Yarian Fatemeh, Bandehpour Mojgan, Seyed Negar, Kazemi Bahram
Department of Biotechnology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran; Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Iran J Microbiol. 2016 Feb;8(1):29-35.
Neisseria meningitidis is a leading cause of meningitis and sepsis worldwide. The factor H binding protein (fHBP) is a key virulence factor of Neisseria meningitidis that is able to selectively bind to human factor H, the key regulator of the alternative complement pathway, which it has important implications for meningococcal pathogenesis and vaccine design. The aims of present research were cloning, expression, purification of fHbp and confirmation of the interaction between serum factor H (fH) and produced factor H binding protein.
A 820 base pairs fhbp gene fragment was amplified by PCR and cloned into expression vector pET28a (+) in Bam HI and SalI restriction enzymes sites. Recombinant DNA was expressed in BL21 (DE3) cell. fHBP protein was purified by Ni-NTA agarose resin. Coupling of recombinant protein into CNBr activated Sepharose 4B resin was carried out for application in serum fH protein purification. (fH-fHBP) interaction was confirmed by SDS-PAGE and far-western blotting.
SDS-PAGE results showed a 35 kDa protein band. 150 kDa fH protein was purified by designed Sepharose 4B resin. Far-western blotting confirmed (fH-fHBP) interaction and proper folding of factor H binding protein.
脑膜炎奈瑟菌是全球范围内脑膜炎和败血症的主要病因。因子H结合蛋白（fHBP）是脑膜炎奈瑟菌的关键毒力因子，能够选择性地与人因子H结合，而人因子H是替代补体途径的关键调节因子，这对脑膜炎球菌的发病机制和疫苗设计具有重要意义。本研究的目的是克隆、表达、纯化fHbp，并证实血清因子H（fH）与所产生的因子H结合蛋白之间的相互作用。
通过PCR扩增出一个820碱基对的fhbp基因片段，并将其克隆到表达载体pET28a（+）的Bam HI和SalI限制性酶切位点。重组DNA在BL21（DE3）细胞中表达。fHBP蛋白通过Ni-NTA琼脂糖树脂纯化。将重组蛋白偶联到CNBr活化的Sepharose 4B树脂上，用于血清fH蛋白的纯化。通过SDS-PAGE和Far-Western印迹法证实（fH-fHBP）相互作用。
SDS-PAGE结果显示出一条35 kDa的蛋白带。通过设计的Sepharose 4B树脂纯化出150 kDa的fH蛋白。Far-Western印迹法证实了（fH-fHBP）相互作用以及因子H结合蛋白的正确折叠。
