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Cloning and expression of bovine prion protein 27-30 gene

Lü Wei, Peng Zhang, Wei Guang-sen

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Abstract

A 420 bp PrP~(27-30) gene fragment of the bovine Prop gene was amplified by PCR from plasmid BoPrnp-T containing the ORF of bovine Prnp gene.The PCR product was cloned into pPIC9K vector to produce recombinant plasmid pPIC9K-boPrP~(27-30) and transformed into E.coli JM109.The pPIC9K-boPrP~(27-30) was linearized with SalⅠand then transfected into Pichia Pastoris GS115 by electroporation.A recombinant strain GS115/pPIC9K-boPrP~(27-30) was obtained by G418 selection.Expression of the recombinant PrP was induced with 1.0% methanol and the expression products were identified by SDS-PAGE and Western blot analysis.The results demonstrated that bovine PrP~(27-30) gene was successfully expressed in Pichia Pastoris as a 27 Ku protein that could be specifically recognized by monoclonal antibody SAF-70.

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What this paper is about

A 420 bp PrP~(27-30) gene fragment of the bovine Prop gene was amplified by PCR from plasmid BoPrnp-T containing the ORF of bovine Prnp gene.The PCR product was cloned into pPIC9K vector to produce recombinant plasmid pPIC9K-boPrP~(27-30) and transformed into E.coli JM109.The pPIC9K-boPrP~(27-30) was linearized with SalⅠand then transfected into Pichia Pastoris GS115 by electroporation.A recombinant strain GS115/pPIC9K-boPrP~(27-30) was obtained by G418 selection.Expression of the recombinant PrP was induced with 1.0% methanol and the expression products were identified by SDS-PAGE and Western blot analysis.The results demonstrated that bovine PrP~(27-30) gene was successfully expressed in Pichia Pastoris as a 27 Ku protein that could be specifically recognized by monoclonal antibody SAF-70.

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Available abstract

A 420 bp PrP~(27-30) gene fragment of the bovine Prop gene was amplified by PCR from plasmid BoPrnp-T containing the ORF of bovine Prnp gene.The PCR product was cloned into pPIC9K vector to produce recombinant plasmid pPIC9K-boPrP~(27-30) and transformed into E.coli JM109.The pPIC9K-boPrP~(27-30) was linearized with SalⅠand then transfected into Pichia Pastoris GS115 by electroporation.A recombinant strain GS115/pPIC9K-boPrP~(27-30) was obtained by G418 selection.Expression of the recombinant PrP was induced with 1.0% methanol and the expression products were identified by SDS-PAGE and Western blot analysis.The results demonstrated that bovine PrP~(27-30) gene was successfully expressed in Pichia Pastoris as a 27 Ku protein that could be specifically recognized by monoclonal antibody SAF-70.

Key concepts: Pichia pastoris, Recombinant DNA, Electroporation, Biology, Molecular biology, Expression vector, Cloning (programming), Plasmid

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