摘要
目的在大肠杆菌中表达人源性β淀粉样蛋白1-42(Aβ1-42),纯化表达蛋白并进行Western blot鉴定。方法根据Gen Bank中人源性Aβ1-42编码序列,分为3个片段人工合成,经退火延伸为全长dsDNA序列,酶切后连接至表达质粒pET-28a(+)构建为重组质粒pET-28a-Aβ1-42,转化至大肠杆菌BL21株中,筛选高表达株,诱导表达,利用Ni+株亲和纯化表达蛋白,SDS-PAGE后,转印PVDF膜,进行Western blot鉴定。结果获得纯化的大肠杆菌表达蛋白Aβ1-42,表达形式为包涵体,经Western blot鉴定为人源性Aβ1-42蛋白。结论本研究获得了纯化的Aβ1-42蛋白,为下一步采用Aβ1-42标记该蛋白作为诊断分子奠定了基础。
Objective To express the human β amyloid 1-42 protein in E. coil,purify the protein and identify it by Western blot. Methods The gene fragment coding the human β amyloid 1-42 region was synthesized with 3 fragments and annealed,then digested by restrictive enzyme and linked into the expression plasmid pET-28a（＋） to construct the expression plasmid pET-28a-Aβ1-42. After identified by restrictive enzyme and DNA sequencing, the expression plasmid was transformed into E. coli BL21 strain,the high expression strain was selected and induced,the expressed recombinant protein was purified by using Ni＋ affinity chromatograph, and the purified protein was transferred to PVDF membrane after SDS PAGE, and then identified by Western blot. Results The expressed recombinant protein was purified, it was expressed as inclusion body form,and the expressed protein was identified to be human β amyloid 1-42 protein by Western blot. Conclusion The human β amyloid 1-42 protein is expressed and purified,and this work provides a basis and material for Aβ1-42 protein marked and used as molecular diagnosis tool for Alzheimer＇s disease.
出处
《重庆医学》
CAS
CSCD
北大核心
2010年第3期262-264,267,I0001,共5页
Chongqing medicine
基金
第三军医大学校管课题资助项目
