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重组带状疱疹gE抗原等电点(毛细管电泳法)检测方法的建立及初步应用
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Abstract:
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.
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