The primer, AT For1, AT For2 and AT Back, are designed and synthesized for adding-PCR that is used to construct the fusion GST-AT gene. Depending on two-time adding-PCR amplification and the insertion of coding sequen...The primer, AT For1, AT For2 and AT Back, are designed and synthesized for adding-PCR that is used to construct the fusion GST-AT gene. Depending on two-time adding-PCR amplification and the insertion of coding sequence of octapeptide carbon skeleton into the 3 terminus of GST gene, the authors get the masculine recon, pGAT-1, confirmed by sequence determination and analysis, whose ORF is read-through. After IPTG induction and partial purification, SDS-PAGE electrophoresis is employed to detect the gene expression. The expressions of total bacterial proteins are about 21.3%, 22.5%, 19.32%, 21.73% in E. coli BL21, DH5α, JM109 and Top10 strains, respectively. Considering the quantity of induced total bacteria, the E. coli BL21 is the best one among the four recipient strains. This article supplies an academic foundation for producing biological active amatoxins.展开更多
文摘The primer, AT For1, AT For2 and AT Back, are designed and synthesized for adding-PCR that is used to construct the fusion GST-AT gene. Depending on two-time adding-PCR amplification and the insertion of coding sequence of octapeptide carbon skeleton into the 3 terminus of GST gene, the authors get the masculine recon, pGAT-1, confirmed by sequence determination and analysis, whose ORF is read-through. After IPTG induction and partial purification, SDS-PAGE electrophoresis is employed to detect the gene expression. The expressions of total bacterial proteins are about 21.3%, 22.5%, 19.32%, 21.73% in E. coli BL21, DH5α, JM109 and Top10 strains, respectively. Considering the quantity of induced total bacteria, the E. coli BL21 is the best one among the four recipient strains. This article supplies an academic foundation for producing biological active amatoxins.
