Tamm-Horsfall蛋白片段的基因克隆、表达、纯化及抗原性鉴定  

Gene Cloning,Expression,Purification and Antigenicity Identification of Tamm-Horsfall Protein Domain

作　　者：朱美财[1] 马红雨[1] 王红[1] 陈涛[1] 

机构地区：[1]空军总医院临床检验中心,北京100142

出　　处：《标记免疫分析与临床》2012年第6期360-363,共4页Labeled Immunoassays and Clinical Medicine

基　　金：首都医学发展基金(编号:2009-2056)

摘　　要：目的克隆并表达尿调节素(Tamm-Horsfall,THP)蛋白片段的基因,检测其抗原性。方法利用RT-PCR技术扩增THP蛋白片段基因,测序后将目的基因克隆入原核表达质粒pET28a载体,转化至大场杆菌BL21(DE3),经IPTG诱导表达,获得重组THP蛋白片段。经NI亲和层析柱纯化,用western-blot检测重组蛋白与THP单克隆抗体结合活性。结果克隆THP蛋白片段基因,其开放阅读框为195 bp编码65个氨基酸。表达的重组THP蛋白片段以包涵体形式存在,菌体蛋白纯化后,经western-blot检测具有较好的抗原性。结论成功地克隆和表达了THP片段的基因,为抗THP单克隆抗体制备和THP检测试剂盒的研发奠定了基础。Objective To investigate the cloning and expression of a part of gene encoding Tamm- Horsfall protein （THP） domain and exam its antigenicity. Methods A part of gene encoding Tamm- Horsfall protein domain was amplified by reverse-translation polymerase chain reaction （RT-PCR）. After sequencing, the intent gene was cloned into expression plasmid vector pET28a and then transformed into Ecoli. BL21 （ DE3 ）. The THP domain was expressed by inducing with IPTG, purified by Ni-affinity chromatography. The combination activity of re- combinant protein with anti-THP monoclonal antibody was detected by western-blot. Results A 195 bp fragment from THP gene encoding 65 amino acids was cloned. The THP domains were expressed as inclusion bodies. Af- ter purification, it could be recognized by anti-THP monoclonal antibody by western-blot. Conclusion A part of gene encoding THP domain was successfully cloned and expressed. It could be used to develop anti-THP mono- clonal antibody and THP test kit.

关 键 词：Tamm—Horsfall蛋白 基因克隆 表达 抗原性 

分 类 号：R392.1[医药卫生—免疫学]