人骨保护素成熟肽表达载体的构建、表达及重组蛋白免疫学鉴定  

Construction and Expression of Recombinant Vector for Mature Peptide of Human Osteoprotegerin and Immunoassay of Its Recombinant Protein

作　　者：王宝利[1] 戴芳[1] 郭刚[1] 邱明才[1] 张镜宇[1] 

机构地区：[1]天津医科大学总医院内分泌科,天津医科大学内分泌所,300052

出　　处：《天津医药》2003年第11期691-693,F002,共4页Tianjin Medical Journal

基　　金：国家自然科学基金 (项目编号 :30271154) ;天津市自然科学基金资助项目 (项目编号 :023609711)

摘　　要：目的 :构建人骨保护素 (OPG)成熟肽cDNA重组表达载体 ,实现其在毕赤酵母中的高效表达。方法 :由人成骨细胞提取总RNA ,经RT -PCR扩增得到人OPG成熟肽cDNA ,克隆入分泌型表达载体 pPIC9K ,转化酵母细胞 ,G418筛选多拷贝整合的重组子进行甲醇诱导表达 ,产物行Westernblotting 鉴定。结果 :成功构建了人OPG成熟肽表达载体 ,该载体能在酵母细胞中获得分泌表达 ,诱导96h的表达量占上清总蛋白的30 %。重组蛋白相对分子质量约55ku ,可被OPG抗体识别。结论 :重组人OPG成熟肽在酵母表达系统获得较高水平表达 ,为进一步研究开发基因工程药物准备了条件。Objective:To study the construction and expression of mature peptide vector of human osteoprotegerin(OPG)and the immunoassay of its recombinant protein.Methods:Total RNA was extracted from primary human osteoblasts.The cDNA encoding the mature peptide of OPG was amplified through RT-PCR and inserted into the secretory expression vector pPIC9K.The recombinant plasmid was then transformed into GS115,a pichia strain.Mut+ phenotypic transformants were selected according to their growth on MM and MD plates.These colonies were further screened for transformants with multicopy inserts integretion by evaluating their growth on YPD plates with different concentration of G418.Finally,the G418-resistant colonies were cultured in BMMY medium at 30 ℃ in a shaking incubator under the induction of 0.5% methanol.Results:The recombinant plasmid was successfully constructed.A transformant resistant to 3.0 g/L G418 was screened out for expression.Induced with methanol,the cells secreted a 55_ku protein that represented about 30% of total protein in the supernatant on SDS-PAGE gel.The 55_ku protein could be recognized,through Western blotting analysis,by the polyclonal antibody against OPG.Conclusion:The construction and expression of the recombinant vector for mature peptide of OPG is of much value since it provides a sound basis for further research on ODF/OPG/PANK system and a promising pharmaceutical tool for osteoporotic and erosive bone disorders.

关 键 词：人骨保护素 成熟肽 载体 重组蛋白 免疫学鉴定 基因表达 

分 类 号：R394[医药卫生—医学遗传学] R392[医药卫生—基础医学]