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刊物信息

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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基因工程单克隆抗体N-端氨基酸序列的测定

Sequencing of amino acids at N-terminus of recombinant antibody

分类号:
出版年·卷·期(页码):2012,32 (6):0-0
DOI: 10.16155/j.0254-1793.2017.01.01
-----摘要:-------------------------------------------------------------------------------------------

目的: 建立了具有焦谷氨酸封闭N-端的重组抗体N-端氨基酸序列分析方法。 方法: 利用高温稳定的焦谷氨酸肽酶,基因工程重组单抗样品在高温变性条件下,去除其N-端焦谷氨酸封闭。经还原SDS-PAGE 和电印迹后,进行N-端氨基酸序列测定,并与电印迹后在PVDF 膜上去封闭处理效果进行比较。 结果: 测定了17种基因工程单克隆抗体,其中有7个为重链去封闭处理后再进行测序的抗体样品,用2种方法都可顺利去除N-端封闭的焦谷氨酸,从而进行N-端氨基酸序列测定;如果不进行去封闭处理,部分样品无法进行N-端氨基酸序列测定。 结论: 2种方法都适于N-端具有焦谷氨酸封闭单抗的氨基酸序列分析。

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

Objective: To establish a analysis of recombinant antibodies N-terminal amino acid sequence blocked by using N-terminal pyroglutamate. Methods: Using stability pyroglutamate peptidase in high-temperature,recombinant monoclonal antibody samples denaturing under high temperature conditions,removing the N-terminal pyroglutamate blocked.By reduced SDS-PAGE and electroblotting,the N-terminal amino acid sequencing and compared with the treatment effect of PVDF membrane after electric blotted. Results: Determination of 17 genetically engineered monoclonal antibodies,including seven for the heavy chain blocked after treatment of the sequencing antibody samples,using the both methods can successfully remove the N-terminal pyroglutamate blocked to the N-terminal amino acid;to sequence N-terminal amino acid sequence.if not to remove deblocking,part of the sample can not existe N-terminal amino acid sequencing. Conclusion: The developed method was suitable for the N-terminal amino acid sequencing of McAb with pyroglutamate blockage.

-----参考文献:---------------------------------------------------------------------------------------
1 PAN meng(潘萌),KONG yun(孔蕴),CHEN chang(陈畅),et al.Research progress of monoclonal antibody drugs(单克隆抗体药物的研究进展).Chin J Biochem Pharm (中国生化药物杂志),2008,29(9):62
2 Moyer M,Harper A,Payne G,et al.In situ digestion with pyroglutamate aminopeptidase for N-terminal sequencing electroblotted proteins.J Protein Chem,1990,9:282
3 Hisashi H,Sersuko K.Deblocking and subsequent microsequence analysis of Na-blocked proteins electroblotted onto PVDF membrane.J Biochem, 1992,111:754
4 Ruedi H,Aebersold,John L,et al.Internal amino acid sequence analysis of proteins separated by one or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose.Proc Natl Acad Sci,1987,84:6970
5 WEI Jing-shuang(魏敬双),CHENG Li-jun(程立均),JIA Qian(贾茜).Sequecing of amino acids at N-terminus of recombinant antibody(重组抗体的N-端氨基酸序列测定).Chin J Biolog(中国生物制品学杂志),2008,21(12):1118
6 William EW.The removal of pyroglutamic acid from monoclonal antibodies without denaturation of the protein chains.Anal Biochem,2005,342:120

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