Decorin Gene Cloning And Its Expression In Pichia Pastoris And Activity Determination

DCN belongs to a member of the expanding gene family encoding the secreted, small, leucine-rich proteoglycans (SLRPs) that affect matrix assembly and cellular proliferation. The cDNA of DCN extron, was composed of 1080 bp of nucleotides and encoded a core protein of 395 amino acids.Objectives: To establish and use the eukaryotic (Pichia Pastoris ) expression system for expressing DCN protein.Methods: ①Full length DCN gene was amplified from cDNA of DCN extron was obtained through human muscle by using surivin specific primers.Then,DCN cDNA wasinserted into pPic9k, and verified by sequencing. ②Verified cDNA gene was subcloned into pPic9k. The recombinant plasmid was linearized by restriction enzyme cutting. The linear DCN gene was introduced into HIS~-/GS115 cells by LiAC.The transformants were transferred onto YPD plates that contained different concentrations of G418 for selecting the positive clones. The integrated cDNA in positive clones was confirmed by PCR. ③Selected transformants were cultured in BMMY medium with 1% methanol for inducing the expression of DCN protein. ④After a period when HepG2 liver cancer cell line were treated with DCN in different concentrations and time, the suppressive effect of DCN on HepG2 liver cancer cell line were detected by counting cell.Results: ①The result of sequencing human cDNA compared with the DCN sequence in the Gen-Bank is the same. ②We have made up an eukaryotic (Pichia pastoris) expressive vector (pPic9k-DCN). After the linear recombinant vector was introduced into HIS~-/GS115 cells,we have selected positice clones against G418(4mg/ml)and confirmed by PCR. ③DCN protein reached highest concentration when expressed for one days in l%methanol BMMY medium. ④After a period when HepG2 liver cancer cell line were treated with DCN in different concentrations and time, the proliferation of HepG2 liver cancer cell line were inhibited by DCN respectively showed counting cell. These results demonstrated that had a dosage and time dependent relationship. There was a statistically significant decreased in the proliferation rate among the three groups with different concentrations (P<0.05).Conclusions: To express DCN core protein in Pichia Pastoris is possible.Improved DCN protein yield may be reached by modifying the experimental conditions. This is necessary for tumor diagnosis, anticancer therapy,and the fundamental research of tumor pathology.