Wheat germ cell-free system-based production of malaria proteins for discovery of novel vaccine candidates
Wheat germ cell-free system-based production of malaria proteins for discovery of novel vaccine candidates
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DOI：
10.1128/iai.01539-07
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发表时间：
2008-04-01
影响因子：
3.1
通讯作者：
Endo, Yaeta
中科院分区：
文献类型：
--
作者：
Tsuboi, Takafumi;Takeo, Satoru;Endo, Yaeta
疟疾研究的主要瓶颈之一是重组蛋白的表达困难。在这里，我们报告了小麦胚细胞无细胞系统在成功生产疟疾蛋白方面的应用。为了验证原理，我们选择了Pfs25、PfCSP和PfAMA1蛋白。这些基因含有很高的A/T序列，也很难以重组蛋白的形式表达。在我们的小麦胚芽无细胞系统中，Pfs25基因的天然版本和密码子优化版本产生了等量的蛋白质。未经密码子优化的PfCSP和PfAMA1基因也得到了表达。这些产物是可溶的，转译混合物的产量在50到200微克/毫升之间，这表明无细胞系统可以用于生产疟疾蛋白，而不需要事先优化其偏向密码子的使用。对小鼠产生的针对每种蛋白质的抗体进行生化和免疫细胞化学分析表明，每种抗体都保持了对所述寄生虫蛋白质的高度特异性。用携带恶性疟原虫配子细胞的病人血喂养蚊体，并添加我们的小鼠抗Pfs25血清，对蚊体内寄生虫的生长有很强的抑制作用，表明Pfs25-3D7/WG和Pfs25-TBV/WG均保持了其免疫原性。最后，我们进行了血液期恶性疟原虫蛋白质的平行表达实验。从现有的数据库中选择124个恶性疟原虫基因的聚合酶链式反应产物直接用于小规模的转录和翻译反应。放射自显影测试显示产生了93种蛋白质。将讨论这一新的基于无细胞系统的方案在发现疟疾候选疫苗方面的应用。
One of the major bottlenecks in malaria research has been the difficulty in recombinant protein expression. Here, we report the application of the wheat germ cell-free system for the successful production of malaria proteins. For proof of principle, the Pfs25, PfCSP, and PfAMA1 proteins were chosen. These genes contain very high A/T sequences and are also difficult to express as recombinant proteins. In our wheat germ cell-free system, native and codon-optimized versions of the Pfs25 genes produced equal amounts of proteins. PfCSP and PfAMA1 genes without any codon optimization were also expressed. The products were soluble, with yields between 50 and 200 mu g/ml of the translation mixture, indicating that the cell-free system can be used to produce malaria proteins without any prior optimization of their biased codon usage. Biochemical and immunocytochemical analyses of antibodies raised in mice against each protein revealed that every antibody retained its high specificity to the parasite protein in question. The development of parasites in mosquitoes fed patient blood carrying Plasmodium falciparum gametocytes and supplemented with our mouse anti-Pfs25 sera was strongly inhibited, indicating that both Pfs25-3D7/WG and Pfs25-TBV/WG retained their immunogenicity. Lastly, we carried out a parallel expression assay of proteins of blood-stage P. falciparum. The PCR products of 124 P. falciparum genes chosen from the available database were used directly in a small-scale format of transcription and translation reactions. Autoradiogram testing revealed the production of 93 proteins. The application of this new cell-free system-based protocol for the discovery of malaria vaccine candidates will be discussed.