CLONING AND EXPRESSION OF STREET RABIES VIRUS GLYCOPROTEIN GENE IN YEAST (Pichia pastoris)
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Date
2009-05-16
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University of Agricultural Sciences GKVK, Bangalore
Abstract
Rabies is a preventable infectious viral disease of mammals, most often
transmitted through the bite of rabid animals and causes significant number of
annual deaths in rural areas of Asia and Africa. Immunization with rabies vaccine
is the most effective means of preventing rabies virus infection. As the presently
available vaccine are expensive and involves risk of handling live virus hence
inexpensive, safe and efficacious vaccines are urgently needed to address the
infectious viral diseases. The yeast, Pichia pastoris, acts as an ideal system for
expression of heterologous genes, as the post translational modifications like
glycosylation, protein folding successfully occurs in the yeast and which secretes
out the heterologous protein into media which makes protein purification simple.
The glycoprotein of rabies virus is most antigenic and immunogenic determinant
present in rabies virion and can serve as effective target for development of
vaccine. Hence the street rabies glycoprotein gene (1.6 Kbp) was cloned into
pBKS+ plasmid vector which was characterized by PCR, restriction digestion and
by sequencing, further the rabies glycoprotein gene was subcloned into yeast
transfer vector (pPICZαA) for expression. Linearised recombinant vector
containing glycoprotein gene was introduced into yeast (Pichia pastoris, GS115).
The recombinant yeast clones were confirmed by PCR analysis. The recombinant
protein expressed in yeast using methanol at 0.5% (v/v) and the expression of
glycoprotein gene product was confirmed by SDS-PAGE, the 70kDa protein
confirmed the antigen specificity by western blot and ELISA. The expressed
recombinant rabies glycoprotein could be used as vaccine after the immunization
trials in animals.
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