| 重组人p75NTR169-Fc抗酶裂嵌合体在大肠杆菌中的表达、纯化及活性鉴定 |
| 投稿时间：2011-07-11 修订日期：2011-07-22 点此下载全文 |
| 引用本文：景丽玲,王欣,陈虹,张俊文.重组人p75NTR169-Fc抗酶裂嵌合体在大肠杆菌中的表达、纯化及活性鉴定[J].医学研究杂志,2012,41(4):48-52 |
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| 中文摘要:目的制备重组人p75NTR169-Fc融合蛋白，其是一种潜在的抗凋亡和止痛的候选药物。方法(1)应用PCR法，以本组保存的pUC12-NGFR和人肝cDNA为模板，扩增出p75NTR(1~169氨基酸)，IgG Fc(216~433氨基酸)基因顺序，再按照重叠PCR的原则和方法，将两组扩增产物混合变性、复性后再扩增，得到“p75NTR (1~169)-IgG Fc(216~433)”融合基因，缩写为p75NTR169-Fc。(2)应用DNA重组技术将p75NTR169-Fc融合DNA片段经NcoⅠ、EcoRⅠ酶切插入到pET28a(+)原核表达载体中，获得pET28a-p75NTR169-Fc重组质粒。(3)利用pET28a(+)/BL21(DE3)原核表达系统诱导表达目的蛋白，经protein A亲和层析纯化，得到纯度约96%的p75NTR169-Fc重组蛋白质。(4)用MTT方法，以PC12细胞检验不同剂量重组蛋白抗β-淀粉样肽（Aβ25~35）细胞毒的作用。结果(1)成功构建了pET28a-p75NTR169-Fc /BL21(DE3)表达系统，其高效表达可溶性重组蛋白。实验表明：该重组蛋白p75NTR169-Fc稳定性好，不再被蛋白酶降解，为单一条带。亲和层析纯化后，分子质量均一。（2）p75NTR169-Fc与p75NTR竞争结合Aβ(25~35），拮抗Aβ(25~35）对PC12的细胞毒作用。结论重组人抗酶裂嵌合体蛋白p75NTR169-Fc具有生物学活性，在临床治疗阿尔茨海默病(Alzheimer′s disease, AD)上具有应用前景，系针对神经退行性疾病的新的候选药物。 |
| 中文关键词:p75NTR169-Fc p75NTR β-淀粉样肽(Aβ) |
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| Purification and Functional Study of the E.coli Expressed Chimeric, Protease-Resisting, Recombinant Human Protein p75NTR169-Fc |
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| Abstract:ObjectiveTo express a chimeric protein p75NTR169-Fc which may be a new type of drug and has the function of anti-apoptosis and pain relief potentially. Methods(1)PCR was used to amplify the gene of p75NTR(1-169) and Fc(216-433) using pUC12-NGFR and human liver cDNA as templates and to join them together by overlapping PCR to get the fusion gene of p75NTR(1-169)-Fc(216-433), abbreviated as p75NTR169-Fc.(2)In order to obtain the recombinant vector pET28a-p75NTR169-Fc, the DNA fragment of p75NTR169-Fc was cleaved by the enzymes NcoⅠand EcoRⅠand then inserted into pET28a(+) vector. (3)The recombinant protein p75NTR169-Fc was expressed in prokaryotic expression system of pET28a(+)/BL21(DE3) and purified by protein A affinity chromatography. (4)The protection effects of different doses of recombinant protein p75NTR169-Fc on PC12 cells from the cytotoxicity induced by Aβ(25-35) were investigated by MTT method. Results(1)Recombinant protein p75NTR169-Fc was expressed with a efficient soluble manner in the prokaryotic expression system of pET28a(+)/BL21(DE3) and showed stability to protease.(2)p75NTR169-Fc could protect PC12 cells from the cytotoxicity induced by Aβ(25-35). ConclusionThe recombinant human p75NTR169-Fc is successfully expressed in E.coli BL21(DE3) and its purity can reach to 96% by protein A affinity chromatography and it is a new anti-protease protein with biological activities and may be a new drug candidate for application in the treatment of neurodegenerative diseases such as Alzheimer′s disease. |
| keywords:p75NTR169-Fc p75NTR β-amyloid（Aβ） |
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