Imam Hossein University, Faculty of Science, Department of Biology, Tehran, Iran.
Imam Hossein University, Faculty of Science, Department of Biology, Tehran, Iran.
Microb Pathog. 2019 Apr;129:176-182. doi: 10.1016/j.micpath.2019.02.004. Epub 2019 Feb 5.
It is about 4 decades from the identification of Enterohemorrhagic Escherichia coli (EHEC) as a food-borne pathogen. There are many shreds of evidence that the bacteria are significant sources of intestinal infections and outbreaks even in developed countries. Developing an effective vaccine against O157 and non-O157 serotypes of EHEC is a good strategy to combat the bacteria. Raising antibody against main toxicity, adhering and colonizing apparatus of the bacteria using a multi-antigenic protein can be hopefully applicable.
A synthetic cassette consists of HcpA-EspA-Tir-Stx2B (HETS) subunit proteins were constructed and sub-cloned in pET32a (+). The protein was expressed and purified with Ni-NTA column and the BALB/c mice were immunized by the purified protein. HETS protein efficacy to elicit immune responses, O157 fecal shedding and immunity against Stx toxin were assessed. In addition, the cellular assays were performed to investigate the immune sera capability for neutralizing of Stx toxin and bacterial attachment apparatus.
The HETS protein (611 amino acid length) was expressed and validated by Western blotting. Exerted EHEC bacteria ratio in the immunized mice was reduced close to 60% in shedding test. Cellular assays revealed that the sera of the immunized animals were able to neutralize Stx holotoxin in an extent of 70%; also, immunized mice were able to tolerate up to 200 LD of the active toxin. Moreover, toxin neutralization assay showed the capability of the immunized sera to block the cell adhesion.
Regarding a lack of an efficient vaccine against EHEC, the proposed candidate immunogen, which consists of main adhesion and invasion factors, can overcome the lack of a vaccine against the bacteria.
肠出血性大肠杆菌（EHEC）被鉴定为食源性病原体已有大约 40 年的历史。有许多证据表明，即使在发达国家，该细菌也是肠道感染和疫情的重要来源。针对 O157 和非-O157 血清型的 EHEC 开发有效疫苗是对抗该细菌的良好策略。使用多抗原蛋白针对细菌的主要毒性、粘附和定植装置产生抗体有望是可行的。
构建了一个由 HcpA-EspA-Tir-Stx2B（HETS）亚单位蛋白组成的合成盒，并在 pET32a（+）中进行了亚克隆。使用 Ni-NTA 柱表达和纯化该蛋白，并使用纯化的蛋白对 BALB/c 小鼠进行免疫。评估了 HETS 蛋白诱导免疫应答、O157 粪便脱落和抗 Stx 毒素的免疫效果。此外，还进行了细胞测定以研究免疫血清中和 Stx 毒素和细菌附着装置的能力。
通过 Western blot 验证了 HETS 蛋白（611 个氨基酸长度）的表达。在脱落试验中，免疫小鼠中的 EHEC 细菌比例降低了近 60%。细胞测定表明，免疫动物的血清能够在 70%的程度上中和 Stx 全毒素；此外，免疫小鼠能够耐受 200LD 的活性毒素。此外，毒素中和测定表明，免疫血清能够阻断细胞粘附。
鉴于缺乏针对 EHEC 的有效疫苗，所提出的候选免疫原，由主要的粘附和入侵因子组成，能够克服针对该细菌的疫苗缺乏问题。
