重组人Neuritin蛋白生物学活性的定量分析方法  

Methods about quantitative analysis of biological activity of recombinant human Neuritin protein

作　　者：王宿洁 宋丹丹 李煜 孟平平 朱金辉 朱礼彦 黄瑾[1] WANG Sujie;SONG Dandan;LI Yu;MENG Pingping;ZHU Jinhui;ZHU Liyan;HUANG Jin(Medical College of Shihezi University,Shihezi,Xinjiang 832000,China;School of Public Health,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）》2022年第5期611-619,共9页Journal of Shihezi University(Natural Science)

基　　金：国家科技重大专项(民口)重大新药创制项目(2019ZX09301-161);浙江省重点研发项目(2019C03060)。

摘　　要：目的建立一种快速高效地定量评价重组人Neuritin(rhNeuritin)蛋白生物学活性的新方法。方法首先建立小鼠海马神经元细胞(HT22细胞)维持培养条件。在此条件下,明确rhNeuritin蛋白作用于HT22细胞的最佳检测时间。根据11组不同浓度梯度的rhNeuritin蛋白对HT22细胞存活的影响,建立标准品rhNeuritin蛋白与HT22细胞存活的量效关系曲线;并确定rhNeuritin蛋白的检测范围。在rhNeuritin蛋白的检测限内,根据标准品rhNeuritin蛋白与HT22细胞存活之间的量效关系的拟合曲线,制订标准品蛋白的活性单位,建立了针对rhNeuritin蛋白生物学活性的评价体系。利用两批测试品rhNeuritin蛋白,对该方法的重复性和准确性进行了验证。结果HT22细胞的维持培养液的浓度为0.4%FBS,rhNeuritin蛋白作用于HT22细胞的最佳检测时间为72 h,rhNeuritin蛋白浓度在62.5~1000 ng·mL^(-1)的检测限内,HT22细胞的存活与rhNeuritin蛋白的浓度呈正相关。在rhNeuritin蛋白的检测线内,拟定标准品的EC50为一个活性单位。两组测试品rhNeuritin蛋白的量效关系曲线与标准品呈现相同趋势,3者R 2均大于0.9,说明该方法的重复性很好。通过拟订的标准品的活性单位,计算得到500 ng·mL^(-1)时测试品1和测试品2的活性单位分别为1.059、1.069,而实验实际测得测试品1和测试品2的活性单位分别为1.074和1.088,与测定的活性单位相比,两组测试品计算得到的活性单位的误差小于2%,说明该实验方法具有较好的准确性。结论成功建立了重组人Neuritin蛋白的定量检测方法,并制定了明确的活性单位。Objective To establish a new method for rapid and efficient evaluation of biological activity of Recombinant human Neuritin protein.Methods Firstly,the maintenance culture conditions of mouse hippocampal neurons(HT22 cells)were established,and the optimal detection time of rhNeuritin protein on HT22 cells was determined under these conditions.According to the effect of rhNeuritin on the survival of HT22 cells at 11 groups of concentration gradients,the dose-response relationship between rhNeuritin and HT22 cell survival was established,and the detection range of rhNeuritin was determined.Within the detection limit of rhNeuritin,according to the fitting curve of dose-effect relationship between rhNeuritin and HT22 cell survival,the activity unit of the standard protein was established,and the evaluation system for biological activity of rhNeuritin was established.Two batches of rhNeuritin proteins were used to verify the repeatability and accuracy of the method.Results The concentration of maintenance medium for HT22 cells was 0.4%FBS.The optimal detection time of rhNeuritin in HT22 cells was 72 h.And the survival of HT22 cells was positively correlated with the concentration of rhNeuritin within the detection range from 62.5 ng·mL^(-1) to 1000 ng·mL^(-1).In the detection line of rhNeuritin,the EC50 of the standard was defined as an active unit.The dose-response curve of the two groups of rhNeuritin protein showed the same trend as that of the standard,and the R 2 of the three samples were all greater than 0.9,indicating that the method was reproducible.Based on the activity unit of the standard,the activity units of test substance 1 and test substance 2 at 500 ng·mL^(-1) were calculated to be 1.059 and 1.069,respectively.In the experiment,the activity units of test substance 1 and test substance 2 were 1.074 and 1.088,respectively.Compared with the measured activity units,the error of the calculated activity units of the two groups of test samples was less than 2%,indicating that the experimental method has a

关 键 词：重组人Neuritin蛋白 小鼠海马神经元HT22细胞 细胞存活率 活性单位 

分 类 号：Q392[生物学—遗传学]