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

Purification and Functional Study of the E.coli Expressed Chimeric,Protease-Resisting,Recombinant Human Protein p75NTR169-Fc

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。

Objective To 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 pUCI2 - NGFR and human liver eDNA as templates and to join thmn together by overlapping PCR to get the fusion gene of p75NTR（ I - 169 ） - Fe （ 216 - 433 ） , abbreviated as p75 NTR169 - Fe. （ 2 ） In order to obtain the recombinant veetor pET28 a - p75 NTR 169 - Fc, the DNA fragment of p75NTR169 -Fe was cleaved by the enzymes Nco I and EcoR I and then inserted into pET28a（ ＋ ） vector. （3）The reeombinant 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 p75 NTR169 - Fe on PC 12 cells fi＇om the eytotoxicity induced by AI3 （ 25 - 35 ） were investigated by MTT method. Results （ 1 ） Recombinant protein p75 NTR169 - Fc was expressed with a efficient soluble manner in the prokaryotie expression system of pET28a（ ＋ ）/BL21 （ DE3 ） and showed stability to protease. （2） p75NTR169- Fc eould protect PCI2 cells from the cytotoxicity induced by Aβ （25 -35 ）. Conclusion The recombinant human p75NTR169 -Fe 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. Key words p75NTR169 -Fe;p75NTR;β -amyloid（Aβ）