阴道毛滴虫黏附蛋白 65（AP65）的分子特征及其免疫保护作用。

The molecular characterization and immune protection of adhesion protein 65 (AP65) of Trichomonas vaginalis.

机构信息

Xinxiang Key Laboratory of Pathogenic Biology, School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang, Henan, 453003, PR China.

出版信息

Microb Pathog. 2021 Mar;152:104750. doi: 10.1016/j.micpath.2021.104750. Epub 2021 Jan 20.

DOI:10.1016/j.micpath.2021.104750

PMID:33484808

Abstract

BACKGROUND

Adherence to the surface of the host cell is the precondition for T. vaginalis parasitism and pathogenicity, causing urogenital infection. The AP65 of T. vaginalis (TvAP65) involves in the process of adhesion. So, the present study was aimed at investigating the molecular characterization and vaccine candidacy of TvAP65 for protecting the host from the onset of Trichomoniasis.

METHODS

The open reading frame (ORF) of TvAP65 was amplified and then inserted into pET-32a (+) to clone recombinant TvAP65 (rTvAP65). The immunoblotting determined the immunogenicity and molecular size of TvAP65, while immunofluorescence staining visualized and the precise localization of TvAP65 in T. vaginalis trophozoites. Animal challenge and enzyme-linked immunosorbent assay (ELISA) test were used to evaluate the immunoprotection and the types of the immune response of TvAP65.

RESULTS

By the sequence analysis, TvAP65 encoded a 63.13 kDa protein that consisted 567 amino acid residues with a high antigenic index. The western blotting revealed that rTvAP65 and native TvAP65 could interact with the antibodies in the rat serums post hoc rTvAP65 immunization and the serums from the mice that were experimentally infected with T. vaginalis, respectively. Immunofluorescence stained TvAP65 on the surface of T. vaginalis trophozoites. Moreover, following emulsification with Freund's adjuvant, rTvAP65 was subsequently administered to BALB/c mice three times at 0, 2, and 4 weeks and the results from this animal challenge experiments showed significant increases in immunoglobulins of IgG2a, IgG1, and IgG, and cytokine of IFN-γ, and IL-2, and 10. Lastly, rTvAP65 vaccinated animals had a prolonged survival time (26.80 ± 4.05) after challenged by T. vaginalis.

CONCLUSIONS

TvAP65 mediated the adhesion of T. vaginalis to the host epithelia for the pathogenesis of the parasite and can be considered as a candidate protein for designing a functional vaccine that induces cell-mediated and humoral immunity against the T. vaginalis infection.

摘要

背景

黏附在宿主细胞表面是阴道毛滴虫寄生和致病的前提条件，导致泌尿生殖系统感染。阴道毛滴虫的 AP65（TvAP65）参与了黏附过程。因此，本研究旨在探讨 TvAP65 的分子特征和疫苗候选性，以保护宿主免受滴虫病的侵袭。

方法

扩增 TvAP65 的开放阅读框（ORF），然后将其插入 pET-32a（+）中克隆重组 TvAP65（rTvAP65）。免疫印迹分析确定了 TvAP65 的免疫原性和分子大小，而免疫荧光染色则可视化并精确定位了 TvAP65 在阴道毛滴虫滋养体中的位置。动物攻毒和酶联免疫吸附试验（ELISA）检测评估了 TvAP65 的免疫保护和免疫反应类型。

结果

序列分析表明，TvAP65 编码一个 63.13kDa 的蛋白质，由 567 个氨基酸残基组成，具有较高的抗原指数。Western blot 分析显示，rTvAP65 和天然 TvAP65 可以与大鼠血清中的抗体相互作用，这些大鼠血清是在 rTvAP65 免疫后获得的，也可以与实验感染阴道毛滴虫的小鼠血清中的抗体相互作用。免疫荧光染色显示 TvAP65 位于阴道毛滴虫滋养体的表面。此外，rTvAP65 与弗氏佐剂乳化后，连续 3 次在 0、2 和 4 周时给 BALB/c 小鼠注射，动物攻毒实验结果显示 IgG2a、IgG1 和 IgG 免疫球蛋白以及 IFN-γ 和 IL-2 细胞因子显著增加，最后，rTvAP65 接种的动物在受到阴道毛滴虫攻击后存活时间延长（26.80±4.05）。