Salavatifar Maryam, Zakeri Sedigheh, Hayati Roodbari Nasim, Djadid Navid Dinparast
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Malaria and Vector Research Group (MVRG), Biotechnology Research Center (BCR), Pasteur Institute of Iran, Tehran, Iran.
Cell J. 2015 Fall;17(3):520-31. doi: 10.22074/cellj.2015.12. Epub 2015 Oct 7.
The apical membrane antigen-1 (AMA-1) is considered as a promising candidate for development of a malaria vaccine against Plasmodium parasites. The correct conformation of this protein appears to be necessary for the stimulation of parasite-inhibitory responses, and these responses, in turn, seem to be antibody-mediated. Therefore, in the present investigation, we expressed the Plasmodium vivax AMA-1 (PvAMA-1) ectodomain in Escherichia coli (E. coli), purified it using standard procedures and characterized it to determine its biological activities for it to be used as a potential target for developing a protective and safe vivax malaria vaccine.
In this experimental investigation, the ectodomain of PvAMA-1 antigen (GenBank accession no. JX624741) was expressed in the E. coli M15pQE30 expression system and purified with immobilized-metal affinity chromatography. The correct conformation of the recombinant protein was evaluated by Western blotting and indirect immunofluorescence antibody (IFA) test. In addition, the immunogenic properties of PvAMA-1 were evaluated in BALB/c mice with the purified protein emulsified in Freund's adjuvant.
In the present study, the PvAMA-1 ectodomain was expressed at a high-level (65 mg/L) using a bacterial system. Reduced and non-reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as well as Western blot analysis confirmed the appropriate conformation and folding of PvAMA-1. The evaluation of immunogenic properties of PvAMA-1 showed that both T helper-1 and 2 cells (Th1 and Th2) responses were present in mice after three immunizations and persisted up to one year after the first immunization. Moreover, the antibodies raised against the recombinant PvAMA-1 in injected mice could recognize the native protein localized on P. vivax parasites.
We demonstrate that our recombinant protein had proper conformation and folding. Also, there were common epitopes in the recombinant forms corresponding to native proteins. These results; therefore, indicate that the expressed PvAMA-1 has the potential to be used as a vivax malaria vaccine.
顶膜抗原1（AMA-1）被认为是开发抗疟原虫疟疾疫苗的一个有前景的候选物。该蛋白的正确构象似乎对于刺激寄生虫抑制反应是必要的，而这些反应反过来似乎是由抗体介导的。因此，在本研究中，我们在大肠杆菌（E. coli）中表达了间日疟原虫AMA-1（PvAMA-1）胞外结构域，使用标准程序对其进行纯化，并对其进行表征以确定其生物学活性，使其能够用作开发安全有效的间日疟疟疾疫苗的潜在靶点。
在本实验研究中，PvAMA-1抗原（GenBank登录号：JX624741）的胞外结构域在大肠杆菌M15pQE30表达系统中表达，并用固定金属亲和色谱法进行纯化。通过蛋白质印迹法和间接免疫荧光抗体（IFA）试验评估重组蛋白的正确构象。此外，用弗氏佐剂乳化的纯化蛋白在BALB/c小鼠中评估PvAMA-1的免疫原性。
在本研究中，使用细菌系统高水平表达了PvAMA-1胞外结构域（65 mg/L）。还原和非还原十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）以及蛋白质印迹分析证实了PvAMA-1的适当构象和折叠。对PvAMA-1免疫原性的评估表明，三次免疫后小鼠体内同时存在辅助性T细胞1和2（Th1和Th2）反应，并且在首次免疫后持续长达一年。此外，注射小鼠中针对重组PvAMA-1产生的抗体能够识别定位于间日疟原虫寄生虫上的天然蛋白。
我们证明我们的重组蛋白具有适当的构象和折叠。此外，重组形式中存在与天然蛋白相对应的共同表位。因此，这些结果表明所表达的PvAMA-1有潜力用作间日疟疟疾疫苗。
