[Objectives]This study was conducted to obtain a Chinese hamster ovary cell line that stably expresses recombinant human coagulation factor X(rhFX),and to induce efficient expression of the target gene with different ...[Objectives]This study was conducted to obtain a Chinese hamster ovary cell line that stably expresses recombinant human coagulation factor X(rhFX),and to induce efficient expression of the target gene with different concentrations of methotrexate(MTX).[Methods]PCR was performed to obtain the rhFX gene,and a recombinant expression plasmid pOptiVEC-rhFX was constructed and subjected to double restriction endonuclease digestion and sequencing identification.CHO-DG44(DHFR-)cells were transfected by the liposome method,and the target protein was purified by affinity chromatography and detected by SDS-PAGE electrophoresis and Western blot.A cell line with efficient and stable expression of the target gene was obtained by increasing the concentration of MTX to select positive clones.[Results]PCR yielded a 1509 bp rhFX sequence,and the results of double digestion and sequencing showed that the constructed pOptiVEC-rhFX plasmid was correct.After transfection of cells,MTX significantly increased protein expression.When MTX reached 1.0μmol/L,the expression efficiency of the target protein was(9±0.27)μg/ml.The purity of the target protein purified by affinity chromatography was 93%,which could be used for subsequent experiments.The expression efficiency of rhFX in eukaryotic mammalian cells was improved by increasing MTX concentration,and an affinity chromatography purification process for the target protein was preliminarily established.[Conclusions]The results of this study provide data support for the expression and purification of rhFX,and will lay a solid foundation for the development of drugs related to rhFX.展开更多
基金Supported by Anhui Provincial Natural Science Foundation of China(2008085MC65)Natural Science Foundation of Anhui Higher Education Institutions of China(KJ2021A0922)+1 种基金China Postdoctoral Science Foundation(2020T130117ZX,2020M671914)Research Activities of Postdoctoral Researchers Foundation of Anhui Province,China(2020B470)。
文摘[Objectives]This study was conducted to obtain a Chinese hamster ovary cell line that stably expresses recombinant human coagulation factor X(rhFX),and to induce efficient expression of the target gene with different concentrations of methotrexate(MTX).[Methods]PCR was performed to obtain the rhFX gene,and a recombinant expression plasmid pOptiVEC-rhFX was constructed and subjected to double restriction endonuclease digestion and sequencing identification.CHO-DG44(DHFR-)cells were transfected by the liposome method,and the target protein was purified by affinity chromatography and detected by SDS-PAGE electrophoresis and Western blot.A cell line with efficient and stable expression of the target gene was obtained by increasing the concentration of MTX to select positive clones.[Results]PCR yielded a 1509 bp rhFX sequence,and the results of double digestion and sequencing showed that the constructed pOptiVEC-rhFX plasmid was correct.After transfection of cells,MTX significantly increased protein expression.When MTX reached 1.0μmol/L,the expression efficiency of the target protein was(9±0.27)μg/ml.The purity of the target protein purified by affinity chromatography was 93%,which could be used for subsequent experiments.The expression efficiency of rhFX in eukaryotic mammalian cells was improved by increasing MTX concentration,and an affinity chromatography purification process for the target protein was preliminarily established.[Conclusions]The results of this study provide data support for the expression and purification of rhFX,and will lay a solid foundation for the development of drugs related to rhFX.
文摘目的探讨STAGO全自动血凝仪在人凝血因子活性测定上的应用。方法对STAGO全自动血凝仪按照GMP要求进行安装确认(installation qualification,IQ):实施信息核查并记录;运行确认(operational qualification,OQ):准确度、精密度(批内精密度及批间精密度)、干扰试验及参考范围的验证确认;性能确认(performance qualification,PQ):按STAGO凝血分析仪相关SOP检测6份人凝血因子Ⅷ样品活性,并计算6份样品检测值间的相对标准偏差(relative standard deviation,RSD)。结果已核实STAGO全自动血凝仪基本信息,安装环境及安装过程均符合要求,并已进行记录。厂家提供的正常及病理值质控血清测定3次的均值均在厂家提供的质控范围内;批内及批间精密性的变异系数(coefficient of variation,CV)均<10%;随机组与对照组相比平均偏差<10%;20份体检样本的均值、标准偏差(standard deviation,SD)及结果在正常参考范围的例数与总标本数的比例(R值)>0.9。6份人凝血因子Ⅷ样品检测值的RSD值为1.95%。结论经全面的设备确认,STAGO全自动血凝仪符合人凝血因子活性测定的要求。
