基于报告基因的重组人促卵泡激素Fc融合蛋白生物学活性测定方法研究  

Study on biological activity assay of fusion protein of rhFSH-Fc based on the reporter gene

作　　者：孙爽[1] 王绿音 李晶[2] 吕萍[2] 徐可铮 梁誉龄 张慧[2] 李湛军[2] 梁成罡[2] SUN Shuang;WANG Lü-yin;LI Jing;Lü Ping;XU Ke-zheng;LIANG Yu-ling;ZHANG Hui;LI Zhan-jun;LIANG Cheng-gang(Shenyang Pharmaceutical University,Shenyang 110016,China;Division of Hormone,National Institutes for Food and Drug Control,NHC Key Laboratory of Research on Quality and Standardization of Biotech Products,Beijing 102629,China)

机构地区：[1]沈阳药科大学,沈阳110016 [2]中国食品药品检定研究院激素室国家卫生健康委员会生物技术产品检定方法及其标准化重点实验室,北京102629

出　　处：《药物分析杂志》2022年第1期60-67,共8页Chinese Journal of Pharmaceutical Analysis

基　　金：国家科技重大专项课题资助项目(2018ZX09101001-003-004)。

摘　　要：目的:利用转基因细胞法建立重组人促卵泡激素Fc融合蛋白(rhFSH-Fc)生物学活性检测方法。方法:利用CHO-K1-FSHR-CRE-Luc转基因细胞,按一定细胞密度种板,培养16~20 h后吸弃培养基,将rhFSH-Fc倍比稀释加入细胞板,药物作用一段时间后加入荧光素酶检测试剂,通过荧光素酶检测系统对rhFSH-Fc进行生物性活性检测,并对各试验条件参数优化及进行方法学验证。结果:rhFSH-Fc在该方法中存在量效关系且符合四参数曲线方程,R;大于0.99;方法优化后确定细胞种板密度为5×10^(5)个·mL^(-1),rhFSH-Fc稀释起始浓度为750 ng·mL^(-1),1∶5倍的稀释倍数,诱导时间为4 h,样品稀释液为DMEM/F12K+10%FBS。该方法具有良好的专属性,9次独立日间、日内精密度检测RSD均小于5%,5个不同稀释组回收率样本经6次测定,相对效价分别为(51±2.00)%、(78±2.05)%、(94±2.23)%、(124±3.46)%、(141±4.45)%;对应回收率平均值分别为(101±3.94)%、(104±3.00)%、(94±2.24)%、(99±2.68)%、(94±3.03)%,RSD均小于4%。结论:本研究利用转基因细胞成功建立rhFSH-Fc生物学活性测定方法,该方法操作简便,重复性好,准确性高,可用于rhFSH-Fc产品的常规检测。Objective:To establish a method to detect the biological activity of recombinant human follicle stimulating hormone Fc fusion protein(rhFSH-Fc)by transgenic cell method.Methods:Using CHO-K1-FSHR-CRE-Luc transgenic cells to seed the plate at a certain cell density.After the strain was cultured for 16-20 h,the medium was abandoned,and rhFSH-Fc was diluted in a multiple and added to the cell plate.After the drug acted for a period of time,the lucifase detection reagent was added,and the biological activity of rhFSH-Fc was detected through the lucifase detection system,and the parameters of each test condition were optimized and the methodology was verified.Results:rhFSH-Fc has a dose-effect relationship in this method and conforms to the four-parameter curve equation,R;was greater than 0.99;after the method was optimized,the cell seed plate density was determined to be 5×10^(5)cells·mL^(-1),and the initial dilution concentration of rhFSH-Fc was 750 ng·mL^(-1),at 1∶5 times dilution factor,induction time was 4 h,sample dilution was DMEM/F12 K+10%FBS.The method has good specificity.The RSD of the precision test within the day was less than 5%in 9 independent days.The recovery samples of 5 different dilution groups were measured 6 times,and the relative titers were(51±2.00)%,(78±2.05)%,(94±2.23)%,(124±3.46)%and(141±4.45)%.The corresponding average recoveries were(101±3.94)%,(104±3.00)%,(94±2.24)%,(99±2.68)%and(94±3.03)%,RSD were all less than 4%.Conclusion:The biological activity assay of rhFSH-Fc was successfully established by transgenic cells.The method is simple,reproducible and accurate.It could be used for routine detection of rhFSH-Fc products.

关 键 词：重组人促卵泡激素Fc融合蛋白 长效重组蛋白药物 报告基因法 环磷酸腺苷 CHO-K1-FSHR-CRE-Luc转基因细胞 生物学活性测定 方法学验证 信号通路 

分 类 号：R917[医药卫生—药物分析学]