用基因免疫方法制备小鼠抗人OAT1多克隆抗体

Generation of Mouse Anti-Human Organic Anion Transporter 1 Polyclonal Antibody by Genetic Immunization

【目的】用基因免疫方法制备小鼠抗人有机阴离子转运蛋白1(hOAT1)多克隆抗体。【方法】提取人肾组织总RNA,用RT-PCR方法扩增hOAT1基因中免疫源性较强的细胞内亲水序列片段,克隆到载体pBQAP-TT中,鉴定正确后用基因枪与辅助载体pCMVi-GMCSF和pCMVi-FIT3L同时免疫小鼠,12周后ELISA方法测定血清抗体滴度,鉴定抗体的特异性和定位表达。【结果】扩增出207 bp的hOAT1细胞内亲水序列片段,构建的基因免疫载体经酶切及序列分析鉴定正确。Western blot结果显示小鼠抗hOAT1多克隆抗体识别人肾皮质膜蛋白55 kD的hOAT1。免疫组化结果提示hOAT1定位表达在人近端肾小管上皮细胞基底侧细胞膜。【结论】用基因免疫的方法可以成功制备高滴度和特异性抗hOAT1多克隆抗体。

[Objective]To generate mouse anti-human organic anion transporterl （hOAT1） polyclonal anti- body by genetic immunization. [Methods] Total RNA of human kidney was isolated and the intracellular high antigenicity fragment of hOAT1 was amplified by RT-PCR, then cloned into pBQAP-TT to construct recombinant vector. Gene gun immunization with this recombinant plasmid and two other adjuvant plasmids, which expressed granulocyte/macrophage colony-stimulating factor and FMS-like tyrosine kinase 3 ligand respectively, induced high level antibody after 12 weeks. The antibodies＇ titer was detected by Enzyme-linked immunosorbent assay, and its specificity was identified by Western blot analysis and immunohistochemistry. [Results]Intracellular high immunogenic fragment of hOAT1 was amplified successfully from total RNA of human kidney by RT-PCR, and cloned into pBQAP-TT for genetic immumization. Recombinant vector was confirmed by restriction digestion and sequencing. The mouse anti-hOAT1 polyclonal antibody could recognize a band of 55 kD hOAT1 protein. Immunohistochemistry indicated that hOAT1 localized at the brush basolateral membrane of human renal proximal tubule cells. [Conclusion]Genetic immunization can offer anti-hOAT1 polyclonal antibody of high throughput and high specificity.