[1]王红梅,陈晔洲,田晶晶,等.新型肿瘤标志物人PF4重组表达、活性鉴定及单克隆抗体制备[J].现代检验医学杂志,2015,30(05):12-16.[doi:10.3969/j.issn.1671-7414.2015.05.005]
　WANG Hong-mei,CHEN Ye-zhou,TIAN Jing-jing,et al.Recombination，Expression，Activity Identification and Monoclonal Antibody Preparation of the Human Platelet Factor 4 as a New Cancer Marker[J].Journal of Modern Laboratory Medicine,2015,30(05):12-16.[doi:10.3969/j.issn.1671-7414.2015.05.005]
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新型肿瘤标志物人PF4重组表达、活性鉴定及单克隆抗体制备()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第30卷
期数:
2015年05期
页码:
12-16
栏目:
论著
出版日期:
2015-12-10

文章信息/Info

Title:
Recombination，Expression，Activity Identification and Monoclonal Antibody Preparation of the Human Platelet Factor 4 as a New Cancer Marker
作者:
王红梅; 陈晔洲; 田晶晶; 刘欢; 段生宝; 丁少华; 蒙青林; 李勇
中国科学院苏州生物医学工程技术研究所，中科院生物医学检验技术重点实验室，江苏苏州215163
Author(s):
WANG Hong-mei; CHEN Ye-zhou; TIAN Jing-jing;  LIU-huan; DUAN Sheng-bao; DING Shao-hua; MENG Qing-lin; LI Yong
CAS Key Lab of Bio-Medical Diagnostics，Suzhou Institute of Biomedical Engineering and Technology，Chinese Academy of Sciences，Jiangsu Suzhou 215163，China
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
分类号:
R392-33
DOI:
10.3969/j.issn.1671-7414.2015.05.005
文献标志码:
A
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。
Abstract:
ObjectiveTo express recombinant human platelet factor 4 (rhPF4) with biological activity in E.coli by constructing a recombinant plasmid pET28a-PF4，and obtain monoclonal antibodies (MAbs) against rhPF4．MethodsThe cDNA of PF4 was amplified from pUC-57-PF4 by PCR，subcloned in the pET28a expression vector and expressed in BL21．The recombinant 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．ResultsThe 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 produce MAbs against rhPF4．ConclusionrhPF4 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．

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备注/Memo

备注/Memo:
基金项目：本课题受苏州市科技计划医疗器械与新医药专项(ZXY2012019)资助。 作者简介：王红梅(1981-)，女，硕士，助理研究员，主要从事血液免疫学与免疫遗传学研究工作，Tel：15295693728，E-mail：wanghm@sibet.ac.cn。
更新日期/Last Update: 1900-01-01