重组人tau441 (P301S)蛋白表达纯化及生物学特性分析

Expression,purification and biological characterization of recombinant human tau441(P301S)

目的初步了解重组人tau441(P301S)蛋白体外聚集、抗原性及免疫原性等生物学性质。方法原核表达带His标签的tau441(P301S)重组质粒,镍亲和层析纯化重组蛋白,BCA测定蛋白浓度,SDS-PAGE凝胶考马斯亮蓝染色确定蛋白的纯度;采用Western blot(WB)和负染透射电镜(transmission electron microscopy,TEM)鉴定;间接酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)检测所得蛋白的抗原性;以重组蛋白免疫小鼠,检测免疫血清特异性抗体滴度来分析其免疫原性。结果所得重组人tau441(P301S)纯度为70%,WB显示在相对分子质量(Mr.×10^(3))64和更高相对分子质量处有特异性条带。TEM显示,tau441(P301S)呈絮状团块聚集,团状面积显著大于tau441野生型蛋白(t=6.439,P=0.003);4℃孵育9 d后,tau441(P301S)形成明显的纤维状结构。间接ELISA结果显示,tau441(P301S)能被tau单抗HT7(1∶80000)识别;小鼠免疫血清tau特异性抗体滴度达到1∶128000,且WB显示,免疫后血清识别阿尔茨海默病(Alzheimer’s disease,AD)转基因小鼠脑裂解抽提物。结论重组人tau441(P301S)蛋白具有体外聚集性增强的特性但其抗原性和免疫原性未改变。

Objective To preliminarily disclose the biological properties of recombinant human tau441(P301S)protein,such as aggregation,antigenicity and immunogenicity.Methods The recombinant plasmid tau441(P301S)was expressed by prokaryotic expression system and purified by nickel column affinity chromatography.The protein concentrations were determined via BCA kit.The purity of protein was determined by SDS-PAGE gel coomassie brilliant blue staining.Western blot(WB)and negative staining transmission electron microscopy(TEM)were used to identify the recombinant proteins.The antigenicity was detected through indirect enzyme linked immunosorbent assay(ELISA),and the immunogenicity was detected by specific antibody titers of mouse immune serum.Results The purity of recombinant human tau441(P301S)was 70%.WB showed specific bands at relative molecular mass(Mr.×10^(3))64 and higher relative molecular mass.Negative staining TEM showed that tau441(P301S)was aggregated,and the area was significantly larger than tau wild-type control protein(t=6.439,P=0.003).After 9 days of incubation at 4℃,tau441(P301S)formed obvious fibrotic structure.Indirect ELISA result showed that tau441(P301S)could be recognized by anti-tau monoclonal antibody HT7(1∶80000).The specific antibody titer of the immunized serum was 1∶128000 and WB showed that the immunized serum recognized the brain lysate extract of Alzheimer’s disease(AD)transgenic mice.Conclusions The recombinant human tau441(P301S)protein had the characteristics of enhanced aggregation in vitro,but its antigenicity and immunogenicity were not changed.