title: Construction of Mycobacterium tuberculosis ESAT-6 fused to human Fcγ of IgG1: To target FcγR as a delivery system for enhancement of immunogenicity.
abstract: In order to prevent spreading of Mycobacterium tuberculosis (Mtb), it is necessary to discover effective vaccines, fast and reliable diagnosis, and appropriate treatment schemes. In the present study, an Fc-tagged recombinant Mtb-ESAT-6 was produced to make a selective delivery system for promoting cellular immunity. To determine 3D structure of the recombinant protein, model building was performed in MODELLER9v13 program. After preparation of Mtb-DNA and Fcγ1 cDNA, they were amplified by specific primers to make ESAT-6 and Fcγ1 products to fuse them in frame using splicing by overlap extension (SOEing)-PCR. After TA cloning, the construct was sequenced to confirm no errors have been introduced. The recombinant DNA was then subcloned into PDR2EF1α eukaryotic expression vector. The plasmid sequenced over the sites at which two DNA fragments were cloned to ensure that the ligation had generated an in-frame fusion of the genes. The CHO cells were then stably transected by PDR2EF1α-ESAT-6:Fcγ1 vector using lipofectamin and the expression and its binding to the Fcγ receptor (FcγRI) on APCs were confirmed by immunofluorescence assay (IFA). The IFA results demonstrated that ESAT6:Fcγ1 was expressed in engineered CHO cells. Semi-scale protein production and purification using HiTrap-PA column showed a high secretion of the recombinant protein by Western blotting method. The molecular weight of the monomer in the SDS-PAGE was equal to a protein of 50kDa, which dimerizes by disulfide bond of Fcγ fragments. Since, ESAT6:Fcγ1 protein dimerizes and bind to FcγRs, therefore, Fc-tagged protein could target APCs for inducing appropriate immune response or using in interferon-based assays.
authors.affiliation: Microbiology & Virology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.
authors.forename: Saman
authors.initials: S
authors.lastname: Soleimanpour
authors.affiliation: Microbiology & Virology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.
authors.forename: Ali
authors.initials: A
authors.lastname: Mohammadi
authors.affiliation: Microbiology & Virology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.
authors.forename: Kiarash
authors.initials: K
authors.lastname: Ghazvini
authors.affiliation: Microbiology & Virology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.
authors.forename: Saeid Amel
authors.initials: SA
authors.lastname: Jamehdar
authors.affiliation: Microbiology & Virology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.
authors.forename: Hamid
authors.initials: H
authors.lastname: Sadeghian
authors.affiliation: Microbiology & Virology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.
authors.forename: Mahboubeh
authors.initials: M
authors.lastname: Taghiabadi
authors.affiliation: Inflammation and Inflammatory Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran. Electronic address: RezaeeR@mums.ac.ir.
authors.forename: S A R
authors.initials: SAR
authors.lastname: Rezaee
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D018014:Gene Transfer Techniques; D006801:Humans; D056724:Immunity, Humoral / Q000235:genetics; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000235:genetics*; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics*; D007167:Immunotherapy / Q000379:methods; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000276:immunology*; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics*; D014376:Tuberculosis / Q000276:immunology / Q000517:prevention & control; D032581:Tuberculosis Vaccines / Q000737:chemistry / Q000235:genetics; D014614:Vaccines, Synthetic / Q000737:chemistry / Q000235:genetics
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D032581:Tuberculosis Vaccines; D014614:Vaccines, Synthetic
keywords: ESAT-6; Fcγ1; Tuberculosis; Vaccine