Lau Y L, Hasan M T, Thiruvengadam G, Idris M M, Init I
Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
Trop Biomed. 2010 Dec;27(3):525-33.
GRA4 of Toxoplasma gondii has been shown to prompt IgG, IgM and IgA responses in previous studies and is thus considered one of the major immunogenic proteins from T. gondii that can be used for both diagnostics purposes and vaccine development. This study seeks to clone and express the GRA4 in Pichia pastoris, which has numerous advantages over other systems for expression of eukaryotic proteins. In order to achieve this, the gene was cloned into the pPICZα A expression vector, which was then incorporated into the P. pastoris genome via insertional integration for expression of the recombinant protein, under the AOX1 promoter. The antigen was expressed along with the prepro sequence of the α-factor of yeast so that it could be excreted out of the P. pastoris cells and obtained from the medium. Upon SDS-PAGE analysis it was found that the recombinant protein was expressed optimally as a 40 kDa protein after 96 hours of induction with 0.75% of methanol. The expressed GRA4 protein showed discrepancy in size with the calculated molecular mass. This may be attributed to the various posttranslational modifications including glycosylation and phosphorylation. Despite the difference in molecular weight, the recombinant protein was able to detect toxoplasmosis in Western blot format. The recombinant GRA4 was expressed with an intact polyhistidine-tag, which could be used for future purification of the antigen.
先前的研究表明，弓形虫的GRA4能引发IgG、IgM和IgA反应，因此被认为是弓形虫的主要免疫原性蛋白之一，可用于诊断和疫苗开发。本研究旨在克隆并在毕赤酵母中表达GRA4，毕赤酵母在表达真核蛋白方面比其他系统有许多优势。为实现这一目标，将该基因克隆到pPICZα A表达载体中，然后通过插入整合将其整合到毕赤酵母基因组中，在AOX1启动子的控制下表达重组蛋白。抗原与酵母α因子的前原序列一起表达，以便其能分泌到毕赤酵母细胞外并从培养基中获得。经SDS-PAGE分析发现，用0.75%甲醇诱导96小时后，重组蛋白以40 kDa蛋白的形式最佳表达。表达的GRA4蛋白大小与计算的分子量存在差异。这可能归因于包括糖基化和磷酸化在内的各种翻译后修饰。尽管分子量不同，但重组蛋白在蛋白质印迹法中能够检测出弓形虫病。重组GRA4表达时带有完整的多组氨酸标签，可用于未来抗原的纯化。
