摘要
目的在大肠杆菌中表达人源性柯萨奇病毒-腺病毒受体胞外区蛋白(exCAR),纯化表达蛋白并进行Western-blot-ting鉴定。方法从Hela细胞株中扩增exCAR基因片段,连接至表达质粒pET-28a(+)构建为重组质粒pET-28a-exCAR,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膜后,进行Western-blotting鉴定。结果获得纯化的大肠杆菌表达蛋白exCAR,表达形式为包涵体,经Western-blotting鉴定为人源性exCAR蛋白。结论本研究获得了纯化的exCAR表达蛋白,为下一步的蛋白复性和腺病毒感染的阻断实验提供了基础。
Objective To express the extra-cellular region of human Coxsaekie-adenovirus receptor（exCAR） in E. coli,to purify the protein and identify it by N-end sequencing. Methods The gene fragment coding the extra-cellular region of CAR was amplified from the eDNA library of Hela cell strain,and linked into the expression plasmid pET-28a（＋） to construct the expression plasmid pET-28a（＋）-exCAR. After identified by restrictive enzyme cut 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,then identified by Western-blotting. Results The expressed recombinant protein was purified, it was expressed as include body form, and the expressed protein was identified to be human exCAR protein by Western-blotting. Conclusion The exCAR recombinant protein was expressed and purified,and this work provides a basis and materials for the following adenovirus-blocking experiment in vitro.
出处
《重庆医学》
CAS
CSCD
北大核心
2009年第21期2691-2694,共4页
Chongqing medicine
基金
重庆市自然科学基金资助(CSTC
2007B5023)
第三军医大学新桥医院人才基金资助
