重组人肝素酶在毕赤酵母中的表达

Expression of Human Heparanase in Pichia yeast Expression System

目的:建立肝素酶表达系统,大量制备人肝素酶,用于肝素酶的深入研究,及其抑制剂筛选模型的建立。方法:采用RT-PCR方法从肝癌细胞HepG2中克隆肝素酶全长cDNA,转入毕赤酵母表达体系,经甲醇诱导表达。结果:四唑蓝活性检测结果表明,表达产物具有肝素酶活性;Western印迹证实表达产物可被肝素酶抗体识别。结论:表达产物中含具有肝素酶活性的重组人肝素酶。

Objective： The expression of human heparanase in large scale is essential in the study of the characterize of heparanase, screening its inhibitor and the producing of the monoclonal antibody. Methods： The full-length cDNA of heparanase was isolated from the HepG2 cDNA library and the truncate of heparanase cDNA was cloned into the pPIC9K vector for the protein expressioon. The expression recombinant protein was identified with Western blot and activity analysis. Results： Activity analysis showed that the recombinant protein was with the endo-β-D-glucuronidase activity. Western blot indicated that the recombinant protein was hybridized with the anti-heparanase monoclone antibody. Conclusion： The recombinant protein was with the biological characterize of mammalian endoglycosidase heparanase.