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A-T cloning and expression of human β -nerve growth factor gene

Shu Zhen Hong

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Abstract

Objective:To analyse and identify subcloning of pro-β-nerve growth factor(β-NGF)gene and expression in Pichia pastoris stain cells.Methods:The pro-β-NGF gene was amplified by PCR.The re-combined plasmid pGEM-Tβ-NGF was obtained by A-T cloning and was detected by enzymolysis,PCR and sequence analysis.Theβ-NGF gene was inserted into pPIC9K vector,and theβ-NGF protein was induced by methanol.Results:The agarose gel electrophoresis results of PCR showed the pro-β-NGF gene appeared at the zone of748bp.The10%SDS-PAGE electrophoresis results of expression protein showed a single band corresponding with monomeric(14.0KD)form of humanβ-NGF protein,which represented30%of the total Pichia pastoris secretive protein approximately.The result of Western Blot showed the immune activity of the expression protein.Conclusions:Recombined plasmid pGEM-Tβ-NGF was successful;the expression pro-tein ofβ-NGF has the immune activity.

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Objective:To analyse and identify subcloning of pro-β-nerve growth factor(β-NGF)gene and expression in Pichia pastoris stain cells.Methods:The pro-β-NGF gene was amplified by PCR.The re-combined plasmid pGEM-Tβ-NGF was obtained by A-T cloning and was detected by enzymolysis,PCR and sequence analysis.Theβ-NGF gene was inserted into pPIC9K vector,and theβ-NGF protein was induced by methanol.Results:The agarose gel electrophoresis results of PCR showed the pro-β-NGF gene appeared at the zone of748bp.The10%SDS-PAGE electrophoresis results of expression protein showed a single band corresponding with monomeric(14.0KD)form of humanβ-NGF protein,which represented30%of the total Pichia pastoris secretive protein approximately.The result of Western Blot showed the immune activity of the expression protein.Conclusions:Recombined plasmid pGEM-Tβ-NGF was successful;the expression pro-tein ofβ-NGF has the immune activity.

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Available abstract

Objective:To analyse and identify subcloning of pro-β-nerve growth factor(β-NGF)gene and expression in Pichia pastoris stain cells.Methods:The pro-β-NGF gene was amplified by PCR.The re-combined plasmid pGEM-Tβ-NGF was obtained by A-T cloning and was detected by enzymolysis,PCR and sequence analysis.Theβ-NGF gene was inserted into pPIC9K vector,and theβ-NGF protein was induced by methanol.Results:The agarose gel electrophoresis results of PCR showed the pro-β-NGF gene appeared at the zone of748bp.The10%SDS-PAGE electrophoresis results of expression protein showed a single band corresponding with monomeric(14.0KD)form of humanβ-NGF protein,which represented30%of the total Pichia pastoris secretive protein approximately.The result of Western Blot showed the immune activity of the expression protein.Conclusions:Recombined plasmid pGEM-Tβ-NGF was successful;the expression pro-tein ofβ-NGF has the immune activity.

Key concepts: Molecular biology, Nerve growth factor, Subcloning, Pichia pastoris, Expression vector, Biology, Gene, Agarose gel electrophoresis

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