白喉毒素无毒突变体CRM197蛋白的结构确证分析

Confirmation analysis of structure of avirulent mutant CRM197 protein of diphtheria toxin

目的对本公司制备的白喉毒素无毒突变体CRM197蛋白进行结构确证分析。方法通过发酵、提纯制备3批CRM197蛋白,经N-末端序列、C-末端序列、质量肽图和肽段覆盖率分析进行一级氨基酸序列结构确证分析,经圆二色谱法进行二级蛋白结构确证分析。结果3批CRM197蛋白N-末端均为GADDVVDSSK,C-末端均为FEIKS,质量肽图的紫外图谱高度一致,肽段覆盖率为100%,一级结构与标准CRM197蛋白氨基酸序列完全一致。3批CRM197蛋白二级结构测算值比例差异不大,批间稳定。结论本公司自制的3批CRM197蛋白与标准CRM197蛋白结构基本一致,为研制以CRM197为载体蛋白的多糖-蛋白结合疫苗及其产业化生产奠定了基础。

Objective To confirm the structure of CRM197 protein,an avirulent mutant of diphtheria toxin prepared by Aimei Weixin Biopharmaceutical(Zhejiang)CO.,LTD.Methods Three batches of CRM197 protein were prepared and purified from the fermentation products of C.diphtheriae.N-terminal sequence analysis,C-terminal sequence analysis,peptide mass mapping and peptide coverage analysis were used to confirm the primary structure,while circular dichroism spectroscopy to confirm the secondary structure of the protein.Results All the three batches of CRM197 protein had GADDVVDSSK sequence at N-terminus and FEIKS sequence at C-terminus,with highly consistent ultraviolet spectra of peptide mass maps,and a coverage rate of peptide segments of 100%.The primary structure,i.e.amino acid sequence of the purified CRM197 protein was completely identical to that of standard CRM197 protein.There was no significant difference in the secondary structures of the three batches of CRM197 protein,indicating the stability between various batches.Conclusion The structures of three batches of prepared CMR197 protein were basically consistent with that of standard CMR197 protein,which laid a foundation of development of polysaccharide conjugate vaccine using CRM197 as a carrier.