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The production of recombinant 63kDa glycoprotein of Leishmania major by methylotropic yeast Pichia Pastoris
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A. A شعبانی   , R. مک مستر , B. کاظمی , M. کریمی , D. شهباززاده , F. مهبودی |
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Abstract: (17008 Views) |
| Introduction: Cut aneo us lesihmahiasis (L) is a world wide infect io us diseas e . Several
approach toward vaccine have been taken around the world . Recombinant vaccines using gp63
in cocta il form is one of the candidates. Because, a significan t protection against a challeng with
L. major was elicited in senssitive BALB/C mice after vaccination with recombinant BCG
producing gp63, as well as , gp63 delivered orally by salmonella typhimurium C an preferably
induce th e development of Th-I subset of CD4+ T cells. Since expression of eukaryotic genes
are similar to Pichia pastoris as a eukaryotic cell, refolding and glycosylation may be an alogous to native form. GP63 gene from L. major (NIH strain) cloned and expressed in Pichia pastoris .
Materials and Methods: Modified gp 63 ge ne th at encoded mature protein o nly (478 aa )
cloned in shuttle vector pHIL-Sl. Under contro l of alcohol oxidase 1 gene promotor (pAOXI)
with yeast acid phosphatase signal sequence (PHO I). KM71 and GSI 15 strains of Pichia pastoris
transformed with it and selected on histidin e minus medium. PCR and Southerbn blotting were
done on chromosomal DNA of transform ant s yeast. Expressio n of rgp63 was evaluated by using Northern blotting, SDS-PAGE, Western-blott ing and immuno electro n micro sco py. Its activity was evaluated by using SDS-PAGE gelatin gel.
Results: PCR and Southern blott ing a na lys is o n chromosomal DNA o f t rnsfo rma nts have
bee n demons tra ted th at gp63 gene in te grated into chro mosomal DNA of ye as t. Expressio n of rgp63 in transformants of Pichia past oris confirmed by using Nor thern blott ing and SDS-PAGE. The findings were cinfirmed by Western blotting analysis and immunoelectron microsco py too.
Conclusion: Rgp63 expressed in Pichia pastori s had glcosylated form too. It was act ive on
SDS-PAGE gelatin gel. Thus, it was more similar to native gp63. |
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| Keywords: Expression, gp63, Leishmania, Yeast, Pichia pastoris |
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Full-Text [PDF 2005 kb]
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Type of Study: Research |
Subject:
General
Received: 2009/01/5 | Published: 2002/02/15
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