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目的采用新型层析技术快速纯化人乳头瘤病毒(human papillomavirus,HPV)L1蛋白病毒样颗粒(virus-like particles,VLPs)。方法应用新型的基于阴离子交换和分子筛原理的色谱填料CaptoCore 700初纯昆虫细胞表达的HPV L1蛋白VLPs,再用高流速、高分辨力的阴离子交换树脂Capto Q ImpRes进一步纯化。各步纯化的样品经SDSPAGE进行分析,采用ImageQuant图像分析软件分析两步纯化后蛋白的纯度,并对初纯蛋白进行质谱分析;改良Lowry法测定初纯蛋白的浓度,并计算杂蛋白去除率。结果昆虫细胞表达的HPV L1蛋白经CaptoCore 700初纯后,目标蛋白组成的VLPs存在于流穿组分中,经质谱分析,为目的蛋白HPV L1;料液处理量为1个柱体积时,杂蛋白去除率达80%,料液处理量为2个柱体积时,杂蛋白去除率为76%。初纯的蛋白经Capto Q ImpRes进一步层析纯化,HPV L1蛋白的纯度接近100%。结论 CaptoCore 700加Capto Q ImpRes两步层析纯化能够快速高效地纯化HPV L1蛋白VLPs,整个工艺过程简单、快速、高效、易放大,适用于大规模生产。
Objective To purify virus-like particles (VLPs) of human papillomavirus (HPV) L1 using novel chromatography. Methods The novel HPV L1 VLPs expressed in the primordial insect cells based on anion exchange and molecular sieve principle were further purified by Capto Q ImpRes, a high-flow and high-resolution anion exchange resin. Samples were purified by SDSPAGE. ImageQuant image analysis software was used to analyze the purity of the two-step purified protein. The purity of the purified protein was determined by modified Lowry method. The impurity removal rate was also calculated. Results The HPV L1 protein expressed by insect cells was purified by CaptoCore 700. The VLPs of the target proteins existed in the flow-through components and were analyzed by mass spectrometry for the HPV L1 protein. When the volume of the liquid was 1 column volume, Protein removal rate of 80%, feed volume of 2 column volume, the hybrid protein removal rate was 76%. Purified proteins were further purified by Capto Q ImpRes. The purity of the HPV L1 protein was approximately 100%. Conclusion CaptoCore 700 plus Capto Q ImpRes two-step purification can rapidly and efficiently purify HPV L1 protein VLPs. The whole process is simple, rapid, efficient and easy to amplify and is suitable for large-scale production.