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Multimerization, codon optimization and characterization of CVS rabies glycoprotein produced in pichia pastoris and its preclinical studies

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Title Multimerization, codon optimization and characterization of CVS rabies glycoprotein produced in pichia pastoris and its preclinical studies
 
Creator R, Madhusudhan
 
Contributor P. H, Ramanjini Gowda
 
Subject fungi, proteins, diseases, genes, cloning, recombination, biological phenomena, genetic processes, enzymes, codons
 
Description Rabies is a viral disease that causes acute encephalitis in warmblooded
animals. Rabies is almost invariably fatal if post-exposure
prophylaxis is not administered prior to the onset of severe symptoms.
Glycoprotein is the major surface protein of rabies virus, responsible for
the production of neutralizing antibodies.
Generally, gene copy number and codon optimization have been
identified as the most important factors influencing the heterologous
protein expression in Pichia pastoris. Hence we have transformed and
screened Pichia GS115 clones with multicopy pPIC9K expression cassette
having CVS rabies glycoprotein gene on YPD plates containing Geneticin
at concentrations of 0.25, 0.5, 1.0, 2.0, 3.0, 4.0 mg/ml. The clones were
validated for multicopy with Southern Blotting and Real-Time PCR. Clone
13 (single copy number) produced 398μg of recombinant protein while
Clone14 (5 copies) produced 893μg of recombinant protein. The
recombinant CVS rabies glycoprotein produced from these clones were
confirmed through Western Blotting and ELISA. The clones with higher
copy number produced more quantity of recombinant CVS rabies
glycoprotein than clone with single copy number. The native CVS rabies
glycoprotein gene sequence was codon optimized and cloned into yeast
expression vector pPICZαA and transformed into P. pastoris strain X-33
and confirmed through PCR. Recombinant protein was confirmed
through Western blot. Pichia clone transformed with codon optimized
rabies glycoprotein gene produced 570μg of glycoprotein while Pichia
clone transformed with native Rabies glycoprotein gene produced 380μg
of glycoprotein. Purified RGP and RGP_Opt produced in P. pastoris were
used for immunization studies in mice. Though RGP and RGP_Opt did
not protect the mice, RIFFIT assay recorded antibody titer value of 0.5
and 1.0 IU for the serum collected from mice immunized with RGP_Opt
and commercial vaccine respectively.
 
Date 2016-05-21T10:11:58Z
2016-05-21T10:11:58Z
2013-09-19
 
Type Thesis
 
Identifier Th-10652
http://krishikosh.egranth.ac.in/handle/1/66190
 
Language en
 
Format application/pdf
 
Publisher University of Agricultural Sciences, Bengaluru