Production of recombinant Human T Lymphotropic Virus type 1 Tax protein in Rosetta (DE3) bacterial host

 HTLV1 is the first detected retrovirus causing disease in human. The physiopathology of HTLV1 related diseases was mainly linked with its Tax protein characteristics. Use of mutant Tax proteins accompanied by immune regulator drugs could help treating HTLV1 associated myelopathy patients as a modulator of potent immune response against this viral protein. Since Tax protein is not commercially available, production of recombinant Tax protein was targeted for this study. Coding sequence of Tax protein (containing R222K mutation) in the pcDNA3.1(+) was digested with BamHI and XhoI restriction enzymes, and then removed and inserted into the expression vector pET32a(+) within the same cutting sites and cloned into E.coli DH5α. Recombinant vector was confirmed with enzymatic digestion, colony PCR, and sequencing of cloned gene. E.coli Rosetta (DE3) was transformed with the recombinant plasmid and the expression was induced. The expression of protein was assayed with SDS-PAGE and western blot using monoclonal antibodies against Tax and 5His epitope. Finally, antigenic characteristic of the recombinant protein was evaluated by western blotting against patient sera. Presence of Tax protein band in the SDS-PAGE and western blot was confirmed. Western blotting of the recombinant protein with patient sera showed the band related to Tax protein. The recombinant protein is well produced and could be detected by patients' sera, making it eligible to be used as a recombinant viral antigen for future purposes

A comparison of recombinant proteins production in CHO and MCF 7 cell lines

Background : The aim of the present study was to identify a suitable cell line for studies of recombinant protein production in eukaryotic system. After transfection, altered expression levels of RNA and its target protein were analyzed. Materials and Methods: To investigate the in vitro expression of E6 protein of human papillomavirus type 16 in cell culture, the plasmid pcDNA3-E6, and two different cell lines MCF7 and CHO were used. Following transfection, RNA expression was determined by RT- PCR and its protein target was evaluated by Western blot using SDS-PAGE gel and Anti-E6 monoclonal antibody. Results: In this study due to the low concentration of proteins in MCF7cells, RT-PCR was positive and Western blotting was negative, while RT-PCR and Western blotting were positive for CHO cell lines. Conclusion: Regarding the results, the use of CHO expression system as a tool for efficient transfection of recombinant protein production is suggested

Construction and Eukaryotic Expression of Recombinant Large Hepatitis Delta Antigen

Background: Hepatitis delta virus (HDV) is a subviral human pathogen that exploits host RNA editing activity to produce two essential forms of the sole viral protein, hepatitis delta antigen (HDAg). Editing at the amber/W site of HDV antigenomic RNA leads to the production of the large form (L-HDAg), which is required for RNA packaging. Methods: In this study, PCR-based site-directed mutagenesis by the overlap extension method was used to create the point mutation converting the small-HDAg (S-HDAg) stop codon to a tryptophan codon through three stages. Results: Sequencing confirmed the desirable mutation and integrity of the L-HDAg open reading frame. The amplicon was ligated into pcDNA3.1 and transfected to Huh7 and HEK 293 cell lines. Western blot analysis using enhanced chemiluminescence confirmed L-HDAg expression. The recombinant L-HDAg localized within the nuclei of cells as determined by immunofluorescence and confocal microscopy. Conclusion: Because L-HDAg requires extensive post-translational modifications, the recombinant protein expressed in a mammalian system might be fully functional and applicable as a tool in HDV molecular studies, as well as in future vaccine research