NDLI: Expression and Solubilization of Insect Cell-Based Rabies Virus Glycoprotein and Assessment of Its Immunogenicity and Protective Efficacy in Mice ▿

Content Provider	PubMed Central
Author	Ramya, R. Subramanian, B. Mohana Sivakumar, V. Senthilkumar, R. L. Rao, K. R. S. Sambasiva Srinivasan, V. A.
Copyright Year	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 in Spodoptera 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.
Related Links	http://dx.doi.org/10.1128/cvi.05258-11
Ending Page	1679
Page Count	7
Starting Page	1673
File Format	PDF
ISSN	15566811
e-ISSN	1556679X
Journal	Clinical and Vaccine Immunology : CVI
Issue Number	10
Volume Number	18
Language	English
Publisher	American Society for Microbiology
Publisher Date	2011-10-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Immunology Immunology and Allergy Microbiology (medical) Clinical Biochemistry Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Immunology and Allergy Clinical Biochemistry Immunology Microbiology (medical)

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