Expression of an antiviral protein from Lonomia obliqua hemolymph in baculovirus/insect cell system

Abstract

The control of viral infections, mainly those caused by influenza viruses, is of great interest in Public Health. Several studies have shown the presence of active properties in the hemolymph of arthropods, some of which are of interest for the development of new pharmacological drugs. Recently, we have demonstrated the existence of a potent antiviral property in the hemolymph of Lonomia obliqua caterpillars. The aim of this study was to produce an antiviral protein in a baculovirus/Sf9 cell system. The resulting bacmid contains the sequence coding for the antiviral protein previously described by our group. Total RNA from L. obliqua caterpillars was extracted with Trizol and used in the reverse transcription assay with oligo(d)T primer followed by polymerase chain reactions (RT-PCR) with specific primers for the cDNA coding for the antiviral protein, based on the sequence deposited in the GenBank database. Restriction sites were inserted in the cDNA for ligation in the donor plasmid pFastBac1™. The recombinant plasmid was selected in Escherichia coli DH5α and subsequently used in the transformation of E. coli DH10Bac for the construction of the recombinant bacmid. This bacmid was used for the expression of the antiviral protein in the baculovirus/Sf9 cell system. After identifying the protein by western blot, activity tests were performed, showing that the purified recombinant protein was able to significantly reduce viral replication (about 4 logs). Studies on the optimization of the expression system for the production of this antiviral protein in insect cells are in progress.

Fig. 1

(A) Agarose gel electrophoresis (1%) confirming amplification of the cDNA that codes for the antiviral protein (line 2 and 3). (B) Agarose gel electrophoresis (1%) confirming amplification of the cDNA that codes for other proteins (line 7 for 8-LOH and line 9 for LOH-19-AY829833), M: molecular marker (1 kb Ladder, New England Biolabs).

Fig. 2

Agarose gel electrophoresis (1%) confirming baculovirus transposition with amplification of the respective products. (Line 1 M: molecular marker (1 kb Ladder, New England BioLabs), line 2 baculovirus without transposition, line 3 and 4 rAVLO 2300 bp + 591 bp protein = 2891 bp and line 5 amplification of sequence of rAVLO with amplification primers.)

Fig. 3

Western blot of samples of a portion of AKTA (ion exchange) showing the presence of the antiviral protein. The proteins present in the nitrocellulose membrane were revealed with an antibody anti histidine. rAVLO: recombinant protein with antiviral activity, negative control and positive control.