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

作　　者：朱礼彦 孟平平 李煜 宋丹丹 孙嘉伟 汪海燕[2] 朱金辉 魏韬艺 杨磊[2] ZHU Liyan;MENG Pingping;LI Yu;SONG Dandan;SUN Jiawei;WANG Haiyan;ZHU Jinhui;WEI Taoyi;YANG Lei(School of Medicine,Shihezi University,Shihezi,Xinjiang 832000,China;Department of Medicine,Hangzhou Normal University,Hangzhou,Zhejiang 310036,China)

机构地区：[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

出　　处：《石河子大学学报（自然科学版）》2024年第2期215-222,共8页Journal of Shihezi University(Natural Science)

基　　金：国家重大新药创制科技重大专项项目(2019ZX09301161);石河子大学自主资助支持校级科研项目(ZZZC202136)。

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0.0094 ng/剂,宿主蛋白残留量占比0.0008%;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。Objective To prepare unlabeled Neuritin wild-type(WT)protein that meets the structural requirements of the pharmacopoeia,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 identified by SDS-PAGE reduction electrophoresis and Western blot.The unlabeled Neuritin WT protein purification method was established.The purified proteins were identified by SDS-PAGE reduction electrophoresis for molecular weight,Western blot for immunogenicity,SEC-HPLC for fine identification,and host DNA and host protein residues for quality inspection.The existence form was identified by SDS-PAGE non-reduction electrophoresis.The activity of Neuritin WT purified protein was detected and analyzed by establishing 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 0.0094 ng/agent and the residual amount of host protein accounted for 0.0008%.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 degradation 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,

关 键 词：Neuritin野生型蛋白 纯度 存在形式 神经元存活 稳定性 

分 类 号：R34[医药卫生—基础医学]