目的:建立重组带状疱疹gE抗原等电点(isoelectric point, pI)的全柱成像毛细管等电聚焦电泳(capillary isoelectric focusing, cIEF)检测方法。方法:通过优化重组带状疱疹gE抗原等电点检测过程中的两性电解质比例、蛋白质浓度、稳定剂...目的:建立重组带状疱疹gE抗原等电点(isoelectric point, pI)的全柱成像毛细管等电聚焦电泳(capillary isoelectric focusing, cIEF)检测方法。方法:通过优化重组带状疱疹gE抗原等电点检测过程中的两性电解质比例、蛋白质浓度、稳定剂比例参数,建立重组带状疱疹gE抗原的等电点检测方法(cIEF法)。在此基础上,对重组带状疱疹gE抗原进行脱糖酶处理后,采用cIEF法检测等电点。结果:优化后的检测条件为2 M尿素、0.35%甲基纤维素(methylcellulose, MC)、4%两性电解质、1%等电点标记物混合溶液作为样品缓冲液,聚焦条件为1500 V 1 min,3000 V 8 min。重组带状疱疹gE抗原经PNGase F酶脱糖后,采用该方法分析等电点,可得到峰型清楚且分离度高的图谱。结论:本研究建立的cIEF方法适用于重组带状疱疹gE抗原的pI检测及分析,可为重组带状疱疹gE抗原的表征以及相关疫苗研发和生产过程中的质量控制提供依据和参考。Objective: To establish a full column imaging capillary isoelectric focusing (cIEF) detection method for recombinant herpes zoster gE antigen isoelectric point (pI). Method: By optimizing the parameters such as reagent ratio, final sample concentration, and sample stabilizer in the process of detecting recombinant herpes zoster gE antigen, a cIEF method for determining recombinant herpes zoster gE antigen was established. Based on this, the recombinant herpes zoster gE antigen was subjected to enzymatic digestion before cIEF detection. Results: The optimized method uses a mixed solution of 2 M urea, 0.35% methylcellulose (MC), 4% zwitterionic electrolyte, and 1% isoelectric point marker as the sample buffer, with focusing conditions of 1500 V for 1 min and 3000 V for 8 min. After PNGase F enzyme deglycosylation, this method can be used to analyze the isoelectric point of recombinant herpes zoster gE antigen, and a clear peak pattern and high separation degree can be obtained. Conclusion: The established cIEF method is suitable for pI detection and analysis of recombinant herpes zoster gE antigen, which can provide a basis for the characterization of recombinant herpes zoster gE antigen and a reference for quality control in the development and production of related vaccines.展开更多
目的用离子交换+疏水层析法代替传统的盐析沉淀技术制备高纯度精制破伤风毒素。方法破伤风毒素浓缩液经Q Sepharose Fast Flow(QFF)离子交换层析去除杂蛋白,将收集到目标蛋白破伤风毒素经2.0 mol/L硫酸铵-磷酸盐缓冲液等量稀释后,再用Ph...目的用离子交换+疏水层析法代替传统的盐析沉淀技术制备高纯度精制破伤风毒素。方法破伤风毒素浓缩液经Q Sepharose Fast Flow(QFF)离子交换层析去除杂蛋白,将收集到目标蛋白破伤风毒素经2.0 mol/L硫酸铵-磷酸盐缓冲液等量稀释后,再用Phenyl Sepharose^(TM)6 Fast Flow(high sub)疏水层析去除大部分杂质及色素制得纯度较高的精制破伤风毒素。结果用离子交换+疏水层析法纯化破伤风毒素实验重复3次,精制破伤风毒素絮状单位平均值达到3600 Lf/mL,纯度平均值为100%,精制破伤风毒素的特异蛋白条带相对分子质量分别在148000~153000、98000~103000、53000~57000范围内。与盐析法相比,离子交换+疏水层析法各项指标均有较大提高。结论离子交换+疏水层析法能有效、稳定地制备符合要求的精制破伤风毒素。在破伤风毒素的纯化中,离子交换+疏水层析法可取代传统的盐析沉淀技术。展开更多
文摘目的:建立重组带状疱疹gE抗原等电点(isoelectric point, pI)的全柱成像毛细管等电聚焦电泳(capillary isoelectric focusing, cIEF)检测方法。方法:通过优化重组带状疱疹gE抗原等电点检测过程中的两性电解质比例、蛋白质浓度、稳定剂比例参数,建立重组带状疱疹gE抗原的等电点检测方法(cIEF法)。在此基础上,对重组带状疱疹gE抗原进行脱糖酶处理后,采用cIEF法检测等电点。结果:优化后的检测条件为2 M尿素、0.35%甲基纤维素(methylcellulose, MC)、4%两性电解质、1%等电点标记物混合溶液作为样品缓冲液,聚焦条件为1500 V 1 min,3000 V 8 min。重组带状疱疹gE抗原经PNGase F酶脱糖后,采用该方法分析等电点,可得到峰型清楚且分离度高的图谱。结论:本研究建立的cIEF方法适用于重组带状疱疹gE抗原的pI检测及分析,可为重组带状疱疹gE抗原的表征以及相关疫苗研发和生产过程中的质量控制提供依据和参考。Objective: To establish a full column imaging capillary isoelectric focusing (cIEF) detection method for recombinant herpes zoster gE antigen isoelectric point (pI). Method: By optimizing the parameters such as reagent ratio, final sample concentration, and sample stabilizer in the process of detecting recombinant herpes zoster gE antigen, a cIEF method for determining recombinant herpes zoster gE antigen was established. Based on this, the recombinant herpes zoster gE antigen was subjected to enzymatic digestion before cIEF detection. Results: The optimized method uses a mixed solution of 2 M urea, 0.35% methylcellulose (MC), 4% zwitterionic electrolyte, and 1% isoelectric point marker as the sample buffer, with focusing conditions of 1500 V for 1 min and 3000 V for 8 min. After PNGase F enzyme deglycosylation, this method can be used to analyze the isoelectric point of recombinant herpes zoster gE antigen, and a clear peak pattern and high separation degree can be obtained. Conclusion: The established cIEF method is suitable for pI detection and analysis of recombinant herpes zoster gE antigen, which can provide a basis for the characterization of recombinant herpes zoster gE antigen and a reference for quality control in the development and production of related vaccines.
文摘目的用离子交换+疏水层析法代替传统的盐析沉淀技术制备高纯度精制破伤风毒素。方法破伤风毒素浓缩液经Q Sepharose Fast Flow(QFF)离子交换层析去除杂蛋白,将收集到目标蛋白破伤风毒素经2.0 mol/L硫酸铵-磷酸盐缓冲液等量稀释后,再用Phenyl Sepharose^(TM)6 Fast Flow(high sub)疏水层析去除大部分杂质及色素制得纯度较高的精制破伤风毒素。结果用离子交换+疏水层析法纯化破伤风毒素实验重复3次,精制破伤风毒素絮状单位平均值达到3600 Lf/mL,纯度平均值为100%,精制破伤风毒素的特异蛋白条带相对分子质量分别在148000~153000、98000~103000、53000~57000范围内。与盐析法相比,离子交换+疏水层析法各项指标均有较大提高。结论离子交换+疏水层析法能有效、稳定地制备符合要求的精制破伤风毒素。在破伤风毒素的纯化中,离子交换+疏水层析法可取代传统的盐析沉淀技术。
