Expression, purification and biological characterization of recombinant human tau441 (P301S)
10.3760/cma.j.cn112866-20210222-00033
- VernacularTitle:重组人tau441 (P301S)蛋白表达纯化及生物学特性分析
- Author:
Jialong JIANG
1
;
Zhenwu LIU
;
Ziqi WANG
;
Zhongliang NIE
;
Peiyun LI
;
He WANG
;
Ying ZHANG
;
Jinsheng HE
;
Tao HONG
Author Information
1. 北京交通大学理学院生命科学与生物工程研究院 100044
- Keywords:
Alzheimer’s disease;
Microtubule-associated proteins;
Amyloid protein aggregation
- From:
Chinese Journal of Experimental and Clinical Virology
2021;35(5):543-547
- CountryChina
- Language:Chinese
-
Abstract:
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∶80 000). The specific antibody titer of the immunized serum was 1∶128 000 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.
