Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS

Development of functional prokaryotic and eukaryotic expression plasmids for making recombinant proteins for use as antigens in immunological assays

The genes encoding MVV EV1 p25, p17, and p14 were cloned and ligated into a commercially available prokaryotic expression vector that expresses the recombinant proteins in E. coli. The proteins were purified using a combination of GST and His tags, with the GST being cleaved from the protein. These recombinant proteins can used as antigens in immunological assays such as T cell proliferation, T cell cytokine production, and ELISA (as in the current project). They could also be used as immunogens in vaccine studies, or as antigens in other immunoassays such as western blotting. All three proteins have been produced before and used in immunoassays and consequently they represent prior knowledge.

The genes encoding the envelope proteins gp150, gp135, and gp46 were ligated into eukaryotic expression vectors and these were transformed into a variety of mammalian cells. No significant expression was observed. Eventually, fragments of sequence from gp135 and gp46 were codon-optimised and the corresponding DNA synthesised. These fragments were ligated into the expression vectors and transformed into mammalian cells and expression was observed. Thus, efficient expression of the env genes requires codon-optimisation.

The end users of the information are other scientists working in the field of SRLV control, or working with other lentiviruses, or working with other plasmid DNA vaccines, or in the general area of vaccines and immune responses, or in the general area of lentivirology.

Commercial companies may be interested in the recombinant proteins, but ELISAs that utilise p25 are currently available commercially and the protein produced in the project is not different in any significant way. The p17 and p14 proteins have been used previously in ELISAs produced for research purposes, but the available information suggests that these proteins do not have an advantage over p25 as diagnostic reagents. The market for these is likely to be restricted and very small. The recombinant env proteins may have potential as vaccine candidates or as diagnostic reagents but further work is required to investigate these possibilities.

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