| Hirulog is a kind of antithrombotic peptides derived from the natural anticoagulant molecule hirudin with strong potential medicine value.A DNA fragment of 100bp had been designed as the purpose gene according to the structure-function relationship of the natural hirudin. Following the insertion of it into vector pBluescript-M13, pBluescript-hir was sequenced to ensure the correct reading fragment.Yeast Pichia pastoris is a powerful host for heterologous protein expression widely used at many laboratories around the world at present. We chose Pichia pastoris GS115 (his4"Mut+) as host and secretory plasmid pPIC9 as vector. First, used plasmid pBluescript-hir as template DNA to amplify insert DNA hir-1 by PCR. Using restriction endonuclease SnaB I and Not I , hirolog DNA in hir-1 was inserted into vector pPIC9 and get the recombinant plasmid pPIC9-hir. After strictly checked, linearized pPIC9-hir with Bgl II and then transformed it into GS115 with electroporation equipment. By cultivating on MM and MD media to screen the his+Muts phenotype transformant, WY-3 had been picked up for furthertesting. Finally, expressed prehirulog from WY-3 supernatant induced by methanol had been analysised and confirmed on Tricine-SDS-PAGE. The expressional prehirulog contains 31 amino acids with molecular weight 3.3kD.Besides, three kinds of electroporation equipments were used and compared. The efficiency of the different protease inhibitor and different kinds of media were researched as well as three kinds of staining method were tried in order to understand the small hetero-peptide expression.In short, a recombinant plasmid pPIC9-hir has been constructed, and WY-3 has been screened out. Some relevant technologies of electroporation and Tricine-SDS-PAGE have been researched.hirulog, Pichia pastoris, gene recombination, inducing expression...
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