摘要
目的 构建重组表达质粒pET28a-PF4,诱导表达重组人PF4 （rhPF4）,制备rhPF4的单克隆抗体.方法 将pUC-57-PF4质粒中的PF4基因克隆到原核表达质粒pET28a上,在BL21菌株中诱导表达,对表达的蛋白进行亲和层析纯化和SDS-PAGE及western blotting鉴定;MTT法检测rhPF4对对数生长期EA.hy926细胞的生长抑制作用;以rhPF4为免疫原,通过细胞融合技术制备抗rhPF4的单克隆抗体.结果 重组质粒在BL21菌株中高效表达,rhPF4占总蛋白的19％,表达蛋白分子量约14 KDa,与商品化人PF4单抗呈阳性反应;rhPF4可抑制内皮细胞EA.hy926的生长繁殖;建立的杂交瘤细胞株能稳定产生抗rhPF4单克隆抗体.结论 建立的BL21菌株可高效表达可溶性的rhPF4,该rhPF4能抑制EA.hy926的生长繁殖,制备的抗rhPF4单克隆抗体可用于新型肿瘤标志物PF4的检测试剂的开发.
Objective To express recombinant human platelet factor 4 （rhPF4） with biological activity in E. coli by construc- ting a recombinant plasmid pET28a-PF4,and obtain monoclonal antibodies （MAbs） against rhPF4. Methods The eDNA of PF4 was amplified from pUC-57-PF4 by PCR, subcloned in the pET28a expression vector and expressed in BL21. The re- combinant protein was purified by his Trap HP and identified by SDS-PAGE and western blotting. The MTT test was used to determine whether the rhPF4 suppress the growth of EA. hy926 cells in exponential phase. The purified rhPF4 was used to immunize BALB/c mice for producing MAbs. Results The recombinant protein, with a molecular weight of about 14KDa,was expressed in a high expression level, 19 % of the total cell protein,and reacted positively with commercial PF4 MAbs. The rhPF4 could suppress the growth of endothelial cell EA. hy926. The hybridoma cell lines could constantly pro- duce MAbs against rhPF4. Conclusion rhPF4 can be expressed with high efficiency in BL21 as a soluble protein,and it can suppress the growth of EA. hy926. The MAbs against rhPF4 be applied in the development of diagnostic reagent for new cancer marker PF4.
出处
《现代检验医学杂志》
CAS
2015年第5期12-16,21,共6页
Journal of Modern Laboratory Medicine
基金
本课题受苏州市科技计划医疗器械与新医药专项（ZXY2012019）资助.
关键词
血小板因子4
基因表达
活性鉴定
单克隆抗体
肿瘤标志物
platelet factor 4 （PF4）
gene expression
activity identification
monoclonal antibody
cancer markers
