Team:Freiburg/Project/pRIG15 17

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pRIG15_17

The Human immunodeficiency virus (HIV) causes acquired immunodeficiency syndrome, AIDS, which leads to major impairments of the immunsystem. ¹⁾ For our experiments we used a sequence coding for a recombinant protein that contains six epitopes from different proteins of the HI virus (HIV-1-trans-activating (tat) encoding region, one epitope of the reverse transcriptase, one of the p24 protein, one of the envelope protein gp41, one of gp120).²⁾

Figure 1: pRIG15_17. BBa_K1621004 inserted into the submission backbone pSB1C3.

To insert the sequence for HIV tat/pol/gag/env into pSB1C3 we designed Gibson primers with compatible overhangs that also included the start codon ATG. This fragment was amplified via PCR (Link zum Labjournal-Eintrag) and then assembled with the digested pSB1C3 backbone using Gibson assembly. To prove correct insertion of our fragment we did a test digest and sent the whole plasmid for sequencing.

We inserted the sequence coding for HIV tat/pol/gag/env into pET_22b+ for overexpression in E.coli. We could show interaction of the HIV tat/pol/gag/env antigen with a polyclonal anti-HIV-1 P24 antibody (Fig. 2)

Figure 2: Western Blot of HIV multi-epitopic antigen. HIV multi-epitopic antigen was analyzed by 12,5% SDS-PAGE. The anti-HIV-1 P24 polyclonal antibody was used in a dilution of 1:5000. The secondary antibody (anti-rabbit HRP) was diluted 1:5000. The expected molecular weigth is 20.5 kDa.

Get the sequence in genebank format: BBa_K1621004.gb.

¹⁾ Douek et al. 2009. Emerging concepts in the immunopathogenesis of AIDS. Annual review of medicine

²⁾ Rahimi et al. 2015. Optimization of multi-epitopic HIV-1 recombinant protein expression in prokaryote system and conjugation to mouse DEC-205 monoclonal antibody: implication for in-vivo targeted delivery of dendritic cells. Iranian journal of basic medical sciences