人Endostatin在毕赤酵母菌中的表达及其抑癌活性研究

目的在毕赤酵母中获得有活性的分泌型重组Endostatin.方法一步法抽提胎肝总RNA,RT-PCR扩增endostatin全基因.用T-A克隆技术构建克隆载体;双酶切回收550bp的endostatin基因,亚克隆入酵母表达载体.重组质粒分别以PCR、酶切及测序鉴定.电转化法将线形化的重组表达DNA转导入GS¨5感受态细胞,SDS-PAGE电泳筛选阳性重组子,G25及Heprin亲和层析柱初步纯化重组蛋白.MTT法测定重组endosatatin对血管内皮细胞增殖的抑制作用.结果RT-PCR获得550bp的特异扩增条带,T-A克隆后,PCR挑选阳性重组子,测序证实序列正确;亚克隆后的表达载体,PCR可得550bp的特异条带,EcoRI、Not I双酶切获得550bp和3kb的条带,与预期一致,DNA测序证明核苷酸序列100%正确.SDSPAGE证实重组菌较空白有一明显条带,Heparin亲和层析后,1mol/INaCl洗脱的蛋白峰在体外可特异性抑制血管内皮细胞的增殖.结论在毕赤酵母菌中成功地获得了分泌表达的活性endostatin蛋白.

EXPRSSION OF HUMAN ENDOSTATIN IN PICHIA PASTORIS

Objective:To gain recombinant endostatin in Pichia pastoris.Methods:The total mRNA was isolated from Chinese fetal liver,endostatin cDNA was synthesized with RT PCR,then cloned into pGEM T vector.The positive clones were screened with PCR,restriction map and DNA sequencing.Endostatin cDNA was subcloned into Pichia expression vector pGAPZa.Pichia pastoris GS115 cells were transformed with recombinant DNA.SDS-PAGE was used to assay the expression of endostatin in recombinant GS115.After purified with G25 and heparin,the recombined protein was used in the proliferation assay of ECV304 cells.Results:DNA sequencing result showed the endostatin cDNA was successfully cloned.After transformation with recombined DNA,GS115 cells expressed recombinant human endostatin.MTT assay implicated the recombinant protein could inhibited the proliferation of ECV304 cell sejectivity.Conclusion:Recombinant human endostatin protein was sa cesefuely expressed in Pichia pastoris.