水通道蛋白4慢病毒表达载体的构建与临床应用  

Construction and clinical application of lentivirus-AQP4 expressing vector

作　　者：廖张元[1] 叶静[1] 关云谦[2] 张愚[2] 尤小凡[1] 安静[2] 张颖[2] 王淑艳[2] 李存江[1] 

机构地区：[1]首都医科大学宣武医院神经内科,100053 [2]首都医科大学宣武医院细胞治疗中心,100053

出　　处：《中华医学杂志》2010年第3期208-212,共5页National Medical Journal of China

基　　金：基金项目：首都医科大学基础临床联合攻关项目（JL2007-10）

摘　　要：目的构建水通道蛋白4（AQP4）慢病毒表达载体，并检测视神经脊髓炎（NMO）患者血清中的抗AQP4抗体。方法从人胶质母细胞瘤中提取RNA，通过RT—PCR获得人AQP4目的片段，将目的片段克隆到慢病毒表达载体中，转染HEK-293T细胞，通过RT-PCR、Western印迹及细胞免疫荧光检测鉴定AQP4表达，并对人血清中抗AQP4抗体初步检测。结果质粒测序结果与Genbank中人AQP4编码片段完全相符，转染人AQP4的HEK-293T细胞能稳定表达AQP4。细胞免疫荧光法检测12例NMO患者血清，11例阳性（91．7％）；34例多发性硬化阳性4例（11．8％）；50名健康人群血清均为阴性。结论HEK-293T细胞经水通道蛋白4慢病毒表达载体转染后能够稳定表达AQP4，可用于临床NMO患者抗AQP4抗体的检测。Objective To construct the human aquaporin-4 （AQP4） expressing vector and detect anti-AQP4 antibody in serum of patients with neuromyelitis optica （NMO）. Methods RNA was extracted from human glioblastoma and AQP4 cDNA obtained through RT-PCR. The fragment was cloned into the lentiviral expressing vector （iDUET101） and transformed into competent strain Hbl01 for later amplification; plasmids were extracted from the amplified positive-bacteria-colony, sequenced and transfected into HEK-293T cells. Expression of AQP4 was identified by RT-PCR, Western blot and immunofluorescence assay. And anti-AQP4 antibody in human serum was tested. Results The sequence of target fragment matched with that of human AQP4 fragment sequences （ NM_001650 ） completely. The constructed AQP4 fragment transfected in HEK-293T cell was tested by immunofluoreseent examination and it exhibited obvious fluorescence located in cell membrane. Western blot test was positive. And the fragment was about 34 KD. Cellular immunofluorescence examination showed 11 examples of 12 NMO patient serums （91.7%） were positive, 4 in 34 multiple sclerosis （ 11.8% ） positive and negative in all 50 serum samples of healthy controls. Conclusion The HEK-293T cell transfected with lentivirus-AQP4 vector can express stably. And the expressed fragment may be applied in clinical examination.

关 键 词：视神经脊髓炎 慢病毒属 水通道蛋白4 免疫荧光技术 

分 类 号：R74[医药卫生—神经病学与精神病学]