FKBP38的原核表达、抗体制备及其应用  

Expression and antibody preparation of FKBP38 and its application

作　　者：刘安玲[1] 赵莉[2] Ma Dong-zhu 贾春宏[2] 李明[2] 温芝华[2] 叶永斌[2] 

机构地区：[1]南方医科大学基因工程研究所,广东广州510515 [2]南方医科大学细胞生物学教研室,广东广州510515 [3]Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA

出　　处：《南方医科大学学报》2010年第6期1226-1229,共4页Journal of Southern Medical University

基　　金：国家自然科学基金(30771027,30870955,30900555)

摘　　要：目的构建人FKBP38 N-末端及C-末端原核表达载体并使其在大肠杆菌中高效表达,纯化基因表达产物,制备兔多克隆抗体并对该抗体用于Western免疫印迹、免疫荧光及免疫组织化学检测进行评价,为研究FKBP38的功能奠定基础。方法以人FKBP38 cDNA为模板,根据人FKBP38全长cDNA系列,设计PCR引物分别扩增N-末端(1-207aa)及C-末端(209-387aa),将扩增产物克隆至大肠杆菌表达载体pGEX-6P1中,经BamH I/Sal I双酶切鉴定。GST-FKBP38-N及GST-FKBP38-C融合蛋白在硫代半乳糖苷(IPTG)诱导下在大肠杆菌BL21中得到表达。利用谷胱甘肽亲和层析纯化融合蛋白,用纯化的GST-FKBP38-N融合蛋白及GST-FKBP38-C融合蛋白免疫家兔制备多克隆抗体并进行纯化。使用纯化的抗FKBP38多抗以Western免疫印迹、免疫荧光及免疫组织化学检测FKBP38的表达与细胞内的定位。结果成功构建FKBP38 N-末端及C-末端原核表达载体,表达并纯化了GST-FKBP38-N融合蛋白及GST-FKBP38-C融合蛋白,制备并纯化了FKBP38特异性抗体。该抗体用于Western免疫印迹检测FKBP38在不同细胞株中的表达,特异性强、效价高;用于免疫荧光检测FKBP38在细胞内主要定位于线粒体上;用于免疫组织化学检测FKBP38在乳腺组织细胞呈现胞浆阳性。结论 FKBP38特异性抗体制备成功,该抗体可用于FKBP38的免疫印迹、免疫荧光及免疫组织化学检测,为深入研究FKBP38的功能奠定基础。Objective To obtain recombinant N-and C-terminal of FKBP38 and prepare anti-FKBP38 polyclonal antibody for Western blotting（WB）,immunohistochemical（IHC） and immunofluorescence（IF） analyses.Methods The N-terminal（1-207 aa） and C-terminal（209-387 aa） cDNA of FKBP38 were sub-cloned from the full-length cDNA of FKBP38 and ligated to prokaryotic expression plasmid pGEX-6P-1 for construction of the recombinant vectors pGEX-6P-1-FKBP38-N and pGEX-6P-1-FKBP38-C.After sequencing,the recombinant vectors were transformed into E.coli BL21 and GST-tagged FKBP38-NT and FKBP38-CT were induced by IPTG.The proteins were purified by Glutathione affinity chromatography column and characterized by SDS-PAGE.Rabbits were immunized with the purified recombinant protein to prepare the antiserum,which were analyzed by WB,IHC and IF.Results The recombinant vectors pGEX-6P-1-FKBP38-N and pGEX-6P-1-FKBP38-C were successfully constructed.After IPTG induction,the E.coli transformed with these plasmids expressed GST-tagged protein,which was successfully purified.Western blotting demonstrated that the purified antibody could specifically bind to FKBP38 in various cell lines.Immunofuorescence assay showed that FKBP38 was located mainly on the mitochondria.Immunohistochemical analysis revealed cytoplasmic location of FKBP38 in breast cells.Conclusion We successfully expressed and purified N-and C-terminal of FKBP38,and FKBP38 polyclonal antibody we prepared can specifically recognize FKBP38 in SB,IF and IHC assays,which facilitates further functional investigation of FKBP38.

关 键 词：FKBP38 多克隆抗体 Western免疫印迹 免疫荧光 免疫组织化学 

分 类 号：R392[医药卫生—免疫学]