Objectives

Shigellosis is one of the infectious diseases causing severe intestinal illness in human beings. Development of an effective vaccine against Shigella is a key to deal with this bacterium. The present study aimed at evaluation of the antibody response as well as the protection of the recombinant chimeric protein containing IpaD, IpaB, StxB, and VirG against Shigella dysentery and flexneri.

Methods

Chimeric protein was expressed and purified by Ni-NTA resin. The identity of the protein was determined by Western blot analysis. Mouse groups were immunized with the recombinant protein and the humoral immune response was measured by Enzyme-Linked Immunosorbent Assay (ELISA). Additionally, neutralization of the bacterial toxin by antibody was assessed by MTT assay. Animal challenge against S.dysentery and S. flexneri was evaluated, as well.

Results

Protein expression and purification were confirmed by SDS-PAGE and western blotting. Analysis of the immune responses demonstrated that the antibody responses were higher in the sera of the subcutaneously immunized mice compared to those immunized intraperitoneally. In vitro neutralization analysis indicated that the 1:10000 dilution of the sera had a high ability to neutralize 0.25 ng/µl (CD50) of the toxin on the Vero cell line. Furthermore, the results of the animal challenge showed that the immunized mice were completely protected against 50 LD50 of the bacterial toxin. Immunization also protected 80% of the mice from 10 LD₅₀ by S. flexneri and S.dysentery. In addition, passive immunization conferred 60% protection in the mice against S. flexneri and S.dysentery. Organ burden studies also revealed a significant reduction in infection among the immunized mice.

Conclusion

This study revealed that the chimeric protein produced in E. coli could be a promising chimeric immunogen candidate against Shigella.

中文翻译：

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一种嵌合亚基免疫原的方法提供了针对志贺氏菌毒性、III 型和 V 型分泌系统的有效保护

目标

志贺氏菌病是引起人类严重肠道疾病的传染病之一。开发针对志贺氏菌的有效疫苗是对付这种细菌的关键。本研究旨在评估抗体反应以及包含 IpaD、IpaB、StxB 和 VirG 的重组嵌合蛋白对志贺氏痢疾和福氏杆菌的保护作用。

方法

Ni-NTA树脂表达和纯化嵌合蛋白。通过蛋白质印迹分析确定蛋白质的身份。用重组蛋白免疫小鼠组，并通过酶联免疫吸附试验（ELISA）测量体液免疫反应。此外，通过MTT测定评估抗体对细菌毒素的中和作用。还评估了针对S.dysentery和S. flexneri的动物攻击。

结果

通过 SDS-PAGE 和蛋白质印迹确认蛋白质表达和纯化。免疫反应的分析表明，与腹膜内免疫的小鼠相比，皮下免疫小鼠血清中的抗体反应更高。体外中和分析表明，1:10000 稀释度的血清对 Vero 细胞系中 0.25 ng/µl (CD50) 的毒素具有很高的中和能力。此外，动物攻击的结果表明，免疫小鼠完全免受 50 LD50 的细菌毒素的侵害。免疫还保护 80% 的小鼠免受S. flexneri和S.dysentery的 10 LD ₅₀. 此外，被动免疫对小鼠福氏链球菌和痢疾链球菌有 60% 的保护作用。器官负担研究还显示免疫小鼠的感染显着减少。

结论

这项研究表明，在 大肠杆菌中 产生的嵌合蛋白可能是一种很有前途的针对志贺氏菌的嵌合免疫原候选物。