【作者】 郑芳； 周新； 夏勇； 赵磊； 胡汉宁； 姜新强； 李霞；

【Author】 ZHENG Fang, Zhou Xin, XIA Yong, ZHAO Lie, Hu Han -ling, JIANG Xin -Qiang.Department of laboratory Medicine, Wuhan University School of Medicine, Wuhan 430071, China

【机构】 武汉大学中南医院基因诊断中心；

【摘要】 目的构建C反应蛋白(CRP)的表达质粒,在大肠杆菌表达,纯化获得C反应蛋白,并鉴定纯化蛋白的免疫反应性。方法:应用逆转录(RT)PCR方法,从人肝细胞文库中扩增人C反应蛋白cDNA基因片段,与表达质粒载体pCRT7/NT TOPO重组,获得表达质粒CRP-pCRT7/NT。用氨苄青霉素平板筛选转化子,双酶切与。DNA测序鉴定CRP基因表达质粒pCRT7/NT TOPO载体的整合状态,用IPTG诱导表达,并经组氨酸亲和层析柱纯化获得CRP的蛋白纯品。采用Western印迹的方法鉴定纯化蛋白的免疫反应性。结论:成功地构建了在大肠杆菌表达CRP的质粒,建立了蛋白纯化的系统,为下一步CRP检测试剂盒的制备打下了基础。更多还原

【Abstract】 AIM: To develop an expression, purification system of recombinant E. coli which can express C reactive protein (CRP) and to identify the immune reactivity of the CRP to anti - CRP specific antibody. METHOD: The CRP cDNA was amplified by RT - PCR from human liver cDNA library and inserted into expression vector pCRT7/NT. The recombined plasmid CRP - pCRT7/NT which expressed the fusion protein of CRP was then transferred into lysogenic host strain E coli. BL21 (DE3). The target protein was identified using SDS - poly-acrylamide gel electrophoresis (SDS -PAGE). Affinity chromatography was used for protein purification. The immune reactivity of purified CRP was identified by Western blot using anti - CRP specific antibody. RESULTS and CONCLUSION: A plasmid expressed CRP protein was constructed and the purification system of CRP protein was established. The immune reactivity of the purified protein was identified by Western blot, which made a good foundation for the preparation of CRP test kit.更多还原