Difference between revisions of "Part:BBa K1621004"

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gag/tat/pol/env - polyepitopic antigen derived from HIV-1

This part contains the coding sequence of a polytopic antigen used for the specific detection antibodies against Human Immunodeficiency Virus-1 (HIV-1). A number of immunogenic epitopes derived from the polyprotein have been combined in a single nucleotide sequence. Thus, the antibody binding properties remain the same as for the individual proteins but the catalytical functions cannot be fulfilled (Jafarpour et al., 2014).

Originally, this sequence was designed for the development of a polytope DNA vaccine strategy. This strategy relies on the concept of combining immunogenic epitopes for vaccination to focus the immune response on conserved and crucial epitopes (Memarnejadian et al., 2009).
Accordingly, those epitopes have been chosen by means of immunogenicity and conservancy as well as the maintenance of their antibody binding affinities without the rest of the protein. Selection of the epitopes was based on bioinformatic analyses (Jafarpour et al., 2014).

The proteins the epitopes derive from contribute to various processes in the viral life cycle. The gag gene encodes for the precursor forms of several cellular and nuclear proteins that are markers for very early stages of an infection. env encoded proteins mediate, for example, the attachment to CD4 T cells and can be found from early stages until the end of the disease. Many enzymes that are crucial for the pathogenesis of the virus are encoded by pol. These include reverse transcriptase and integrase, important for integration of viral genes into the host genome, among others. The tat gene encodes for the TAT protein which increases viral transcription (Goepfert, 2013).
All in all, the combination of epitopes derived from those proteins enables to target several different stages of the viral life cycle and, thus, bears great potential for next generation vaccines (Goepfert, 2013). Additionally, it is a useful tool for the detection of antibodies developed in response to HIV-1 infections at multiple stages.

Cloning the parts sequence into an expression vector enables efficient overexpression of this polyepitopic peptide in Escherichia coli. Table 1 shows the conditions that were used to grow the cells and induce the expression. The protein solution before and after His-tag based affinity purification were analyzed by SDS PAGE to confirm successful overexpression (figure 1).

Sequence and Features