Rabies virus glycoprotein ectodomain expressed in Drosophila Expression System (Drosophila melanogaster Schneider 2)

Abstract

Background: Rabies is a viral neglected disease considered fatal, that affects mammals and with only 12 cases of survival reported in humans. Despite the existence of preventive vaccines, rabies still represents a serious public health problem, with a major impact in vulnerable populations around the world, causing about 55,000 human deaths per year. The membrane glycoprotein of the rabies virus (RVGP), specifically the ectodomain portion, has been intensively studied for the development of recombinant proteins, due to its ability to interact with neutralizing antibodies and induce immune responses. This project aims to construct a plasmidial vector containing only the ectodomain portion and detect the soluble RVGP expressed by recombinant Drosophila. Methods & Materials: The gene encoding ectodomain was synthesized in pUC57 (pRVGPecto) and subcloned into pMtBipV5HisC using EcoRI and NotI enzymes. Products of pMtBip-RVGPecto constructions were screened after DH5a transformation. Then plasmid constructed was transfected into S2cells using Lipofectamine® 2000 to obtain the recombinant stable lineage (S2MTBip_RVGP-ecto). Schott flasks were inoculated with 1 × 105 cells/ml of recombinant population. Cultures were induced at different concentrations of CuSO4 and sampled at different times. Protein detection was done by qualitative Dot and Western blotting analysis using mouse anti-rabies virus Glycoprotein Antibody (LS-C75309) non-conformational monoclonal antibody. Results: Transformation of pMtBip-RVGPecto into DH5a generated two colonies. After mini preparation, those samples had a banding pattern analyzed by enzyme digestion, confirming the presence of ectodomain insert. Recombinant cell population (S2MTBip_RVGP-Ecto) was capable to express rabies virus glycoprotein in soluble form under different induction and sampling conditions, although, part of RVGP is still detected into the cells. The highest RVGP expressions were observed for cultures induced with 0.7 mM (24, 48 and 72 hours), 0.5 mM and 1 mM (24 hours). Western Blotting results showed the presence of a band corresponding to a molecular mass of approximately 50 kDa in culture media, that correspond with expected value since the recombinant protein does not present complete RVGP. Conclusion: It is possible to produce a membrane-independent RVGP using an insect cells expression system (Drosophila melanogaster Schneider 2) contributing to the advancement expected in the vaccine production process.