Grothaus G D, Kreier J P
Infect Immun. 1980 Apr;28(1):245-53. doi: 10.1128/iai.28.1.245-253.1980.
Soluble material was obtained from sonically freed plasmodiae by three procedures. Two procedures, cryo-impacting and freeze-thawing, were evaluated for their ability to disrupt the parasites and release soluble material. The soluble materials obtained by these procedures were compared to materials washed from the surfaces of sonically freed parasites. Between 35 and 40% of the total parasite protein was solubilized by freeze-thawing or cryo-impacting. One cycle of freeze-thawing released nearly as much protein as could be released by this method, and additional cycles of freeze-thawing had little additional effect. Cryo-impacting solubilized only a small amount of protein in addition to that which was released by the cycle of freeze-thawing inherent in the procedure. Reductions in the packed cell volume of the material remaining after freeze-thawing or cryo-impacting indicate that the insoluble fragments are broken into smaller pieces as treatment is extended. Electron microscopy of 30-s cryo-impacted and three-times freeze-thawed parasites revealed membrane fragments similar in appearance. Patterns obtained by polyacrylamide gel electrophoresis of the soluble material from freeze-thawed and cryo-impacted parasites were also similar, and approximately 13 protein bands were demonstrated. The material washed from the surfaces of the free parasites, on the other hand, resolved into only two to four major bands on the gel columns. In immunization studies, the soluble and insoluble fractions obtained by freeze-thawing or cryo-impacting and the material washed from the surfaces of the parasites all stimulated a protective immune response. On the basis of the amount of protein required to stimulate roughly comparable immunity, the soluble fraction obtained by freeze-thawing or cryo-impacting free parasites was about twice as potent an immunogen as was the insoluble fraction. The material obtained by gentle washing of the freed parasites was approximately 20 times as potent an immunogen as were the freed parasites and about 7 times as potent as the soluble material obtained by freeze-thawing or cryo-impacting.
通过三种方法从经超声处理释放的疟原虫中获得可溶性物质。对两种方法，即冷冻冲击法和冻融法，评估了它们破坏寄生虫并释放可溶性物质的能力。将通过这些方法获得的可溶性物质与从经超声处理释放的寄生虫表面洗下的物质进行比较。通过冻融或冷冻冲击可溶解35%至40%的寄生虫总蛋白。一个冻融循环释放的蛋白量几乎与该方法所能释放的量相同，额外的冻融循环几乎没有额外效果。冷冻冲击除了溶解该过程中固有的冻融循环所释放的蛋白外，仅溶解少量蛋白。冻融或冷冻冲击后剩余物质的红细胞压积降低表明，随着处理时间延长，不溶性碎片被分解成更小的碎片。对经30秒冷冻冲击和三次冻融的寄生虫进行电子显微镜观察，发现膜碎片外观相似。对冻融和冷冻冲击后的寄生虫可溶性物质进行聚丙烯酰胺凝胶电泳得到的图谱也相似，显示出约13条蛋白带。另一方面，从游离寄生虫表面洗下的物质在凝胶柱上仅分离出两到四条主要条带。在免疫研究中，通过冻融或冷冻冲击获得的可溶性和不溶性部分以及从寄生虫表面洗下的物质均能刺激保护性免疫反应。基于刺激大致相当的免疫力所需的蛋白量，通过冻融或冷冻冲击游离寄生虫获得的可溶性部分作为免疫原的效力约为不溶性部分的两倍。通过温和洗涤游离寄生虫获得的物质作为免疫原的效力约为游离寄生虫的20倍，约为通过冻融或冷冻冲击获得的可溶性物质的7倍。
