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目的:建立稳定表达水通道蛋白-4(AQP4)的HEK293细胞株,并以其检测12例视神经脊髓炎患者血清中是否存在AQP4抗体。方法:从颞叶癫痫患者脑组织提取总RNA,RT-PCR扩增AQP4的cDNA,构建AQP4-绿色荧光蛋白(GFP)融合表达重组体pEGFP-N1-AQP4,转染HEK293细胞,抗生素筛选联合流式细胞仪分选筛选稳定表达细胞株,RT-PCR和间接免疫荧光法鉴定AQP4的表达,激光共聚焦显微镜观察AQP4在细胞中的定位,并以已建立的细胞为底物间接免疫荧光法检测12例视神经脊髓炎患者血清中是否存在AQP4抗体。结果:经PCR、双酶切、测序鉴定证实成功获取AQP4的cDNA,AQP4-GFP融合表达重组质粒pEGFP-N1-AQP4构建成功,抗生素筛选联合流式细胞仪分选筛选稳定表达细胞株,稳定表达率达90%以上,RT-PCR和间接免疫荧光法鉴定存在AQP4的表达,激光共聚焦显微镜进一步确认AQP4-GFP融合蛋白主要表达于细胞膜上,11例典型视神经脊髓炎患者血清中检出AQP4抗体,对视神经脊髓炎诊断敏感性为91.7%,特异性为94.7%。结论:成功建立稳定表达AQP4的HEK293细胞株,以其作为检测底物检出视神经脊髓炎患者血清中存在AQP4自身抗体。
OBJECTIVE: To establish a HEK293 cell line stably expressing AQP4 and to detect the presence or absence of AQP4 antibody in 12 patients with optic neuromyelitis. Methods: The total RNA was extracted from the brain tissue of patients with temporal lobe epilepsy. The cDNA of AQP4 was amplified by RT-PCR. The recombinant plasmid pEGFP-N1-AQP4 fused with AQP4-GFP was constructed and transfected into HEK293 cells. The expression of AQP4 was identified by RT-PCR and indirect immunofluorescence. The localization of AQP4 in the cells was observed by confocal microscopy. The established cells were detected by indirect immunofluorescence AQP4 antibody was detected in serum of 12 patients with optic neuritis. Results: The cDNA of AQP4 was obtained successfully by PCR, double enzyme digestion and sequencing. The recombinant plasmid pEGFP-N1-AQP4 with AQP4-GFP fusion protein was constructed successfully. The stable cell line was stably expressed by antibiotic selection combined with flow cytometry The expression of AQP4 was identified by RT-PCR and indirect immunofluorescence. Confocal laser scanning microscopy confirmed that the AQP4-GFP fusion protein was mainly expressed on the cell membrane. AQP4 antibody was detected in serum of 11 patients with typical optic neuromyelitis , The diagnostic sensitivity of optic neuromyelitis was 91.7%, the specificity was 94.7%. CONCLUSION: HEK293 cell line stably expressing AQP4 has been successfully established, and the presence of AQP4 autoantibodies in the serum of patients with optic neuromyelitis was detected by using it as a detection substrate.