Cloning, expression and characterization of zebrafish ferroportin in Hek293T cell line

Ferroportin [Fpn], a regulator of iron homeostasis is a conserved membrane protein that exports iron across the enterocytes, macrophages and hepatocytes into the blood circulation. Fpn has also critical influence on survival of microorganisms whose growth is dependent upon iron, thus preparation of Fpn is needed to study the role of iron in immunity and pathogenesis of micoorganisms. To prepare and characterize a recombinant ferroportin, total RNA was extracted from Indian zebrafish duodenum, and used to synthesize cDNA by RT-PCR. PCR product was first cloned in Topo TA vector and then subcloned into the GFP expression vector pEGFP-Nl. The final resulted plasmid [pEGFP-ZFpn] was used for expression of Fpn-EGFP protein in Hek 293T cells. The expression was confirmed by appearance of fluorescence in Hek 293 T cells. Recombinant Fpn was further characterized by submission of its predicted amino acid sequences to the TMHMM V2.0 prediction server [hidden Markov model], NetOGlyc 3.1 and NetNGlyc 3.1 servers. The obtained Fpn from indian zebrafish also contained eight transmembrane domains with N- and C-termini inside the cytoplasm and harboured 78 O-glycosylated amino acids. The recombinant Fpn from Indian zebra fish was successfully expressed in Hek 293 cell line. Although the discrepancy in two amino acids was observed in our produced Fpn and resulted in an additional O-glycosylation site, but had no effect on the topology of the protein compared to other Fpn described by other researchers. Therefore this construct can be used in future iron studies

[Study the capacity of recombinant HCV core+1 protein to induce immune responses in combination with different adjuvants]

Hepatitis C virus [HCV] genome contains a large open reading frame coding for a polyprotein that is cleaved into ten proteins. Recently, a new protein, named core+1, has been described to be expressed through a ribosomal frame shift within the capsid-encoding sequence. To address these possibilities, core+1 was produced in E.coli and the purified protein was evaluated for the immunological properties. The core+1 corresponding nucleotide sequence was created by PCR-based induction of a +1 frame shift mutation within the core gene template. The amplicon was cloned into the pET-24a vector and expressed in E.coli host. The expressed protein was purified under denaturing condition and after refolding steps was characterized by SDS-PAGE and Western Blotting. The immunization potential of core+1 with various adjuvant [Freunds [C/IFA], Montanide ISA 206 and IMS 1312, pluronic acid [F127] and imiquimod [IMIQ] was assessed in Balb/c mice. ELISA-based assays were used to analyze the humoral immune responses. The yield of E.coli-derived core+1 was 5 mg/ L of culture media and antigenicity of this protein was confirmed by western blotting. All the core+1/adjuvant formulations significantly developed the anti-core+1 IgG responses in the immunized mice but C/IFA and ISA206 elicited the highest antibody titers. ISA 206 and IMS 1312 formulation of core+1 induced strong Th1/Th2 responses. Our results indicated that core+1 formulation with various adjuvants may elicit the different immune response profiles [Th1/Th2]. Thus core+1 might be a potential component of future HCV vaccine too

[Designing and producing polytope DNA vaccine containing HBsAg gene for the induction of protective immunity against hepatitis C]

Background: considering the immunosuppressive effects and prevalent mutations in some HCV antigens, induction of CD8[+] T cell responses is focused on conserved and critical epitopes which as a multi-epitope vaccine can prevent the chronic nature of the disease

Materials and Methods: two immunodominant HLA-A2-restricted human epitopes [E2[614- 622] and NS3[1406-1415]] and two H-2[d]-restricted mouse epitopes [core[132-142] and E2[405-414]] were designed in a sequential tandem, predicted by immunoinformatic analyses. Following the synthesis, related nucleotide sequence was cloned into the pcDNA3.1 vector with and without the fusion of hepatitis B surface antigen [HBsAg]. Two constructed plasmids [pcDNA3.1.HPOL and pcDNA3.1.POL, respectively] were evaluated for the protein expression and secretion in Cos-7 cell line. After the vaccination of BALB/c mice [n=6 in each group] with different DNA and peptide immunization regimens, CD8[+] T cell activity as well as the type and protective potency of the induced responses were evaluated

Results: despite the induction of epitope-specific responses in pcDNA3.1.POL injected mice, the group immunized with pcDNA3.1.HPOL indicated a significant increase in the number and activity of CD8[+] T cells [P<0.05]. Peptide boosting of this group [formulated in two human-compatible adjuvant] still led to the more activation of CD8[+] cells, induction of Th1 response and the inhibition of tumor model growth [P<0.05]

Conclusion: fusion of HBsAg as a particle-forming sequence and the source of helper epitopes along the DNA-prime/peptide-boosting immunization regimen are proposed as two promising strategies to improve the CTL multi-epitope vaccines against HCV