摘要
为了在毕赤酵母中表达鸡骨保护素(chOPG),采用DNA重组技术将鸡OPG成熟肽cDNA片段插入毕赤酵母表达载体pPICZα-A中,以电穿孔法转化酵母X-33,用Zeoc in平板筛选阳性重组子,经甲醇诱导,实现了OPG在毕赤酵母中的分泌表达。SDS-PAGE电泳分析结果显示,表达产物存在两种形式,其相对分子质量分别为43000和53000,表达量约为200 mg.L-1,经免疫印迹验证,有较好的抗原性。表达产物经处理后加入到体外培养的鸡胚破骨细胞上,能显著抑制成熟破骨细胞的骨吸收活性,减少骨吸收陷窝的个数与面积。
To express chicken osteoprotegerin （chOPG） in Pichia pastoris and determine its inhibitive effects on mature osteoclastic bone resorptive function in vitro, the chOPG cDNA encoding for 382 amino acid without signal peptide was subcloned into Pichia pastoris expression vector pPICZα-A. The constructed recombinant plasmid was transformed into yeast host cell X-33 by electropora- tion. The positive recombinant transformants were selected with PCR from Zeocin YPDS plate. Induced by the addition of methanol every 24 hours, mainly owing to heterogeneous glycosylation, the product relative moleculer mass was about 43 000 and 53 000 respectively at a yield of 200 mg per litter of culture. The result of Western-blot indicated that the recombinant protein had specific antigenicity. The chOPG recombinant protein at certain concentration could restrain the activity of mature osteoclasts in vitro, that is to say, the number and area of the bone resorptive lacunae both decreased.
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2009年第2期88-91,共4页
Journal of Nanjing Agricultural University
基金
国家自然科学基金项目(30671546)
教育部重点项目(107059)
江苏省自然科学基金项目(BK2004099)
关键词
鸡骨保护素
毕赤酵母
分泌表达
破骨细胞
chicken osteoprotegerin
Pichia pastoris
secretive expression
osteoclast
