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。
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)
出处
《医学研究杂志》
2012年第4期48-52,共5页
Journal of Medical Research