Faezi Sobhan, Bahrmand Ahmad Reza, Siadat Seyed Davar, Nikokar Iraj, Sardari Soroush, Mahdavi Mehdi
Departments of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran.
Microbiology Research Center (MRC), Pasteur Institute of Iran, Tehran, Iran.
Iran J Basic Med Sci. 2017 May;20(5):458-466. doi: 10.22038/IJBMS.2017.8667.
Infection with has been a long-standing obstacle for clinical therapy due to the complexity of the genetics and pathogenesis, as well for widespread resistance to antibiotics, thus attaching great importance to explore effective vaccines for prevention and treatment. This paper focuses on the introduction of novel type IV pili (T4P)-based fusion protein containing the secretin domain of PilQ and tandem PilA-related peptides.
We surveyed the expression of the PilQ-PilA fusion protein in-frame with pET26b vector in which a rigid linker was used between two polypeptides and flexible linkers were inserted between the three tandem repeats and each domains. The transformants were expressed in BL21. The reactivity of specific antisera to the fusion protein was assessed by ELISA. The biological activities of this candidate vaccine were evaluated by western blotting, opsonophagocytosis, and twitching inhibition assays.
The fusion protein was purified in high yield by osmotic shock method using HisTrap affinity column. The protein was confirmed by immunoblot analysis. The checkerboard titration showed that the optimal dilution of the antibody to react with antigen is 1:128. Results of opsonophagocytosis assay revealed that the antibodies elevated to the fusion protein promoted phagocytosis of the PAO1 and 6266E strains, so that the twitching immobilization test confirmed these results.
Due to excellent killing activity mediated by opsonic antibodies and efficient immobilization of the strains, it seems that PilQ-PilA fusion protein could be a reliable candidate vaccine against infection.
由于其遗传学和发病机制的复杂性以及对抗生素的广泛耐药性，感染一直是临床治疗的长期障碍，因此探索有效的预防和治疗疫苗至关重要。本文重点介绍了一种新型的基于IV型菌毛（T4P）的融合蛋白，该蛋白包含PilQ的分泌素结构域和串联的PilA相关肽段。
我们研究了与pET26b载体读框内的PilQ-PilA融合蛋白的表达情况，其中在两个多肽之间使用了刚性接头，在三个串联重复序列和每个结构域之间插入了柔性接头。转化体在BL21中表达。通过ELISA评估特异性抗血清与融合蛋白的反应性。通过蛋白质印迹、调理吞噬作用和抽动抑制试验评估该候选疫苗的生物学活性。
使用HisTrap亲和柱通过渗透休克法高产率纯化融合蛋白。通过免疫印迹分析确认了该蛋白。棋盘滴定法表明，与抗原反应的抗体的最佳稀释度为1:128。调理吞噬作用试验结果显示，针对融合蛋白产生的抗体促进了PAO1和6266E菌株的吞噬作用，抽动固定试验证实了这些结果。
由于调理抗体介导的出色杀伤活性和菌株的有效固定，PilQ-PilA融合蛋白似乎可能是一种可靠的抗感染候选疫苗。
