Back to Search
Start Over
Expression and Solubilization of Insect Cell-Based Rabies Virus Glycoprotein and Assessment of Its Immunogenicity and Protective Efficacy in Mice
- Source :
- Clinical and Vaccine Immunology. 18:1673-1679
- Publication Year :
- 2011
- Publisher :
- American Society for Microbiology, 2011.
-
Abstract
- Rabies is a fatal zoonotic disease of serious public health and economic significance worldwide. The rabies virus glycoprotein (RVG) has been the major target for subunit vaccine development, since it harbors domains responsible for induction of virus-neutralizing antibodies, infectivity, and neurovirulence. The glycoprotein (G) was cloned using the baculovirus expression vector system (BEVS) and expressed inSpodoptera frugiperda(Sf-9) cells. In order to obtain a soluble form of G suitable for experimentation in mice, 18 different combinations of buffers and detergents were evaluated for their ability to solubilize the insect cell membrane-associated G. The combination that involved 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) detergent in lysis buffer 1, formulated with Tris, NaCl, 10% dimethyl sulfoxide (DMSO), and EDTA, gave the highest yield of soluble G, as evidenced by the experimental data. Subsequently, several other parameters, such as the concentration of CHAPS and the duration and temperature of the treatment for the effective solubilization of G, were optimized. The CHAPS detergent, buffered at a concentration of 0.4% to 0.7% (wt/vol) at room temperature (23 to 25°C) for 30 min to 1 h using buffer 1, containing 10% DMSO, resulted in consistently high yields. The G solubilized using CHAPS detergent was found to be immunogenic when tested in mice, as evidenced by high virus-neutralizing antibody titers in sera and 100% protection upon virulent intracerebral challenge with the challenge virus standard (CVS) strain of rabies virus. The results of the mice study indicated that G solubilized with CHAPS detergent retained the immunologically relevant domains in the native conformation, thereby paving the way for producing a cell-free and efficacious subunit vaccine.
- Subjects :
- Microbiology (medical)
Tris
Insecta
Rabies
Detergents
Genetic Vectors
Clinical Biochemistry
Immunology
Gene Expression
Buffers
Spodoptera
Biology
Antibodies, Viral
medicine.disease_cause
Virus
Cell Line
Mice
chemistry.chemical_compound
Viral Envelope Proteins
Chaps
Lysis buffer
medicine
Animals
Immunology and Allergy
Cloning, Molecular
Antigens, Viral
Glycoproteins
Infectivity
Vaccines, Synthetic
Immunogenicity
CHAPS detergent
Rabies virus
Vaccine Research
Antibodies, Neutralizing
Survival Analysis
Molecular biology
Rabies Vaccines
Solubility
chemistry
Baculoviridae
Subjects
Details
- ISSN :
- 1556679X and 15566811
- Volume :
- 18
- Database :
- OpenAIRE
- Journal :
- Clinical and Vaccine Immunology
- Accession number :
- edsair.doi.dedup.....7f36d35bce99c9dc0ed43dcfe975a125