【作者】 杨霄鹏；

【导师】 刘斌； 吕同德； 郭建巍；

【作者基本信息】 兰州大学 ， 病理学及病理生理学， 2008， 硕士

【摘要】 目的构建重组人脂联素(adiponectin,ADPN)原核表达载体,获取同源性、可溶性、高纯度的人重组脂联素融合蛋白,为进一步脂联素的深入研究及Ⅱ型糖尿病的早期诊断奠定基础。方法从人脂肪组织中提取RNA,经RT-PCR扩增,克隆出脂联素基因;用EcoRⅠ和HIndⅢ对人脂联素基因和载体pET32进行双酶切,构建pET32—ADPN重组质粒;将重组质粒转化BL21感受态细胞,通过氨苄青霉素抗性、PCR和重组质粒双酶切筛选重组子转化菌落,经DNA测序及BLAST比对确认脂联素基因序列正确性以及插入pET32质粒的位点和方向;用IPTG诱导表达、Western blotting鉴定,用镍离子金属螯合层析柱纯化重组融合蛋白。结果(1)通过RT-PCR,经1%的琼脂糖凝胶电泳观察,扩增的脂联素DNA片段与预期大小一致。(2)经双酶切及DNA测序鉴定脂联素DNA定向插入了pET32质粒。(3)用重组质粒转化大肠杆菌BL21、诱导表达和纯化目的蛋白。经Western—Blotting鉴定所诱导的蛋白为重组脂联素融合蛋白。结论成功构建了人重组脂联素蛋白原核表达载体、表达和纯化了脂联素融合蛋白,为进一步研究其生学物学功能和在Ⅱ型糖尿病中的作用机制奠定了基础。更多还原

【Abstract】 Objective: To construct a prokaryotic expression vector in which the maximum level of homogeneous, soluble, and fully bioactive of recombinant human adiponectin fusion protein was obtained from E.coli.Methods: Total RNA were extracted from human adipose tissue and the adiponectin gene were fished by reverse transcription PCR; the adiponectin gene were amplified by a pair of special primer including endonucleases sites of EcoR I and Hind III by PCR and inserted into the prokaryotic expression vector pET 32a(+) . After being selected by Ampicillin resistance, identified by PCR and double digestion of endonucleases, the sequenced DNA of adiponectin was analyzed by BLAST. The Adiponectin/Trx fusion protein expressed in E.coli. BL21 (DE3) was induced with IPTG, identified by western blot and purified with Ni—NTA agarose respectively.Results: Through RT-PCR and 1% agarose gel electrophoresis, the products of 1000bp were obtained as expected . After double digestion with Endonucleases, the Adiponectin gene has been inserted into the prokaryotic expression vector PET 32a (+). After detected the DNA sequence, the constructed expression plasmid was transformed into competent E.coli. BL21(DE3). The recombinant Adiponectin/Trx fusion protein was specific against adiponectin antibody identified by western blot .The purity of the protein was over 80% after been purified by Ni—NTA agarose.Conclusion:The prokaryotic expression plasmid PET 32a (+)/Adipoenctin issuccessfully constructed and high purified Adiponectin/Trx fusion proteinis obtained ether.更多还原