J].Chin J Biol,2017,30(8):833
[7] BRORSON K,JIA AY.Therapeutic monoclonal antibodies and consistent ends:terminal heterogeneity,detection,and impact on quality[J].Curr Opin Biotechnol,2014,30C:140
[8] RENZ A,SCHIKORA S,SCHMID R,et al.cDNA sequence and heterologous expression of monomeric spinach pullulanase:multiple isomeric forms arise from the same polypeptide[J].1998,331(Pt 3):937
[9] VIGUERA AR,SERRANO L.Stable proline box motif at the N-terminal end of alpha-helices[J].Protein Sci,1999,8(9):1733
[10] 何冰芳,米兰,陈文华.大肠杆菌蛋白质分泌机理及其重组蛋白分泌表达新进展[J].食品与生物技术学报,2012,31(6):561 HE BF,MI L,CHEN WH.Research advances in mechanism of protein secretion and secretory expression of recombinant prote in E.coli[J].J Food Sci Biotechnol,2012,31(6):561
[11] RAUCCI R,COSTANTINI S,CASTELLO G,et al.An overview of the sequence features of N-and C-terminal segments of the human chemokine receptors[J].Cytokine,2014,70(2):141
[12] HESS AD,THOBURN C,CHEN W,et al.The N-terminal flanking region of the invariant chain peptide augments the immunogenicity of a cryptic "self" epitope from a tumor-associated antigen[J].Clin Immunol,2001,101(1):67
[13] EL-SHAMY A,SHOJI I,KIM SR,et al.Sequence heterogeneity in NS5A of hepatitis C virus genotypes 2a and 2b and clinical outcome of pegylated-interferon/ribavirin therapy[J].PLoS One,2012,7(2):e30513

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