Team:UFMG Brazil/Results

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DNA coding sequence of Mouse interferon-beta (IFN-beta) linked to signal peptide that allow protein secretion and latency-associated peptide (LAP) that possibilite IFN activation only in inflammatory site was designed with optimized codons for expression in Leishmania host (Figure 1).

Figure 1 - LAP-IFN-beta synthetic gene synthetized. Highlighted in green is signal peptide to secretion and highlighted in purple is LAP sequence.

The final sequence was synthesized by IDT (California, USA) in pUC_IDT standard vector with resistance to ampicillin. E. coli strain DH10b/TOP10 was transformed with the plasmid and resistant colonies were observed after incubation by 16 hours at 37 oC (Figure 2).

Figure 2 - Plate of E. coli colonies transformed with pUC_IDT vector containing recombinant IFN-beta. Red arrows label positive colonies.

Miniprep to recover the plasmids were carried out and the quality of extraction was confirmed by 1.2% agarose gel stained with SYBR Safe DNA Gel Stain from Invitrogen (Waltham, USA) (Figure 3).

Figure 3 - Electrophoresis in 1.2% agorose gel of plasmid pUC_IDT containing recombinant IFN-beta. Labels of lanes: 1) Molecular weight markers; 2) Negative control; 3 and 4) Plasmids recovered by miniprep protocol.

Plasmids containing IFN-beta were used as DNA template in a PCR with specific primers pair to the synthetic gene (Figure 4). The forward primer had restriction site of enzyme XbaI in the 5’ region and the reverse oligonucleotide sequence had site of BamHI in the 5’ region.

Figure 4 - Electrophoresis in 1.2% agarose gel of IFN-beta PCR product containing restriction sites to XbaI and BamHI enzymes. The molecular weight of correct PCR product should be 1461 base pairs. Labels of lanes: 1) Molecular weight markers; 2, 3 and 4) Positive PCR for three transformed E. coli colonies; 5) Negative control.

The PCR product was joined with pGEM-T easy vector from Promega (Fitchburg, USA). E. coli DH10b/TOP10 were transformed with the ligated product (Figure 5) or pLpNEO vector specific to Leishmania expression. After resistant bacteria selection and cultivation in liquid medium, miniprep protocol was performed to recovered the plasmids (Figures 6 and 7).

Figure 5 - Plate of E. coli colonies transformed with pGEM-T easy vector containing recombinant IFN-beta. Red arrows label positive colonies.

Figure 6 - Electrophoresis in 1.2% agarose gel of plasmid pGEM-T easy containing synthetic IFN-beta. Labels of lanes: 1) Molecular weight markers; 2, 3, 4 and 5) Plasmids recovered by miniprep protocol.

Figure 7 - Electrophoresis in 1.2% agarose gel of pLpNEO vector. Labels of lanes: 1) Molecular weight markers; 2 and 3) Plasmids recovered by miniprep protocol.