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Isolation and cloning of the β subunit of human follicle stimulating Hormone (hFSHβ) with its native gene’s signal sequence in methylotroph yeast Pichia pastoris pPIC9 shuttle vector
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M. R. اکبریعیدگاهی   , R. نصر , A. A. شعبانی , M. مقبلی |
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Abstract: (12527 Views) |
| Introduction: Follicle Stimulating Hormone (FSH) is one of the pituitary glycoproteines that it consists of two subunits alpha and beta. The beta subunit is responsible for the biological activity of FSH.
The aim of present study was isolation of the β subunit coding sequence containing its signal sequence from human genome and then cloning of the isolated sequence in pPIC9 shuttle vector under the control of AOX1 promoter and α factor signal sequence.
Material and Methods: the gene sequence of interest was isolated as a 2kb DNA fragment and cloned in pTZ57R vector resulting to pTV-2019 plasmid. The construct was used as template for modification of 5΄ region of gene upstream to ATG codon using PCR. Finally, amplicon was cloned in pPIC9 and the new construct named pPIC9F1.
Results: The sequence of FSHβ gene in pTV-2019 was confirmed by restriction analysis and DNA sequencing. In addition, restriction analysis and AOX1 primer-mediated PCR showed that pPIC9F1 has correct construction.
Conclusion: The new construct, pPIC9F1, contains the coding sequence of FSHβ gene and its signal sequence (E2-IVS2-E3). Therefore, this construct can be used for integration of FSHβ gene into yeast genome exactly downstream to AOX1 promoter. Under this condition, a fusion protein is produced that it contains two signal peptides, α factor and FSH signal peptides. Yeast expression system is able to cleavage α factor. It seems this is the first attempt for cloning of human FSHβ in yeast expression system. |
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| Keywords: Pichia pastoris, pPIC9, follicle stimulating hormone |
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Full-Text [PDF 549 kb]
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Type of Study: Applicable |
Subject:
General
Received: 2008/09/2 | Published: 2006/11/15
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