重组抗体的N-端氨基酸序列测定

Sequencing of Amino Acids at N-terminus of Recombinant Antibody

目的建立具有焦谷氨酸封闭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,在重组人源抗狂犬病病毒单抗样品高温变性条件下,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。结果用该方法去封闭后的抗体样品N-端焦谷氨酸去除效果较好,可顺利进行N-端氨基酸序列测定;而电印迹后在PVDF膜上去封闭处理,N-端焦谷氨酸不能充分去除,无法进行N-端氨基酸序列测定。结论该方法适于具有焦谷氨酸封闭N-端的单抗的N-端氨基酸序列分析。

Objective To develop a method for sequencing of amino acids at N-terminus of recombinant antibody with pyroglutamate blockage. Methods The pyroglutamate at N-terminus of recombinant human anti-rabies virus McAb was removed at high temperature by using thermal-stable Pfu pyroglutamate aminopeptidase, based on which the McAb was identified by reduced SDS- PAGE and eleetroblotting and analyzed for sequence at N-terminus. The result was compared with that of McAb deblocked on PVDF membrane after electroblotting. Results The pyroglutamate at N-terminus of recombinant human anti-rabies virus McAb was removed effectively by the developed method, and the N-terminal amino acids of McAb after deblocking was sequenced successfully. However, the pyroglutamate at N-terminus of McAb deblocked on PVDF membrane after electroblotting could not be removed effectively, and the N-terminal amino acids could not be sequenced. Conclusion The developed method was suitable for the N-terminal amino acid sequencing of McAb with pyroglutamate blockage.