Expression, purification and immunodetection of a recombinant fragment (residues 179-281) of the G protein from rabies virus ERA strain.
The glycoprotein (G) of rabies virus (RV) is important for virus infectivity and induction of the protective immunity. In this study, the region comprising linear epitopes (residues 179-281, ERA strain), named rGERA179-281, was cloned in frame with a hexahistidine tag coding sequence at its N-terminal end and overexpressed in Escherichia coli Rosetta strain. The expression under transcriptional regulation of T7 promoter yielded insoluble protein aggregates in the form of inclusion bodies. The inclusion bodies were solubilized with 6M guanidine HCl and the protein was purified to homogeneity under denaturing conditions. Mass spectrometry data confirmed the identity of the protein. The purified protein (13.8kDa) showed significant reactivity with antibodies present in a therapeutic human rabies immune globulin (HRIG), as demonstrated by immunoblotting analysis. In addition, by in vitro competitive neutralization assay, rGERA179-281 led to a measurable reduction in the ability of HRIG to neutralize rabies virus. These results, along with the good yield obtained, encourage further studies on the more detailed immunological properties of rGERA179-281, such as the ability to induce rabies virus neutralizing antibodies and the production of anti-G monoclonal antibodies, which together, might be useful for the development of new diagnostic methods.
Bassi
Enio J
EJ
Laboratório de Imunologia Aplicada, Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, SC, Brazil.
Vernal
Javier
J
Zanluca
Camila
C
Terenzi
Hernán
H
Zanetti
Carlos R
CR
D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007120:Immunochemistry; D016056:Immunodominant Epitopes / Q000096:biosynthesis* / Q000276:immunology* / Q000302:isolation & purification; D007136:Immunoglobulins / Q000276:immunology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010446:Peptide Fragments / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000276:immunology* / Q000302:isolation & purification; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*
D000956:Antigens, Viral; D006023:Glycoproteins; D016056:Immunodominant Epitopes; D007136:Immunoglobulins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C064951:glycoprotein G, Rabies virus