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人脂联素球状结构域的表达、纯化和功能鉴定 被引量：2

Expression,purification and characterization of recombinant human globular domain of adiponectin
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摘要 目的表达和纯化人脂联素(adiponectin,APN)球状结构域并观察其生物学活性。方法用PCR方法复制人脂联素球状结构域(globular domain of adiponectin,gAPN)的cDNA,并克隆于pET28a(+)原核表达载体中。将重组表达质粒pET28a(+)-gAPN转化大肠埃希菌BL21(DE3),37℃培养至A600达到0.6时,加入异丙基-β-D-硫代半乳糖苷(IPTG)诱导目的蛋白的表达。细菌经超声破菌,得到粗提的包涵体洗涤变性后经镍柱亲和层析柱一步纯化,获得纯化的gAPN重组蛋白,复性后冻干保存。然后,用链佐星(streptozocin,STZ)诱导的小鼠高血糖模型检测其生物活性。结果IPTG诱导后有Mr约为17 000大小的目的蛋白表达,免疫印迹结果进一步表明,其具有人gAPN的抗原性。表达蛋白主要以包涵体形式存在,占菌体蛋白的30%以上,表达产物经变性、纯化、复性,可获得纯度90%以上的目的蛋白。该重组蛋白能够降低STZ诱导的小鼠高血糖模型的血糖水平。结论在大肠埃希菌BL21(DE3)中成功表达了人gAPN蛋白,经变性、纯化、复性后得到具有生物学活性的蛋白。 Purpose To obtain bioactive globular domain of human adiponectin. Methods cDNA encoding human globular domain of adiponectin （gAPN） was amplified by PCR, and cloned in the expression vector pET28a（ ＋ ）. The recombinant expression plasmid pET28a（ ＋ ）-gAPN was transformed into E. coli BL21 （DE3）. When A600 （nm） reached 0.6, this transformant was induced for expression of recombinant protein by IPTG. Recombinant protein was obtained after denaturation, purification by Ni^＋ -affinity chromatography and renaturation. The bioactivity of recombinant protein was evaluated by Streptozocin （STZ）-induced hyperglycemia model. Results After induction for 3 hours by IPTG, the recombinant protein was over 30% of total bacterial protein. The recombinant protein was expression in form of inclusion bodies. After purification, SDS-PAGE analysis indicated that molecular weight of recombinant protein was about 17 000 and the purity was up to 90M. Western blot indicated that recombinant protein could react with anti-APN polyclonal antibody. The recombinant protein could significantly reduce the level of blood glucose in STZ-induced hyperglycemia model. Conclusions We successfully expressed human globular domain of adiponectin （gAPN） in E. coli BL21 （DE3） and obtained the bioactive protein.
作者 刘宏磊 李希 宋后燕 汤其群
机构地区 复旦大学上海医学院生物化学与分子生物学系-分子医学教育部重点实验室
出处 《复旦学报（医学版）》 CAS CSCD 北大核心 2006年第1期97-100,共4页 Fudan University Journal of Medical Sciences
基金 863项目(2004AA215131) 上海市重点科技攻关项目(044319203) 上海市重点实验室合作开放资金(04d205607)资助
关键词 脂联素 重组蛋白 表达与纯化 高血糖模型 adiponectin recombinant protein expression and purification hyperglycemia model
分类号 Q57 [生物学—生物化学] Q78 [生物学—分子生物学]
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参考文献13

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同被引文献22

  • 1常冰梅,王勇,解军,张悦红,程牛亮,牛勃,胡晓年,向前.表达人肝细胞生长因子突变体——NK4工程菌的发酵工艺研究[J].中国生物制品学杂志,2005,18(2):132-134. 被引量：1
  • 2李琳,季海生.SEB诱导活化的淋巴细胞及其细胞因子对乳腺癌细胞杀伤作用的研究[J].山东医药,2006,46(2):50-51. 被引量：2
  • 3赵嘉惠,张华屏,王春芳.MTT法在检测细胞增殖方面的探讨[J].山西医科大学学报,2007,38(3):262-263. 被引量：92
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  • 7Du W,Hattori Y,Yamada T,et al.NK4,an antagonist of hepatocyte growth factor (HGF),inhibits growth of multiple myeloma cells:molecular targeting of angiogenic growth factor[J].Blood,2007,109(7):3 042-3 049.
  • 8Kubota T,Taiyoh H,Matsumura A,et al.NK4,an HGF antagonist,prevents hematogenous pulmonary metastasis by inhibiting adhesion of CT26 cells to endothelial cells[J].Clin Exp Metastasis,2009,26(5):447-456.
  • 9Nakamura T,Sakai K,Nakamura T,et al.Anti-cancer approach with NK4:bivalent action and mechanisms[J].Anticancer Agents Med Chem,2010,10(1):36-46.
  • 10Thapa A,Shahnawaz M,Karki P,et al.Purification of inclusion body-forming peptides and proteins in soluble form by fusion to Escherichia coli thermostable proteins[J].Biotechniques,2008,44(6):787-796.

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复旦学报（医学版）
2006年 第1期

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