Medical Parasitology, Toxoplasmosis Research Center and Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran.
Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Microb Pathog. 2019 Jul;132:275-281. doi: 10.1016/j.micpath.2019.05.013. Epub 2019 May 9.
Toxoplasma gondii is an obligate intracellular parasite that causes one of the most common parasitic infections in humans and other warm-blooded animals. Currently, there are no effective treatments for inhibiting the formation of chronic tissue cysts in infected hosts. Thus, the development of a vaccine to protect against toxoplasmosis is an attractive option for avoiding infection. The aim of this study was to design an epitope-based vaccine for T. gondii. In the present study, an in silico approach was used to predict and analyze B-cell and T-cell epitopes and the transmembrane domain of proteins SAG1, MIC3, and ROP8. We also predicted the antigenicity, allergenicity, secondary and tertiary structures, and physicochemical characteristics of a chimeric protein. Next, codon optimization and mRNA structure prediction were conducted using bioinformatics tools, and the designed construct was chemically synthesized and cloned into the pET28a vector. SAG1 (amino acid positions 85-235), MIC3 (30-180), and ROP8 (85-185) were found to have several strong immunodominant epitopes that were joined with a rigid linker A(EAAAK)A. Although the resultant protein called MRS (MIC3, ROP8, and SAG1) did not turn out to be an allergen, its antigenicity was estimated to be 0.7983. Additionally, MRS was selected as the best vaccine candidate on the basis of its secondary and tertiary structures. The number of amino acids, molecular weight, and numbers of negatively and positively charged residues of MRS were 427 and 45,661.31 Da, 45, and 50, respectively. ΔG of the best-predicted structure was -413.0 kcal/mol, and the first nucleotides at the 5' end did not form a stable hairpin or pseudoknot. Finally, successful expression and verification of the expressed MRS protein showed that in silico analysis was almost accurate. This vaccine candidate selected by in silico tools should be validated in experimental studies.
刚地弓形虫是一种专性细胞内寄生虫，可引起人类和其他温血动物中最常见的寄生虫感染之一。目前，尚无有效的方法来抑制感染宿主中慢性组织囊肿的形成。因此，开发一种疫苗来预防弓形虫病是避免感染的一种有吸引力的选择。本研究旨在设计一种基于表位的弓形虫疫苗。在本研究中，我们使用计算机方法预测和分析了 SAG1、MIC3 和 ROP8 蛋白的 B 细胞和 T 细胞表位和跨膜结构域。我们还预测了嵌合蛋白的抗原性、变应原性、二级和三级结构以及理化特性。接下来，我们使用生物信息学工具进行了密码子优化和 mRNA 结构预测，并对设计的构建体进行了化学合成和克隆到 pET28a 载体中。SAG1（氨基酸位置 85-235）、MIC3（30-180）和 ROP8（85-185）被发现具有几个强免疫显性表位，与刚性接头 A(EAAAK)A 相连。尽管所得的蛋白质称为 MRS（MIC3、ROP8 和 SAG1）不是过敏原，但它的抗原性估计为 0.7983。此外，根据 MRS 的二级和三级结构，选择 MRS 作为最佳疫苗候选物。MRS 的氨基酸数、分子量和带负电荷和带正电荷的残基数分别为 427 和 45,661.31Da、45 和 50。最佳预测结构的ΔG 为-413.0kcal/mol，5'端的第一个核苷酸没有形成稳定的发夹或假结。最后，成功表达和验证了表达的 MRS 蛋白表明，计算机分析几乎是准确的。应该在实验研究中验证通过计算机工具选择的这种疫苗候选物。
