水通道蛋白－9表达载体的构建及视神经脊髓炎患者血清中抗体的检测

摘要

Objective To construct a eukaryotic aquaporin -9 (AQP9) expressing vector for AQP9 antibody de-tection in patients with neuromyelitis optica (NMO).Methods The recombinant vector was constructed by subcloning human AQP9 cDNA into pcDNA3.1+vector, with which human embryonic kidney (HEK) 293T cells was transfected to establish an AQP9 protein expressing cell line .The cell line expression AQP 9 was confirmed by reverse transcription poly-merase chain reaction ( RT-PCR) and Western blot analysis .Serum antibody was tested with cell -based assay by indi-rect immunofluorescence assay .Results The relative expression level of AQP 9 mRNA was significantly higher than the empty vector, and Western blotting analysis showed the expression content at 32 kD bands was significantly higher than the empty vector and 293T cells.The cell line expressing AQP9 was successfully constructed and confirmed by reverse tran-scription polymerase chain reaction ( RT-PCR) and Western blot analysis .The serum antibody was positive in all 4 pa-tients with NMO and negative in one with cerebral infarction and one normal adult .Conclusion The transfected cell mod-el is simple and advantageous in AQP 9 serum antibody detection for research on NMO .%目的：构建一种能稳定表达人水通道蛋白－9（ AQP9）的真核细胞载体，并检测视神经脊髓炎（NMO）患者血清中的AQP9抗体。方法将人AQP9基因克隆入pcDNA3．1＋质粒中构建AQP9－pcDNA3．1＋载体，并转染HEK－293T细胞中。 AQP9蛋白的表达通过RT－PCR和Western blot法验证。通过间接免疫荧光法检测NMO患者血清中的AQP9抗体。结果 AQP9的mRNA相对表达量明显高于空载体，且蛋白表达量在32 kD条带附近明显高于空载和293T细胞。在4例NMO患者血清中均检测到AQP9抗体，1例脑梗死和1例健康人中抗体均为阴性。结论这种转染表达AQP9的细胞模式相对简捷，可用来检测NMO患者中的AQP9抗体，对NMO研究有一定意义。