Process for producing a prophylactic and therapeutic dna immunological composition for hpv and cancers associated with the virus, hybrid protein, expression vector, immunological composition and uses thereof

A Process for producing prophylactic and therapeutic vaccine against HPV and cancers associated with the virus characterized by the fact that it consists of the following steps:

• a) selection of epitopes and construction of a vaccine vector containing peptides selected from HPV16 L2 and E6 proteins, wherein there were used HPV16 L2 and E6 gene sequences which were obtained from the international GenBank(r) database, wherein the access code of the E6 gene used was AAD33252 and the reference for access to L2 was NC_001526.2, preferentially, the peptides selected from the E6 gene are among the amino acid residues 50 to 57 as established in SEQ ID NO:
• 5;
• b) amplification of the vaccine vector, wherein amplification of the vaccine vector occurs in bacterial cultures of Escherichia coli DH5α;
• c) bacterial transformation, wherein was carried out from the competent bacteria, where a mixture of 10 µL of plasmideal DNA, approximately 15 g, and an aliquot of chemo-competent bacteria was incubated for 30 minutes on ice, subjected to a thermal shock for 2 minutes at 42° C. and incubated again on ice for 5 minutes, then 350 µL of S.O.C medium (Tryptone 2%, Yeast extract 0.5%, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM Glucose), the mixture being incubated for 90 minutes at 37° C., under agitation of 250 rpm, then the transformed bacteria were seeded in plates of LB-agar medium containing or not 100 µg/ml ampicillin and incubated at 37° C. for 18 hours, preferentially, colonies grown on LB-agar plates containing ampicillin identify the positive clones that were selected by colony PCR and seeded in tubes containing 3 mL of LB medium, incubated at 37° C., under stirring for approximately 18 hours;
• d) selection of bacterial colonies, wherein for the selection of bacterial colonies after transformation, the colony PCR technique was used, where 2 specific primers synthesized from the L2E6 gene sequence were designed, EAK03 and EAK02, and capable of generating a 144 bp product, preferentially, the initiators are as established by SEQ ID NO 6 and SEQ ID NO:
• 7, and the primers were resuspended in TE buffer (10 mM Tris / HCl, 1 mM EDTA) at a stock concentration of 100 µM, in which for PCR, each solution was prepared separately in microtubes containing 25 µL of PCR-Mix solution (1.5 mM MgCl2, 0.5U Taq, 200 5 M dNTPs – LGC Biotecnologia, Cotia, SP, Brazil), 0.5 µL of each primer at 25 µM and ultrapure water for a final solution volume of 50 µL, and as template DNA, a sample of each transformed bacterial colony was used, selected and grown in LB medium with ampicillin, wherein the amplification was performed in a thermocycler occurring as the protocol:
• 95° C. for 5 minutes, 25 cycles of 1 minute at 95° C., 30 seconds at 56° C. and 1 minute at 72° C., followed by final extension at 72° C. for 7 minutes, which the detection of PCR products occurred through electrophoresis in 0.8% agarose gel in 0.5X TAE buffer, The samples were prepared with the addition of 0.5 µL of Blue Green Loading Dye I and subjected to a run at 80 A for 30 minutes;
• e) extraction of plasmideal DNA;
• wherein the positive clones selected by the colony PCR were cultured in 5 mL of LB medium, at 37° C., for approximately 18 hours, under agitation, in the presence of the antibiotic of selection, then they were centrifuged at room temperature, for 1 minute at 12000xg to sediment the intact bacteria, and for the purification of plasmideal DNA, the GeneJet Plasmid Miniprep Kit (Thermo Fisher Scientific) was used, where the purified DNA was analyzed in 0.8% agarose gel in 0.5X TAE buffer and submitted to the sequencing technique to confirm the insertion of the gene of interest to the vector, quantified in a spectrophotometer and stored at -20° C.;
• f) DNA sequencing;
• wherein plasmideal DNA samples were sequenced to confirm the correct insertion of the L2E6 gene and its integrity, where the DNA samples purified from the gel were quantified by spectrophotometry, and a volume of 5 µL of the plasmid was aliquoted and 2.5 µL of the EAK03 primer was added at a concentration of 5 µM;
• g) cell transfection;
• wherein the transient transfection of HEK 293T cells was performed by lipofection using the GeneJuice® Transfection Reagents, where the transfection reagent was mixed by vortex to an aliquot of DMEM medium, being incubated for 5 minutes at room temperature;
• to the mixture, the plasmideal DNA of interest was added, in the appropriate proportion, gently homogenized and incubated for 15 minutes at room temperature, then the mixture Medium/transfection reagent/DNA was added to the cells with complete culture medium, and incubated in oven at 37° C. and 5% CO2, where after 8 hours the culture medium was changed, now with a complete medium containing antibiotics and 10% SBF, wherein the incubation continued for approximately 48 hours when the cells were collected and analyzed in immunofluorescence and in SDS-PAGE electrophoresis gel, and in step (g) for 293F cells, the transfection assays were carried out using the 293fectin transfection reagent;
• h) expression and characterization of recombinant proteins;
• wherein HEK 293T cells were grown on 13 mm diameter round coverslips in 12-well culture plates (TPP®), where the easily adherent strains were grown directly on the coverslips in D10 medium, kept in a humid atmosphere, containing 5% CO2, for approximately 18 hours, and in step (h), for the assays using the 293F cells already transfected, said cells were centrifuged in microtubes and the supernatant discarded;
• and
• i) Protein synthesis.