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Cloning expression and immunization studies of cvs rabies virus glycoprotein gene in yeast (pichia pastoris)

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Title Cloning expression and immunization studies of cvs rabies virus glycoprotein gene in yeast (pichia pastoris)
 
Creator T. M, Ningaraju
 
Contributor P. H, Ramanjini Gowda
 
Subject proteins, diseases, fungi, biological phenomena, genes, cloning, viruses, recombination, pcr, enzymes
 
Description Rabies is an important disease, fatal in 100 per cent of cases if no treatment is
administered. The disease spreads through domestic and wildlife animals. Despite the
existence of effective pre and post-exposure treatments, at least 60,000 deaths occur
worldwide annually from rabies. Most human rabies vaccines currently used in the
developed countries are highly attenuated virus strains obtained by multiple passages
through the chick embryos or cells in culture, during which multiple mutations are
expected to accumulate in the viral gene(s) involved in the virulence. Hence safe,
inexpensive and efficacious vaccines are needed to address the infectious viral diseases.
The most effective approaches to develop new rabies vaccines is, to use the cloned viral
genes to which the genetic engineering technologies are applied. The yeast, Pichia
pastoris has become a highly popular expression host for the production of a wide variety
of intracellular and extracellular recombinant proteins of interest. The glycoprotein of
rabies virus is most antigenic and immunogenic determinant protein in rabies virion and
can serve as effective target for development of vaccine. Hence the CVS rabies
Glycoprotein gene was cloned into TA cloning vector (pTZ57R/T) by amplifying the full
length Glycoprotein from isolated total RNA of BHK -21 cells infected with CVS rabies
virus and named the construct as pNRCRG1506. Rabies Glycoprotein was amplified
from pNRCRG1506 with specific primer having restriction sites and cloned into Pichia
pastoris integration vector pPICZαA and confirmed by PCR and restriction digestion and
named this construct as pNRCRG1510. The Glycoprotein construct is transformed into
Pichia pastoris (X-33) by linerizing the construct pNRCRG1510 and confirmed by PCR.
Glycoprotein was induced with 0.5% methanol and Glycoprotein was confirmed by
running SDS gel and western blotting. Immunization trails was conducted in mice with
induced Glycoprotein and it was found to be immunogenic when it was challenged
against live CVS rabies virus. For increasing the Glycoprotein, Invitro multimerization of
Glycoprotein was carried out by cloning rabies Glycoprotein into pPIC9K vector and
transformed into Pichia pastoris (GS115), and confirmed by Real time PCR. This results
indicates that the Glycoprotein produced in Pichia pastoris can be used as subunit
vaccine for rabies.
 
Date 2016-05-24T11:47:43Z
2016-05-24T11:47:43Z
2011-11-18
 
Type Thesis
 
Identifier Th-9824
http://krishikosh.egranth.ac.in/handle/1/66264
 
Language en
 
Format application/pdf
 
Publisher University of Agricultural Sciences, Bengaluru