title: In silico design, cloning and high level expression of L7/L12-TOmp31 fusion protein of Brucella antigens.
abstract: Globally, Brucella melitensis and B. abortus are the most common cause of human brucellosis. The outer membrane protein 31 (Omp31) and L7/L12 are immunodominant and protective antigens conserved in human Brucella pathogens which are considered as potential vaccine candidates. We aimed to design the fusion protein from Brucella L7/L12 and truncated Omp31proteins, in silico, clone the fusion in pET28a vector, and express it in Escherichia coli host. Two possible fusion forms, L7/L12-TOmp31 and TOmp31-L7/L12 were subjected to in silico modeling and analysis. Analysis and validation of the fusion proteins with three dimensional (3D) models showed that both models are in the range of native proteins. However, L7/L12-Tomp31 structure was more valid than the TOmp31-L7/L12 model and subjected to in vitro production. The major histocompatibility complex (MHC II) epitope mapping using IEDB database indicated that the model contained good MHC II binders. The L7/L12-TOmp31 coding sequence was cloned in pET28a vector. The integrity of the construct was confirmed by polymerase chain reaction, restriction enzyme mapping, and sequencing. The fusion was successfully expressed in E. coli BL21 (DE3) by induction with isopropyl β-D-thiogalactopyranoside. The rL7/L12-TOmp31 was purified with Ni-NTA column. The yield of the purified rL7/L12-TOmp31 was estimated by Bradford method and found to be 40 mg/L of the culture. Western blotting with anti-His antibody revealed a specific reactivity with purified rL7/L12-TOmp31 produced in E. coli and showed the functional expression in the prokaryotic system. In this study, a new protein vaccine candidate against brucellosis was constructed with the help of bioinformatics tools and the construct was expressed in the bacterial host. Studies evaluating the immunogenicity and cross-protection of this fusion protein against B. melitensis and B. abortus are underway.
authors.affiliation: Department of Molecular Biology, Pasteur Institute of Iran, Tehran, I.R. Iran.
authors.forename: Maryam
authors.initials: M
authors.lastname: Golshani
authors.affiliation: Department of Molecular Immunology and Vaccine Research, Pasteur Institute of Iran, Tehran, I.R. Iran.
authors.forename: Sima
authors.initials: S
authors.lastname: Rafati
authors.affiliation: Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences and Isfahan Pharmaceutical Sciences Research center, Isfahan University of Medical Sciences, Isfahan, I.R. Iran.
authors.forename: Ali
authors.initials: A
authors.lastname: Jahanian-Najafabadi
authors.affiliation: Bacterial Vaccine Branch, Pasteur Institute of Iran, Karaj, I.R. Iran.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Nejati-Moheimani
authors.affiliation: Department of Bacteriology, Pasteur Institute of Iran, Tehran, I.R. Iran.
authors.forename: Seyed Davar
authors.initials: SD
authors.lastname: Siadat
authors.affiliation: Department of Bacteriology, Pasteur Institute of Iran, Tehran, I.R. Iran.
authors.forename: Fereshteh
authors.initials: F
authors.lastname: Shahcheraghi
authors.affiliation: Department of Molecular Biology, Pasteur Institute of Iran, Tehran, I.R. Iran.
authors.forename: Saeid
authors.initials: S
authors.lastname: Bouzari
keywords: Brucella; Cloning; Fusion protein; In silico design; L7/L12; Omp31