Neuritin野生型蛋白的制备及活性、稳定性的检测及分析
Preparation and activity,stability detection and analysis of Neuritin wild-type protein
朱礼彦 1孟平平 1李煜 1宋丹丹 2孙嘉伟 1汪海燕 2朱金辉 1魏韬艺 1杨磊2
作者信息
- 1. 石河子大学医学院,新疆 石河子 832000
- 2. 杭州师范大学公共卫生学院,浙江 杭州 310036
- 折叠
摘要
目的 制备符合药典结构要求的无标签 Neuritin野生型(wild type,WT)蛋白,并完成其存在形式、活性及稳定性鉴定及分析.方法 利用基因重组技术构建符合药典结构要求的 Neuritin WT 酵母重组子,利用 SDS-PAGE 还原电泳及Western blot对其进行高表达筛选及鉴定;摸索建立无标签Neuritin WT蛋白纯化方法,对纯化后的蛋白进行SDS-PAGE还原电泳分子量鉴定、Western blot免疫原性鉴定、SEC-HPLC 精细鉴定及宿主 DNA、宿主蛋白残留量检测;利用 SDS-PAGE非还原电泳完成存在形式的鉴定;通过参照药典建立的重组 Neuritin 蛋白活性鉴定方法,检测并分析 Neuritin WT纯化蛋白的活性,并观察 37℃下不同时间Neuritin WT纯化蛋白的稳定性.结果 制备了纯度高达 98%以上的 Neuritin WT纯化蛋白,蛋白相对分子质量为 9.7 kDa,宿主 DNA 残留量为 0.009 4 ng/剂,宿主蛋白残留量占比 0.000 8%;经检测,该纯化蛋白存在单体、二聚体 2 种形式.活性鉴定结果表明该蛋白具有维持神经元存活的生物学活性;且 37℃下 3 d内降解率小于 10%、7 d内未到达半衰期(蛋白降解率小于 35%).结论 成功制备了纯度高达 98%、杂质含量及结构符合药典要求的无标签 Neuritin WT 蛋白,分析了 Neuritin WT 蛋白的存在形式,并明确了该蛋白具有较好的生物学活性及稳定性,为 Neuritin进一步功能、药学研究及生物制品的研制提供了物质基础.
Abstract
Objective To prepare unlabeled Neuritin wild-type(WT)protein that meets the structural requirements of the pharmacopoe-ia,and complete its identification and analysis of its form,activity,and stability.Methods Neuritin WT yeast recombinants conforming to pharmacopoeia structure were constructed by gene recombination technology,and the high-expression strains was screened and iden-tified by SDS-PAGE reduction electrophoresis and Western blot.The unlabeled Neuritin WT protein purification method was estab-lished.The purified proteins were identified by SDS-PAGE reduction electrophoresis for molecular weight,Western blot for immunoge-nicity,SEC-HPLC for fine identification,and host DNA and host protein residues for quality inspection.The existence form was identi-fied by SDS-PAGE non-reduction electrophoresis.The activity of Neuritin WT purified protein was detected and analyzed by establis-hing a method for identifying the activity of recombinant Neuritin protein in accordance with the requirements of the pharmacopoeia,and the stability of Neuritin WT purified protein at different times at 37℃was observed.Results Neuritin WT purified protein with a purity of more than 98%was prepared,the relative molecular weight of protein was 9.7 kDa,the residual amount of host DNA was 0.009 4 ng/agent and the residual amount of host protein accounted for 0.000 8%.The purified protein was detected in the form of monomer and dimer.The activity identification results indicate that the protein has biological activity to maintain neuronal survival;The degrada-tion rate within 3 days at 37℃is less than 10%,and the half-life is not reached within 7 days(the protein degradation rate is less than 35%).Conclusion Neuritin WT unlabeled protein with purity up to 98%,impurity content and structure meeting pharmacopoeia requirements was successfully prepared.The existence form of Neuritin WT protein was analyzed,and it was confirmed that the protein has good biological activity and stability.Neuritin provides material basis for further functional,pharmaceutical and biological products development.
关键词
Neuritin野生型蛋白/纯度/存在形式/神经元存活/稳定性Key words
Neuritin wild-type protein/purity/existence form/neuron survival/stability引用本文复制引用
基金项目
"重大新药创制"科技重大专项(2019ZX09301161)
石河子大学自主资助支持校级科研项目(ZZZC202136)
出版年
2024
NETL
NSTL
万方数据