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2005•Chinese Journal of VeterinaryRequires access

Expression of Bovine Interleukin-2 Gene in Pichia pastoris

Fang Chen, Sun Hong-li, Xiangrong Cao, Zhen Li, Ruisong Yu

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Abstract

The Interleukin-2 gene cDNA was cloned into the Pichia pastoris expression vector pPICZB,which is under the control of the alcohol oxidase promoter AOX1.The linearized recombinant plasmid of BoIL2-pPICZB,digested by SacⅠ,was transformed into X-33 strains by electroporation.The multi-copy insert transformants were screened by Zeocin-resistance and induced by 1% methanol.The intracellular expression products were tested by SDS-PAGE analysis and Western blotting.Purified recombinant BoIL2 was gained by metal-chelating affinity chromatographic (MCAC).Assay with murine CTLL-2 cells showed that the recombinant BoIL2 exhibited the biological activity.

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What this paper is about

The Interleukin-2 gene cDNA was cloned into the Pichia pastoris expression vector pPICZB,which is under the control of the alcohol oxidase promoter AOX1.The linearized recombinant plasmid of BoIL2-pPICZB,digested by SacⅠ,was transformed into X-33 strains by electroporation.The multi-copy insert transformants were screened by Zeocin-resistance and induced by 1% methanol.The intracellular expression products were tested by SDS-PAGE analysis and Western blotting.Purified recombinant BoIL2 was gained by metal-chelating affinity chromatographic (MCAC).Assay with murine CTLL-2 cells showed that the recombinant BoIL2 exhibited the biological activity.

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Available abstract

The Interleukin-2 gene cDNA was cloned into the Pichia pastoris expression vector pPICZB,which is under the control of the alcohol oxidase promoter AOX1.The linearized recombinant plasmid of BoIL2-pPICZB,digested by SacⅠ,was transformed into X-33 strains by electroporation.The multi-copy insert transformants were screened by Zeocin-resistance and induced by 1% methanol.The intracellular expression products were tested by SDS-PAGE analysis and Western blotting.Purified recombinant BoIL2 was gained by metal-chelating affinity chromatographic (MCAC).Assay with murine CTLL-2 cells showed that the recombinant BoIL2 exhibited the biological activity.

Key concepts: Pichia pastoris, Recombinant DNA, Alcohol oxidase, Electroporation, Molecular biology, Complementary DNA, Expression vector, Insert (composites)

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