离子交换和疏水层析法代替盐析沉淀技术精制破伤风毒素初探  

Study on purification of tetanus toxin by ion exchange and hydrophobic chromatography instead of salt precipitation

作　　者：田贝贝 崔晓峰 郭旺 孙伟 李津 安文珏 潘若文 高新灿 TIAN Beibei;CUI Xiaofeng;GUO Wang;SUN Wei;LI Jin;AN Wenjue;PAN Ruowen;GAO Xincan(Vaccine Chamber 2,Hualan Biological Bacterin Inc.,Xinxiang 453003,Henan Province,China;不详)

机构地区：[1]华兰生物疫苗股份有限公司菌苗二室,河南新乡453003 [2]河南省生物医药产业研究院,河南新乡453003 [3]河南省预防型疫苗工程技术研究中心,河南新乡453003

出　　处：《微生物学免疫学进展》2023年第4期25-29,共5页Progress In Microbiology and Immunology

摘　　要：目的用离子交换+疏水层析法代替传统的盐析沉淀技术制备高纯度精制破伤风毒素。方法破伤风毒素浓缩液经Q Sepharose Fast Flow(QFF)离子交换层析去除杂蛋白,将收集到目标蛋白破伤风毒素经2.0 mol/L硫酸铵-磷酸盐缓冲液等量稀释后,再用Phenyl Sepharose^(TM)6 Fast Flow(high sub)疏水层析去除大部分杂质及色素制得纯度较高的精制破伤风毒素。结果用离子交换+疏水层析法纯化破伤风毒素实验重复3次,精制破伤风毒素絮状单位平均值达到3600 Lf/mL,纯度平均值为100%,精制破伤风毒素的特异蛋白条带相对分子质量分别在148000~153000、98000~103000、53000~57000范围内。与盐析法相比,离子交换+疏水层析法各项指标均有较大提高。结论离子交换+疏水层析法能有效、稳定地制备符合要求的精制破伤风毒素。在破伤风毒素的纯化中,离子交换+疏水层析法可取代传统的盐析沉淀技术。Objective To prepare purified tetanus toxin by ion exchange and hydrophobic chromatography instead of traditional salting-out precipitation.Methods Concentrated fermentation broth of tetanus toxin was subjected to Q Sepharose Fast Flow(QFF)ion-exchange chromatography to remove protein impurity.QFF-purified tetanus toxin was collected and 1:1 diluted with 2.0 mol/L ammonium sulphate-phosphate buffer.Then hydrophobic chromatography with Phenyl Sepharose^(TM) 6 Fast Flow(high sub)was adopted to remove most impurities and pigments to prepare therefined tetanus toxin.Results Tetanus toxin was purified through ion-exchange and hydrophobic chromatography 3 times repeatedly.The average flocculent unit value of the refined tetanus toxin was 3600 Lf/mL and the average purity was 100%for 3 consecutive experimental samples.Relative molecular weights of the specific protein bands of the purified tetanus toxin were within the range of 148000-153000,98000-103000 and 53000-57.000,respectively.Compared with salting-out method,the indices of ionexchange and hydrophobic chromatography were improved greatly.Conclusion Ion-exchange and hydrophobic chromatography can be applied to prepare tetanus toxin effectively and repeatablely.In the purification of tetanus toxin,ion-exchange and hydrophobic chromatography can replace traditional salting-out precipitation technique.

关 键 词：破伤风毒素 离子交换层析 疏水层析 纯度 纯化 

分 类 号：R378[医药卫生—病原生物学]