Construction of Mycobacterium tuberculosis ESAT-6 fused to human Fcγ of IgG1: To target FcγR as a delivery system for enhancement of immunogenicity

• 3D-structure of the ESAT-6:Fcγ1 protein was constructed via homology modeling.
• The PCR products of ESAT-6 and Fcγ1 fragments were then recombined using SOEing PCR.
• Binding of the ESAT-6:Fcγ1 protein to FcγRI on APCs was confirmed using direct IFA.
• ESAT6:Fcγ1 protein was dimerized by disulfide bond of Fcγ fragments.

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 50 kDa, 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.

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