绿色荧光标记C-反应蛋白的原核表达和毛细管电泳检测  

The Prokaryotic Expression of Human C-reactive Protein Labelled with Green Fluorescence and Its Detection with Capillary Electrophoresis

作　　者：陈腾祥[1] 夏高晓[2] 陈丽[2] 刘亚伟[2] 刘靖华[2] 姜勇[2] 

机构地区：[1]贵阳医学院生理学教研室,贵州贵阳550004 [2]南方医科大学病理生理学教研室和广东省蛋白质组学重点实验室,广东广州510515

出　　处：《贵阳医学院学报》2008年第5期441-445,共5页Journal of Guiyang Medical College

基　　金：国家自然科学基金资助(NO.30670829;30670828;30572151)

摘　　要：目的:表达纯化重组的His-EGFP-CRP蛋白,通过毛细管电泳技术对该重组蛋白的生物活性进行评价。方法:用pET14b/EGFP-hCRP原核表达质粒转化到大肠杆菌BL21(DE3)中诱导表达,表达的重组蛋白通过亲和色谱纯化、梯度透析,获得复性的His-EGFP-CRP蛋白;并用高效毛细管电泳(HPCE)技术对复性的蛋白进行检测,了解蛋白质的等电点及活性。结果:转化实验发现,该质粒pET14b/EGFP-hCRP在BL21(DE3)中能够被诱导表达;通过包涵体变性溶解、亲和色谱纯化可获得纯度较高的His-EGFP-CRP蛋白;HPCE分离发现复性后的His-EGFP-CRP蛋白峰为多个,其在电泳液pH值低于6时易检出,His-EGFP-CRP与THP-1细胞裂解物孵育后的检测峰增多。结论:成功地在大肠杆菌BL21(DE3)中诱导表达重组蛋白His-EGFP-CRP,通过包涵体变性溶解及亲和色谱纯化获得该重组蛋白,复性后的His-EGFP-CRP等电点接近于6,以单体或多聚体等结构形式存在,具有结合活性,能与细胞内相关分子结合成复合物,可应用于CRP功能和内化机制的研究。Objective： To express the purified recombinant protein, His-EGFP-CRP, and to evaluate the feasibility of its application in the research of function and internalization mechanism of human C- reactive protein （CRP）. Methods： The re-constructed vector pET14b/EGFP-hCRP was transformed into Escherichia. coli BL21 （DE3）, induced by isopropyl-β-D-thiogalactopyranoside （IPTG）. The expressed protein, His-EGFP-CRP, was purified with affinity chromatography method and refolded with gradient filtration. The renatured His-EGFP-CRP was analyzed with high performance capillary electrophoresis （HPCE） to evaluate its isoelectric point （pI） and bio-activity. Results： Fusion protein His- EGFP-CRP successfully expressed in transformed E. coli cells after the induction, and then was purified with inclusion lysis and affinity chromatography. His-EGFP-CRP was detected at several peaks of the fraction profile of HPCE when the pH of electrophoresis buffer was under 6. The number of His- EGFP-CRP detection peaks increased after the protein was incubated with lysate of THP-1 cells. Conclusions： The pI of His-EGFP-CRP is about 6, and its structures may be monomer or polymer, which have the ability to bind relative molecles in lysate of THP-1 to form complex. These results suggest the recombinant protein could be used in exploiting the function and internalization mechanism of human CRP.

关 键 词：C反应蛋白 基因表达 蛋白质复性 电泳 毛细管 

分 类 号：Q51[生物学—生物化学] TQ936.22[化学工程]