Behrouzi Ava, Bouzari Saeid, Siadat Seyed Davar, Jafari Anis, Irani Shiva
Department of Biology, School of Basic Science, Science and Research Branch, Islamic Azad University, Tehran, IR Iran.
Department of Molecular Biology, Pasteur Institute of Iran, Tehran, IR Iran.
Jundishapur J Microbiol. 2015 Aug 29;8(8):e23218. doi: 10.5812/jjm.23218. eCollection 2015 Aug.
Nontypeable Haemophilus influenzae (NTHi) is a significant pathogen in children, causing otitis media, sinusitis, conjunctivitis, pneumonia, and occasionally invasive infections. Protein D (PD) belongs to the minor outer-membrane proteins of H. influenza. Moreover, it has been shown that this protein is one of the most potent vaccine candidates against the NTHi strain.
In the present study, a new truncated form of PD was designed based on conserved areas, and recombinant truncated PD was expressed.
Truncated PD was designed using bioinformatics tools, and a 345 bp fragment of the truncated hpd gene was amplified by polymerase chain reaction (PCR) from H. influenzae and subsequently cloned into the prokaryotic expression vector pBAD-gIIIA. In addition, for the expression of the recombinant protein, the pBAD-truncated PD plasmid was transformed into competent TOP10 cells. The recombinant protein was expressed with Arabinose. The expressed protein was purified by affinity chromatography using Ni-NTA resin.
The cloning of PD was confirmed by colony-PCR and enzymatic digestion. Arabinose 0.2% was able to efficiently induce protein expression. The SDS-PAGE analysis showed that our constructed pBAD-PD-TOP10 efficiently produced a target recombinant protein with a molecular weight of 16 kDa. A high concentration of the recombinant protein was obtained via the purification process by affinity chromatography. The recombinant PD was reacted with peroxidase-conjugated rabbit anti-mouse immunoglobulins.
Our results showed that the recombinant protein produced by the pBAD vector in the Escherichia coli system was very efficient.
不可分型流感嗜血杆菌（NTHi）是儿童的重要病原体，可引起中耳炎、鼻窦炎、结膜炎、肺炎，偶尔还会引发侵袭性感染。蛋白D（PD）属于流感嗜血杆菌的次要外膜蛋白。此外，研究表明该蛋白是针对NTHi菌株最有效的候选疫苗之一。
在本研究中，基于保守区域设计了一种新的截短形式的PD，并表达了重组截短型PD。
使用生物信息学工具设计截短型PD，通过聚合酶链反应（PCR）从流感嗜血杆菌中扩增出截短型hpd基因的345 bp片段，随后将其克隆到原核表达载体pBAD-gIIIA中。此外，为了表达重组蛋白，将pBAD-截短型PD质粒转化到感受态TOP10细胞中。用阿拉伯糖表达重组蛋白。通过使用Ni-NTA树脂的亲和层析法纯化表达的蛋白。
通过菌落PCR和酶切鉴定确认了PD的克隆。0.2%的阿拉伯糖能够有效诱导蛋白表达。SDS-PAGE分析表明，我们构建的pBAD-PD-TOP10能够高效产生分子量为16 kDa的目标重组蛋白。通过亲和层析纯化过程获得了高浓度的重组蛋白。重组PD与过氧化物酶偶联的兔抗小鼠免疫球蛋白发生反应。
我们的结果表明，pBAD载体在大肠杆菌系统中产生的重组蛋白非常高效。
