Malaei Fatemeh, Rasaee Mohammad Javad, Latifi Ali Mohammad, Rahbarizadeh Fatemeh
Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Iran J Allergy Asthma Immunol. 2019 Jun 8;18(3):269-280. doi: 10.18502/ijaai.v18i3.1120.
Dickkopf (DKK) family of proteins are known as antagonists for the Wnt-β-catenin signaling pathway. It is suggested that the Dickkopf-1 (DKK-1) has a role in several diseases such as hepatocellular carcinomas, hepatoblastomas, Wilms' tumors, lung cancer and Myeloma bone disease. The aim of the present study was to produce a chimeric-recombinant DKK-1 protein in order to induce immune response against the antigen. The recombinant Dickkopf-1 (rDKK-1) protein was designed using bioinformatics analysis. The standard methods were used for cloning, expression and purification. The structure of recombinant protein was analyzed by spectroscopy methods. Enzyme-linked immunosorbent assay (ELISA) and Western blotting were performed to confirm the recombinant protein using a commercial anti-DKK-1 (whole protein) polyclonal antibody. The immunogenicity of the recombinant DKK-1 was assessed by immunizing, intraperitoneally, BALB/C mice four times with the 31-kDa and 45-kDa purified rDKK-1 cloned in pET28a and pET32a vectors respectively. The antibody titer was measured in due course of time. Stronger immunogenic parts of the protein were selected based on in-silico predictions and recombinant protein was successfully designed. The chimeric gene was sub-cloned, expressed, purified and refolded. The purified protein was confirmed by Western blotting and ELISA. The three dimensional structural was confirmed by CD spectrum and predicted structures by bioinformatics tools, revealed the stability of helix structures. rDKK-1 protein was capable of inducing immune response with high titer antibody and excessive humoral immune response. No significant difference was observed between immunization by 31-kDa and 45-kDa antigen.
Dickkopf（DKK）蛋白家族是Wnt-β-连环蛋白信号通路的拮抗剂。据推测，Dickkopf-1（DKK-1）在多种疾病中发挥作用，如肝细胞癌、肝母细胞瘤、肾母细胞瘤、肺癌和骨髓瘤骨病。本研究的目的是制备一种嵌合重组DKK-1蛋白，以诱导针对该抗原的免疫反应。利用生物信息学分析设计重组Dickkopf-1（rDKK-1）蛋白。采用标准方法进行克隆、表达和纯化。通过光谱学方法分析重组蛋白的结构。使用商业抗DKK-1（全蛋白）多克隆抗体进行酶联免疫吸附测定（ELISA）和蛋白质印迹法，以确认重组蛋白。分别用克隆于pET28a和pET32a载体中的31-kDa和45-kDa纯化rDKK-1对BALB/C小鼠进行腹腔注射免疫4次，评估重组DKK-1的免疫原性。在适当的时候测量抗体滴度。根据计算机模拟预测选择蛋白中免疫原性更强的部分，并成功设计了重组蛋白。将嵌合基因进行亚克隆、表达、纯化和复性。通过蛋白质印迹法和ELISA对纯化的蛋白进行确认。通过圆二色谱（CD）光谱确认三维结构，并通过生物信息学工具预测结构，揭示了螺旋结构的稳定性。rDKK-1蛋白能够诱导产生高滴度抗体的免疫反应和过度的体液免疫反应。用31-kDa和45-kDa抗原免疫之间未观察到显著差异。
