人骨保护素在毕赤酵母中的分泌表达及表达产物的生物活性分析(英文)

为了在毕赤酵母表达系统中分泌表达人骨保护素 (osteoprotegerin ,OPG) ,以人骨肉瘤细胞系MG6 3的mRNA为模板 ,采用RT PCR法得到人OPG编码区cDNA ,克隆入毕赤酵母表达载体pPICZ B ,电转化毕赤酵母GS115 (Mut+) ,经 3%甲醇诱导分泌表达人OPG与组氨酸的融合蛋白 .SDS PAGE及Western印迹分析表明 ,有分子量约 6 6kD的目的蛋白表达 .纯化后的表达产物加入体外培养的小鼠骨髓细胞培养基中 ,当浓度为 10 0ng ml时 ,象牙片上骨吸收陷窝的数量及玻片上的TRAP阳性多核细胞的数量均减少 (P <0 0 5 ) .而同时加入人OPG的多克隆抗体后 ,这一抑制作用可被拮抗 ,在浓度为 5 0ng ml时则无此作用 .人OPG蛋白在酵母系统的成功表达 ,为该蛋白的进一步应用研究提供了依据 .

Secretory Expression of Human Osteoprotegerin in Pichia pastoris and Bioactivity Analysis of the Recombinant Protein

To express human osteoprotegerin (OPG) in Pichia pastoris and determine its inhibitive effects on osteoclast differentiation and bone resorptive function in vitro. Synthetic oligonucleotides were used to amplify human osteoprotegerin gene by RT-PCR from total RNA of human osteosarcoma cell line MG63. The OPG cDNA coding for 410 amino acid residues with native signal peptide was inserted into Pichia pastoris expression vector pPICZ-B containing AOX1 promoter and 18-nucleotide fragment encoding hexahistidine residues (6×His) at the 3'end, a recombinant expression plasmid pPICZB-OPG was constructed and transformed to yeast host strain GS115, and human OPG was expressed under the induction of 3% methanol for 5 days. SDS-PAGE and Western blot showed that the expressed product existed in supernatant in the form of soluble molecule, and molecular weight of recombinant protein was about 66 kD and it could react specifically with anti-OPG antibody. The addition of purified OPG-6His protein (100 ng/ml) could decrease the number of dentine resorption pits and tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in vitro(P<0.05). A highly efficient recombinant expression system was developed which produced a native and functional form of human OPG in the Pichia pastoris.