Molecular Cloning and Expression of Novel Fibroblast Growth Factor-2 Conjugated with Immunodominant Domains of Pseudomonas exotoxin

Hamid Haghighatfard^1,2, Nader Mansour Samaei¹, Toraj Farazmandfar¹, Mazdak Ganjalikhani Hakemi³, Ahad Yamchi⁴ , Farhad Jadidi-Niaragh^5,6 and Yaghoub Yazdani^*2,1

1Department of Medical Biotechnology, Faculty of Advanced Medical Technologies, Golestan University of Medical Sciences, Gorgan, Iran. 2Infectious Diseases Research Center , Golestan University of Medical Sciences, Gorgan, Iran. 3Cellular and Molecular Immunology Research Center, Isfahan University of Medical Science, Isfahan, Iran. 4Department of Plant Biotechnology, Gorgan University of Agricultural Science and Natural Resources, Gorgan, Iran. 5Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. 6Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.

Abstract: Angiogenesis is very important in cancer growth and metastasis. Basic fibroblast growth factor (bFGF) as one of the most important angiogenesis factors is an attractive target for cancer vaccine. Due to low immunogenicity, it cannot stimulate an effective immune response. Theoretically, pseudomonas exotoxin (PE) as a potent immunogenic carrier protein when fused to low immunogenic antigens such as bFGF significantly increased immunogenicity of it. In this study, we tried to molecular cloning and expression of bFGF conjugated with immunodominant domains of pseudomonas exotoxin. The coding sequence of fusion protein composed of bFGF linked to PE domains 1b and 2 using EAAAK poly linker. The KDEL sequence was also used in C-terminal coding sequence. It was synthesized and expressed using recombinant DNA technology in the bacterial expression system. Expression of recombinant protein verified using SDS-PAGE and western blot analyses. Finally, it purified using Ni-affinity chromatography. The band close to 37 kDa in SDS-PAGE and western blot analyses was aligned completely to designed sequence. Purified recombinant protein also showed as a clear single band near to 37 kDa.

Keywords: Basic fibroblast growth factor (bFGF); Cloning; Pseudomonas exotoxin (PE)

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