抗人有机阴离子转运蛋白4多克隆抗体的制备及其在肾脏的定位研究  

Generation of Mouse Anti-human Organic Anion Transporter4 Antibody and its Localization in Human Kidney

作　　者：吴镝[1] 许国双[1] 陈香美[1] 师锁柱[1] 洪权[1] 张萍[1] 吕扬[1] 

机构地区：[1]中国人民解放军总医院肾科,中国人民解放军肾病中心暨重点实验室,北京100853

出　　处：《中国中西医结合肾病杂志》2005年第6期321-324,i001,共5页Chinese Journal of Integrated Traditional and Western Nephrology

基　　金：国家自然科学基金资助项目 (No .3 0 10 0 0 62 ) ;国家重点基础研究发展规划项目 (No .G2 0 0 0 0 5 70 0 0 );国家自然科学基金青年创新研究群体基金资助项目 (No .3 0 12 10 0 5 )

摘　　要：目的:拟采用一套新的基因免疫系统制备高特异性和高效价的小鼠抗有机阴离子转运蛋白4(hOAT4 )多克隆抗体,并用该抗体对hOAT4在人肾小管上皮细胞的表达进行定位研究。方法:应用Accelrys软件分析hOAT4抗原表位,选择其中免疫源性较强的细胞内表位(E2 78～R345 ) ,从人肾组织总RNA中用反转录-聚合酶链反应(RT -PCR)方法扩增其2 0 4bp的cDNA序列,克隆到pBQAP -OVA构建基因免疫载体,鉴定正确后与辅助载体pCMVi-GMCSF和pCMVi -FlT3L同时免疫小鼠,5周后取血清抗体,ELISA方法测定抗体滴度,人肾组织Westernblot和免疫组化鉴定抗体的特异性和定位表达。结果:成功构建基因免疫载体pBQAP -OVA -hOAT4 ,经酶切及序列分析鉴定完全正确。ELISA方法测得抗体滴度高达1∶30 0 0 ,该抗体识别人肾皮质膜蛋白和肾小管刷状缘蛋白6 5kD糖基化的hOAT4。人肾组织免疫组化结果提示该抗体识别定位在近段肾小管上皮细胞刷状缘的hOAT4。结论:用基因免疫方法可以成功制备高效价和高特异性抗hOAT4多克隆抗体。Objective:Ogranic anion transporter 4 (OAT4),which expresses in human kidney, plays a principal role in secreting organic anion metabolized from both endogenous and exogenous compounds.Antibody is an important tool to study hOAT4.This study is aimed to produce mouse anti-hOAT4 polyclonal antibody with high throughput and high specificity by genetic immunization and to detect the localization fo hOAT4 in human kidney.Methods:Intracellular high antigenicity fragment (E278～R345) of hOAT4 was chosen by protein antigenicity prediction software and its cDNA was amplified from human renal total RNA by RT-PCR, which was subsequently cloned into pBQAP-OVA plasmid to construct recombinant plasmid pBQAP-OVA-hOAT4 for genetic immunization. The mice were inoculated with this recombinant plasmid and two other adjuvant plasmids, pCMVi-GMCSF and pCMVi-FlT3L, which helped to enhance the antibody's generation. After 5 weeks, mice were sacrificed to obtain anti-hOAT4 antibody. Antibody titer was measured by ELISA. The antibody was identified by Western blot analysis and immunohistochemistry.Results:The cDNA of hOAT4 intracellular high immunogenic fragment were amplified successfully. Recombinant plasmid pBQAP-OVA-hOAT4 for genetic immunization was confirmed by restriction digestion and sequencing. ELISA assay indicated that mouse anti-hOAT4 antibody has high titer and could recognize a band of 65 kD glycosylated hOAT4 from human renal membrane protein and brush border membrane protein in Western blot.Immunohistochemistry results indicated that hOAT4 located at the brush border membrane of renal proximal tubular cells.Conclusion:Genetic immunization can generate anti-hOAT4 polyclonal antibody with high throughput and specificity.

关 键 词：抗人有机阴离子转运蛋白4 多克隆抗体 制备方法 肾脏定位 免疫系统 

分 类 号：R392[医药卫生—免疫学]