■弓形虫(T.gondii)是一种广泛的,人畜共患原生动物细胞内寄生虫具有复杂的生命周期,会导致弓形虫病,一种潜在的严重疾病.在入侵过程中,弓形虫蛋白首先与相关的宿主细胞受体结合,如糖胺聚糖分子(GAG结合基序),是寄生虫或病毒感染宿主细胞的主要受体之一。然而,TGME49_216510(T.gondiiTrx21),一种来自弓形虫的蛋白质,是有限的。
■首先进行Trx21蛋白的生物信息学分析。然后使用保守结构域和GAG结合基序设计特异性引物进行扩增,快递,并纯化Trx21蛋白的片段。纯化的Trx21-GST蛋白用于抗氧化和细胞粘附实验。同时,用Trx21-His免疫小鼠以产生用于亚细胞定位分析的特异性多克隆抗体。
■Trx21蛋白,由774个氨基酸组成,包括一个跨膜区,三个GAG结合基序,和一个硫氧还蛋白样结构域。重组Trx21-His蛋白的分子量约为31kDa,而Trx21-GST蛋白的分子量约为55kDa,通过SDS-PAGE和Western印迹分析。通过IFA的亚细胞定位分析显示Trx21主要分布在弓形虫的细胞质中。此外,Trx21对超螺旋DNA对金属催化的氧化(MCO)具有保护作用,并表现出对Vero细胞的粘附能力。
■这些结果表明,Trx21在宿主细胞相互作用和氧化损伤中起重要作用。
UNASSIGNED: Toxoplasma gondii (T. gondii) is a widespread, zoonotic protozoan intracellular parasite with a complex life cycle, which can cause toxoplasmosis, a potentially serious disease. During the invasion process, T. gondii proteins first bind to the relevant host cell receptors, such as
glycosaminoglycan molecule (GAG-binding motif), which is one of the main receptors for parasites or virus to infect host cells. However, research on TGME49_216510 (T. gondii Trx21), a protein from Toxoplasma gondii, is limited.
UNASSIGNED: Bioinformatics analysis of the Trx21 protein was performed firstly. And specific primers were then designed using the conserved domain and GAG-binding motif to amplify, express, and purify a fragment of the Trx21 protein. The purified Trx21-GST protein was used for antioxidant and cell adhesion experiments. Simultaneously, mice were immunized with Trx21-His to generate specific polyclonal antibodies for subcellular localization analysis.
UNASSIGNED: The Trx21 protein, consisting of 774 amino acids, included a transmembrane region, three GAG-binding motifs, and a Thioredoxin-like domain. The recombinant Trx21-His protein had a molecular mass of about 31 kDa, while the Trx21-GST protein had a molecular mass of about 55 kDa, which was analyzed by SDS-PAGE and Western blot. Subcellular localization analysis by IFA revealed that Trx21 is predominantly distributed in the cytoplasm of T. gondii. Furthermore, Trx21 exhibited a protective effect on supercoiled DNA against metal-catalyzed oxidation (MCO) and demonstrated adhesion abilities to Vero cells.
UNASSIGNED: These results indicate that Trx21 plays an important role in host cell interaction and oxidative damage.