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Construction of Recombinant Pichia pastoris-rat glutamic acid decarboxylase(rGAD)

Baoli Wang

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Abstract

Objective:To Construct the recombinant Pichia pastoris containing rat glutamic acid decarboxylase(rGAD)gene,thus establish a new way to express rGAD effectively in euˉkaryotic cells.Methods:The encoding fragment of rGAD65was multified by PCR from plasmid pUC18_rGAD,and then cloned into a secretary expression vector pPIC9K.Linearized plasmid pPIC9K/rGAD with SalI was transformed into Pichia pastoris strain GS115through method of PEG1000.Finally,prospective transfermants with phenotype His + Mut + were selected via PCR assay.Results:DNA sequencing and PCR analysis indicated that the recombinant Pichia pastoris strain GS115/pPIC9K/rGAD was construced successfully.Conclusion:Constructed recombinant Pichia pastoris made a basis for expression of rGAD by genetic engineering as an important antigen.

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Objective:To Construct the recombinant Pichia pastoris containing rat glutamic acid decarboxylase(rGAD)gene,thus establish a new way to express rGAD effectively in euˉkaryotic cells.Methods:The encoding fragment of rGAD65was multified by PCR from plasmid pUC18_rGAD,and then cloned into a secretary expression vector pPIC9K.Linearized plasmid pPIC9K/rGAD with SalI was transformed into Pichia pastoris strain GS115through method of PEG1000.Finally,prospective transfermants with phenotype His + Mut + were selected via PCR assay.Results:DNA sequencing and PCR analysis indicated that the recombinant Pichia pastoris strain GS115/pPIC9K/rGAD was construced successfully.Conclusion:Constructed recombinant Pichia pastoris made a basis for expression of rGAD by genetic engineering as an important antigen.

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Available abstract

Objective:To Construct the recombinant Pichia pastoris containing rat glutamic acid decarboxylase(rGAD)gene,thus establish a new way to express rGAD effectively in euˉkaryotic cells.Methods:The encoding fragment of rGAD65was multified by PCR from plasmid pUC18_rGAD,and then cloned into a secretary expression vector pPIC9K.Linearized plasmid pPIC9K/rGAD with SalI was transformed into Pichia pastoris strain GS115through method of PEG1000.Finally,prospective transfermants with phenotype His + Mut + were selected via PCR assay.Results:DNA sequencing and PCR analysis indicated that the recombinant Pichia pastoris strain GS115/pPIC9K/rGAD was construced successfully.Conclusion:Constructed recombinant Pichia pastoris made a basis for expression of rGAD by genetic engineering as an important antigen.

Key concepts: Pichia pastoris, Recombinant DNA, Pichia, Molecular biology, Plasmid, Expression vector, Gene, Chemistry

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