Study On The Recombinant Ginseng Peptide

Ginseng peptide consists of 14 amino acid residues. It has the potential of declining blood sugar and is used to treat diabetes.A DNA fragment had been designed as the purpose gene according to the structure-function relationship of the natural ginseng peptide. Following the insertion of it into vector pBluescript, pBluescript-gsp 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, dealt plasmid pBluescript-gsp and pPIC9 with Hind IQ and Not I, gsp DNA in gsp-1 was inserted into vector pPIC9 and get the recombinant plasmid pPIC9-gsp. After strictly checking, linearized pPIC9-gsp with Bgl II and then transformed it into GS115 with electroporation equipment. By cultivating on MM and MDmedia to screen the his+Muts phenotype transformant, positive transformants had been picked up for further testing. Finally, expressed protein from fermentation supernatant induced by methanol had been analyzed and confirmed on Tricine-SDS-PAGE. The expressed protein contains 59 amino acids with molecular weight 5.9kD. Dealt with trypsin and carboxypeptidase B then analyzed by Tricine-SDS-PAGE, the construction of expressed protein had been confirmed too.After the optimization of nitrogen source in induced media culture, the expression of purpose protein had been improved greatly. Batch fermentation had proved the recombinant yeast was adapted to express foreign protein continuously.In short, a recombinant plasmid pPIC9-gsp has been constructed, and positive transformants have been screened out. Some relevant technologies of electroporation and Tricine-SDS-PAGE have been researched. The optimization of fermentation and related areas has been carried out.