ULBP4原核表达、生物学功能鉴定及其单克隆抗体的制备和初步鉴定  

Expression and biological function of ULBP4 in Rosetta-gamiTMB(DE3) and mAb preparation

作　　者：曹伟[1] 郝志勇[1] 郗雪艳[1] 孔燕[1] 马驰[1] 崔莲仙[1] 何维[1] 

机构地区：[1]中国医学科学院基础医学研究所中国协和医科大学基础医学院免疫系,北京100005

出　　处：《细胞与分子免疫学杂志》2007年第3期242-245,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家重点基础研究发展规划(973)资助项目(2001CB510009;2004CB518706);国家自然基金委重大项目(30490244);国家自然基金委面上项目(30400391)

摘　　要：目的:在大肠杆菌中表达ULBP4重组蛋白,并制备其特异性的单克隆抗体(mAb),为γδT细胞对ULBP4的识别机制研究奠定基础。方法:根据GenBank中ULBP4的基因序列设计相应的引物,用RT-PCR方法从HO-8910细胞中扩增得到ULBP4片段,构建重组原核表达质粒pET22b(+)/ULBP4(前225aa胞外段),在Rosetta-gamiTMB(DE3)大肠杆菌中获得表达且主要位于包含体中,经纯化透析复性后,分析其对NK细胞IFN-γ细胞因子分泌的影响。以ULBP4为抗原,免疫BALB/c小鼠,取免疫小鼠脾细胞和Sp2/0骨髓瘤细胞常规融合,依次经HAT选择培养、间接ELISA法、克隆化培养、荧光免疫检测和流式细胞术以及Western blot筛选和鉴定抗ULBP4 mAb的杂交瘤细胞株。结果:构建了pET22b(+)/ULBP4(225aa)原核表达体系,并检测到重组蛋白的有效表达;纯化的重组蛋白在体外培养体系中可以刺激NK细胞IFN-γ细胞因子的分泌。此外,还获得了4株可稳定分泌抗人ULBP4 mAb的杂交瘤细胞株。结论:成功地构建并表达了具有生物活性的ULBP4,为γδT细胞的识别机制研究建立抗原制备平台,同时所制备的抗人ULBP4 mAb,为后续研究奠定了基础。AIM： To express human ULBP4 in RosettagamiTMB（DE3） and to prepare monoclonal antibody against ULBP4 for the research of γδT cells recognition mechanism. MBTHODS： DNA fragments of ULBP4 were derived from HO-8910 RNA by reverse-transcriptase polymerase chain reaction（RT-PCR）. The fragments encoding the former 22.5 amino acids of ULBP4 were cloned into Histag fusion protein expression vector pET22b（ ＋ ）. The C-Histag fusion ULBP4 （225a） protein was expressed in inclusion body and purified step by step according to manufactory＇s protocol and renatured in bag filter. It’s functional effect on NK cells was evaluated by NKG2D binding assay and IFN-γ secretion experiments. Prokaryotic expressed human ULBP4 was used as an antigen to prepare monoclonal antibodies by means of the B lymphocyte hybridoma technique, RESULTS： ULBP4 recombinant protein can stimulate NK cells to secrete IFN-γ. Through PEG fusion and screening by limited dilution, we obtained four strains of hybridoma cells secreting anti- ULBP4 antibodies. CONCLUSION： The fusion protein was expressed successfully and functional. At the same time, the anti-ULBP4 mAbs were prepared successfully. Both of them provide a platform for further research.

关 键 词：ULBP4 NKG2D IFN-Γ Rosetta-gami^TMB(DE3)大肠杆菌 表达 生物学功能 单克隆抗体 

分 类 号：R392.11[医药卫生—免疫学]