[Construction and clinical application of lentivirus-AQP4 expressing vector].
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.


CONCLUSIONS
The HEK-293T cell transfected with lentivirus-AQP4 vector can express stably. And the expressed fragment may be applied in clinical examination.
Liao
Zhang-yuan
ZY
Department of Neurology, Xuanwu Hospital, Capital Medical University, Beijing 100053, China.
Ye
Jing
J
Guan
Yun-qian
YQ
Zhang
Yu
Y
You
Xiao-fan
XF
An
Jing
J
Zhang
Ying
Y
Wang
Shu-yan
SY
Li
Cun-jiang
CJ
D051401:Aquaporin 4 / Q000235:genetics* / Q000276:immunology*; D001323:Autoantibodies / Q000097:blood; D045744:Cell Line, Tumor; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors / Q000096:biosynthesis*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D016086:Lentivirus / Q000235:genetics / Q000276:immunology; D009103:Multiple Sclerosis / Q000175:diagnosis; D009471:Neuromyelitis Optica / Q000175:diagnosis*; D014162:Transfection
D051401:Aquaporin 4; D001323:Autoantibodies; D007074:Immunoglobulin G