Multimerization, codon optimization and characterization of CVS rabies glycoprotein produced in pichia pastoris and its preclinical studies
KrishiKosh
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Title |
Multimerization, codon optimization and characterization of CVS rabies glycoprotein produced in pichia pastoris and its preclinical studies
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Creator |
R, Madhusudhan
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Contributor |
P. H, Ramanjini Gowda
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Subject |
fungi, proteins, diseases, genes, cloning, recombination, biological phenomena, genetic processes, enzymes, codons
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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. |
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Date |
2016-05-21T10:11:58Z
2016-05-21T10:11:58Z 2013-09-19 |
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Type |
Thesis
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Identifier |
Th-10652
http://krishikosh.egranth.ac.in/handle/1/66190 |
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Language |
en
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Format |
application/pdf
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Publisher |
University of Agricultural Sciences, Bengaluru
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