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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。
OBJECTIVE: To establish a method for the analysis of N-terminal amino acid sequence of recombinant antibody with pyroglutamic acid blocked N-terminal. Methods: The pyroglutamic acid peptidase was immobilized at high temperature, and the genetically engineered recombinant monoclonal antibody was used to remove the N-terminal pyroglutamic acid at high temperature. After reduction SDS-PAGE and electroblotting, the N-terminal amino acid sequence was determined and compared with the effect of deblocking PVDF membrane after electroblotting. RESULTS: Seventeen genetically engineered monoclonal antibodies were tested, of which seven were heavy chain-deprotected antibody samples that were successfully sequenced. Both methods were successful in removing N-terminal blocked pyroglutamic acid N-terminal amino acid sequence determination; N-terminal amino acid sequence determination can not be carried out on some samples without de-blocking treatment. Conclusion: Both of these methods are suitable for the amino acid sequence analysis of pyroglutamic acid-blocked mAb at the N-terminus.