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作 者:陈楠 吴同鑫 章汝 李晓月 顾欣雨 唐香平 李云[1] 易永祥 CHEN Nan;WU Tong-xin;ZHANG Ru;LI Xiao-yue;GU Xin-yu;TANG Xiang-ping;LI Yun;YI Yong-xiang(Nanjing Hospital Affiliated to Nanjing University of Chinese Medicine,Nanjing 210003,China;School of Public Health,Nanjing Medical University,Nanjing 211166,China)
机构地区:[1]南京中医药大学附属南京医院,江苏南京210003 [2]南京医科大学公共卫生学院,江苏南京211166
出 处:《生物技术》2023年第5期544-550,共7页Biotechnology
基 金:南京市科技计划项目(ZX20200009);江苏省研究生科研与实践创新计划项目(SJCX22-0894)。
摘 要:[目的]构建Fiber Knob原核表达载体,在大肠杆菌(Escherichia coli)BL21(DE3)中诱导其表达,纯化具有良好生物活性的Fiber Knob重组蛋白。[方法]从人55型腺病毒(HAdV-55)中通过PCR扩增目的基因片段Fiber Knob,将该片段与原核表达载体pET15b-His连接,构建重组原核表达质粒pET15b-Fiber Knob-His,转化大肠杆菌BL21(DE3)中,IPTG诱导表达目的蛋白,通过镍柱亲和层析对目的蛋白进行纯化,SDS-PAGE和Western Blotting检测目的蛋白的表达与纯度。ELISA检测目的蛋白的生物学活性。[结果]重组表达质粒经双酶切和测序鉴定证明构建正确。重组蛋白主要以包涵体形式存在,上清中也有少量表达,重组蛋白相对分子质量约为25 kDa且纯度95%以上,ELISA结果显示制备的Fiber Knob蛋白与CD46胞外域蛋白之间存在直接的相互作用,并且这种相互作用是剂量依赖性的。[结论]成功构建了原核表达质粒pET15b-Fiber Knob-His,在大肠杆菌中成功诱导表达了Fiber Knob蛋白,纯化的Fiber Knob蛋白具有明显结合CD46胞外域蛋白的生物学活性,为其单克隆抗体的制备及人55型腺病毒新的细胞受体的发现奠定了基础。[Objective]To construct Fiber Knob prokaryotic expression vector,induce its expression in Escherichia coli BL21(DE3),and purify Fiber Knob recombinant protein with good biological activity.[Method]The target gene fragment Fiber Knob was amplified from human adenovirus type 55(HAdV-55)by PCR,and the fragment was ligated with the prokaryotic expression vector pET15b-His to construct a recombinant prokaryotic expression plasmid pET15b-Fiber Knob-His,which was transformed into Escherichia coli BL21(DE3),and the target protein was induced by IPTG.The target protein was purified by metal chelate affinity chromatography,and the expression and purity of the target protein were detected by SDS-PAGE and Western Blotting.ELISA was performed to detect the biological activity of the target protein.[Result]The recombinant expression plasmid was identified by double digestion and sequencing.The relative molecular mass of the recombinant protein was about 25 kDa and the purity was more than 95%.ELISA results showed that there was a direct interaction between the prepared Fiber Knob protein and CD46 extracellular domain protein,and this interaction was dose-dependent.[Conclusion]The prokaryotic expression plasmid pET15b-Fiber Knob-His was successfully constructed,and the Fiber Knob protein was successfully induced in Escherichia coli.The purified Fiber Knob protein had obvious biological activity of binding CD46 extracellular domain protein,which laid the foundation for the preparation of its monoclonal antibody and the discovery of a new cellular receptor for human type 55 adenovirus.
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