摘要
目的基于紫外分光光度法,建立一种测定白喉毒素无毒突变体CRM197蛋白含量的方法。方法在天然折叠状态和非折叠状态(CRM197蛋白经盐酸胍变性)下,分别测定相同含量的CRM197蛋白A_(280 nm)值,再根据朗伯-比尔定律c=A/εL计算其折叠状态消光系数。同时,使用当前建立的方法与BCA法分别测定CRM197蛋白的含量,并对2种方法的差异和检测特点进行比较分析。结果根据CRM197蛋白的氨基酸一级序列与相对分子质量计算出其理论消光系数为0.934 mL/(mg·cm),在280 nm处的实验消光系数为(0.959±0.006)mL/(mg·cm)。2种方法测定同一蛋白样本的结果一致,分别为(2.17±0.02)mg/mL和(2.14±0.09)mg/mL;本方法检测值的CV为0.70%,BCA法检测值的CV为4.15%。结论本研究建立的CRM197蛋白含量测定方法准确性高、线性范围宽、获取数据快,可用于CRM197蛋白含量的测定,同时为其他载体蛋白的含量测定提供了参考。
Objective To establish a method for determining the content of CRM197,a non-toxic mutant of diphtheria toxin by ultraviolet spectrophotometry.Methods The same content(c)of CRM197 protein in the folded and the unfolded state(CRM197 denatured in guanidine hydrochloride)were measured by using absorbance at 280 nm,respectively.Then,its extinction coefficient in the folded state was calculated according to the Lambert-Beer law c=A/εL.Also,the content of CRM197 protein was determined by this method and BCA method,respectively.The differences and detection characteristics of the two methods were compared and analyzed.Results According to the amino acid sequence and relative molecular mass of CRM197 protein,its theoretical extinction coefficient was calculated to be 0.934 mL·mg^(-1)·cm^(-1),and the experimental extinction coefficient at 280 nm was(0.959±0.006)mL·mg^(-1)·cm^(-1).The results of the two methods for determining the same protein sample were consistent,which were(2.17±0.02)mg/mL and(2.14±0.09)mg/mL,respectively.The coefficient of variation of this method was 0.70%,and that of BCA was 4.15%.Conclusion The method for determining the content of CRM197 protein has the advantages of high accuracy,wide linear rang and rapid data acquisition,which can be used for the determination of CRM197 protein content and provides a reference for the determination of other carrier proteins.
作者
何刚
黄杰
冯树宏
于健
孙俊
魏鑫
侯文礼
HE Gang;HUANG Jie;FENG Shu-hong;YU Jian;SUN Jun;WEI Xin;HOU Wen-li(Research and Development Center,Chengdu Kanghua Biological Products Co.,Ltd.,Chengdu 611130,Sichuan Province,China)
出处
《微生物学免疫学进展》
CAS
2022年第6期47-51,共5页
Progress In Microbiology and Immunology
基金
四川省科技创新人才项目(2022JDRC0048)。
关键词
白喉毒素
无毒突变体
CRM197蛋白
消光系数
紫外分光光度法
BCA法
蛋白含量
Diphtheria toxin
Non-toxic mutant
CRM197 protein
Extinction coefficient
Ultraviolet spectrophotometry
Bicinchonininc acid method
Protein content
