CAJ | 학술논문

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.
목적 구건수통도단백4(AQP4)만병독표체재체,병검측시신경척수염(NMO)환자혈청중적항AQP4항체.방법 종인효질모세포류중제취RNA,통과RT-PCR획득인AQP4목적편단,장목적편단극륭도만병독표체재체중,전염HEK-293T세포,통과RT-PCR、Western인적급세포면역형광검측감정AQP4표체,병대인혈청중항AQP4항체초보검측.결과 질립측서결과여Genbank중인AQP4편마편단완전상부,전염인AQP4적HEK-293T세포능은정표체AQP4.세포면역형광법검측12례NMO환자혈청,11례양성(91.7%);34례다발성경화양성4례(11.8%);50명건강인군혈청균위음성.결론 HEK-293T세포경수통도단백4만병독표체재체전염후능구은정표체AQP4,가용우림상NMO환자항AQP4-항체적검측.
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 Hb101 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 immunofluorescent 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.