Construction and identification of Stathmin gene Pichia pastoris expression system / 国际检验医学杂志

ABSTRACT

Objective To provide the experimental basis for the further research of the interacting proteins with Stathmin ,the Stathmin gene Pichia pastoris expression system was constructed ,the expressed Stathmin product was purified and identified .Meth‐ods Stathmin gene was amplified from tumor cell line of SKBR3 by PCR method and cloned into the yeast expression vector pPIC3 .5K .The recombinant vector pPIC3 .5K‐Stathmin was constructed and transformed into Pichia pastoris GS115 .The positive clones were screened by YPD medium containing Geneticin 600 μg/mL .Expression was induced with 0 .5% methanol and expres‐sion products were identified by SDS‐PAGE and Western Blotting .Results DNA sequencing result showed that the gene fragment was consistent with Stathmin gene sequence .pPIC3 .5K‐Stathmin was selected from YPD culture medium containing Geneticin ,and the positive clones were identified by PCR .SDS‐PAGE showed that a 37 × 103 protein band could be seen on the PAGE gel after Coomassie Blue staining ,which was further confirmed and identified as Stathmin protein by Western Blotting .Conclusion Stathmin yeast expression vector is successfully constructed and expressed in Pichia pastoris ,which laid the foundation for the study of inter‐acting proteins with Stathmin ,and for the preparation of the biological treatment drugs of Stahtmin target .