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GIOVANNI, D. N. S. Cloning and expression of the antiviral protein present in the hemolymph of Lonomia obliqua by recombinant DNA technology in Escherichia coli. 2014. 116 p. PhD Thesis (Biotechnology) Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, 2014. Viral infections are of great public health interest. Insect hemolymph studies demonstrated the presence of active principles of interest for the development of new pharmacological drugs such as antivirals. In 2009, we demonstrated the existence of a potent antiviral in hemolymph of L. obliqua. This purified protein has reduced the viral titer (TCID50 ml) of measles 157 times, 61 times that of poliovirus and 64 times that of the H1N1 influenza virus. Recently, we constructed a recombinant baculovirus / insect cell for the expression of antiviral protein, showing good antiviral activity because it reduced the title herpes virus 10 times, rubella and EMC 10 times in the system. However, this expression system is very expensive and laborious. Thus, cloned and expressed this protein with antiviral activity in bacterial system. The antiviral sequence was obtained by PCR based on the sequence previously cloned in our lab and the specific primers used restriction sites (BamHI and HindIII) have been added to the connection with the pET28a plasmid. The PCR product and the vector pET28a are digested with the same enzymes. The connection, where the resulting construction was transformed by heat shock into E. coli BL21 DE3 pLysS was taken. The antiviral protein was induced with 1.0 mM IPTG, 0.7 DO and 37 °C for 4 hours. Following expression, the protein was detected on western blot using anti-His antibody and the bacteria were then sonicated. The crude supernatant was affinity purified by histidine tail by magnetic beads and the HisTrap FF 5 ml column, the resulting material was shown to be toxic to the present tests because imidazole. However the expressed protein showed a cell protection, the material only with imidazole destroying the cell carpet much faster than the same dose containing antiviral along with imidazole. Despite the protection, we chose to remove the imidazole of antiviral testing and evaluating this cell protection separately. We used a dialysis run under refrigeration and gel filtration in the Akta to remove imidazole. Antiviral tests were performed and a titration (TCID50 ml) was made with EMC virus, where 20 μg/ml protein showed a protection of 10 000 times compared to the control. Using qPCR and showed a reduction in the levels of RNA transcript of herpes virus and rubella virus at all dilutions and completely inhibited from dilutions 10 and 10, respectively, showing a potent antiviral effect.

Clonagem e expressão de proteína antiviral presente na hemolinfa de Lonomia obliqua por tecnologia de DNA recombinante em Escherichia coli.