【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。更多还原

【Abstract】 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.更多还原