Mellado Maria Candida M, Mena Jimmy A, Lopes António, Ramírez Octavio T, Carrondo Manuel J T, Palomares Laura A, Alves Paula M
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
Biotechnol Bioeng. 2009 Nov 1;104(4):674-86. doi: 10.1002/bit.22430.
Virus-like particles constitute potentially relevant vaccine candidates. Nevertheless, their behavior in vitro and assembly process needs to be understood in order to improve their yield and quality. In this study we aimed at addressing these issues and for that purpose triple- and double-layered rotavirus-like particles (TLP 2/6/7 and DLP 2/6, respectively) size and zeta potential were measured using dynamic light scattering at different physicochemical conditions, namely pH, ionic strength, and temperature. Both TLP and DLP were stable within a pH range of 3-7 and at 5-25 degrees C. Aggregation occurred at 35-45 degrees C and their disassembly became evident at 65 degrees C. The isoelectric points of TLP and DLP were 3.0 and 3.8, respectively. In vitro kinetics of TLP disassembly was monitored. Ionic strength, temperature, and the chelating agent employed determined disassembly kinetics. Glycerol (10%) stabilized TLP by preventing its disassembly. Disassembled TLP was able to reassemble by dialysis at high calcium conditions. VP7 monomers were added to DLP in the presence of calcium to follow in vitro TLP assembly kinetics; its assembly rate being mostly affected by pH. Finally, DLP and TLP were found to coexist under certain conditions as determined from all reaction products analyzed by capillary electrophoresis. Overall, these results contribute to the design of new strategies for the improvement of TLP yield and quality by reducing the VP7 detachment from TLP.
病毒样颗粒构成了潜在的相关疫苗候选物。然而，为了提高它们的产量和质量，需要了解它们在体外的行为和组装过程。在本研究中，我们旨在解决这些问题，为此，使用动态光散射在不同的物理化学条件下，即pH、离子强度和温度下，测量了三层和双层轮状病毒样颗粒（分别为TLP 2/6/7和DLP 2/6）的大小和zeta电位。TLP和DLP在pH值为3至7以及5至25摄氏度的范围内均稳定。在35至45摄氏度时发生聚集，在65摄氏度时它们的解体变得明显。TLP和DLP的等电点分别为3.0和3.8。监测了TLP解体的体外动力学。离子强度、温度和所使用的螯合剂决定了解体动力学。甘油（10%）通过防止TLP解体使其稳定。解体的TLP能够在高钙条件下通过透析重新组装。在有钙存在的情况下，将VP7单体添加到DLP中以跟踪体外TLP组装动力学；其组装速率主要受pH影响。最后，通过毛细管电泳分析所有反应产物确定，发现DLP和TLP在某些条件下共存。总体而言，这些结果有助于设计新策略，通过减少VP7从TLP上的脱离来提高TLP的产量和质量。
