Asadi Karam Mr, Oloomi M, Habibi M, Bouzari S
Department of Molecular Biology, Pasteur Institute of Iran, Tehran, Iran.
Iran J Microbiol. 2012 Jun;4(2):55-62.
Urinary tract infection (UTI) is one of the most common infections in the world. The majority of UTIs are caused by Uropathogenic Escherichia coli (UPEC) strains. FimH and FliC are the most important virulence factors of UPEC. To date, any ideal vaccine against UTI has not been approved for human use and we need to test new targets to develop an ideal vaccine against UTI. In this study, we constructed fusion fimH/fliC of UPEC as a novel vaccine candidate against UTI.
PCR amplification of fimH and fliC genes of the UPEC isolates was performed by specific primers designed for this purpose. Construction of fimH/fliC hybrid gene was performed by overlap PCR. The fimH, fliC and fimH/fliC were cloned in pET28a vector. The confirmation of expression of the proteins was done by SDS-PAGE and Western blot.
The fliC and fimH genes were amplified in all of the UPEC isolates tested. The fimH showed significant homology with the sequences in GenBank. We generated a fusion consisting of the fimH linked to the N-terminal end of fliC. Sequencing of the fusion fimH/fliC showed that fusion was constructed correctly. SDS-PAGE and western blot confirmed the expression of the proteins in optimized condition.
Urinary tract infection is a huge burden on healthcare system in many countries. UPEC is isolated in around 80% of UTI cases. Antibiotic therapy resulted in the emergence of antibiotic resistance in UPEC strains. This is the major cause for an increasing requirement for a vaccine to prevent UTI. This work describes for the first time the construction of a novel fusion protein from Iranian UPEC isolates. Further immunological studies are required for evaluation of this protein as a novel and safe vaccine candidate against UTI caused by UPEC.
尿路感染（UTI）是全球最常见的感染之一。大多数尿路感染由尿路致病性大肠杆菌（UPEC）菌株引起。FimH和FliC是UPEC最重要的毒力因子。迄今为止，尚无针对尿路感染的理想疫苗获批用于人类，我们需要测试新的靶点来研发针对尿路感染的理想疫苗。在本研究中，我们构建了UPEC的融合蛋白fimH/fliC作为一种新型的抗尿路感染疫苗候选物。
使用为此设计的特异性引物对UPEC分离株的fimH和fliC基因进行PCR扩增。通过重叠PCR构建fimH/fliC杂交基因。将fimH、fliC和fimH/fliC克隆到pET28a载体中。通过SDS-PAGE和蛋白质免疫印迹法确认蛋白质的表达。
在所有测试的UPEC分离株中均扩增出fliC和fimH基因。fimH与GenBank中的序列具有显著同源性。我们生成了一个由连接到fliC N末端的fimH组成的融合体。融合蛋白fimH/fliC的测序表明融合体构建正确。SDS-PAGE和蛋白质免疫印迹法证实在优化条件下蛋白质的表达。
尿路感染给许多国家的医疗保健系统带来了巨大负担。在约80%的尿路感染病例中分离出UPEC。抗生素治疗导致UPEC菌株出现抗生素耐药性。这是对预防尿路感染疫苗需求增加的主要原因。这项工作首次描述了来自伊朗UPEC分离株的新型融合蛋白的构建。需要进一步的免疫学研究来评估该蛋白作为一种针对由UPEC引起的尿路感染的新型安全疫苗候选物。
