Construction Of Recombinant Plasmid Encoding Human AQP4and Its Stable Expression In HKE293Cells

Objective: NMO (neuromyelitis optica, NMO), also known as Devic,sdisease, is an idiopathic inflammatory demyelinating disease of the centralnervous system, which mainly affects the optic nerves and spinal cord,generally follows a relapsing course, acute or subacute onset and a poorprognosis. For a long time, there has been controversy over whether NMO is aseparate disease entity, or a special clinical subtype of multiple sclerosis(multiple sclerosis, MS). Many patients showing initial symptoms ofneuromyelitis optica are diagnosed with multiple sclerosis. But the prognosisand optimum treatments for the two diseases differ. Compared with MS, NMOhas more severe clinical manifestations and prognosis. Within five years,monocular vision of about half of NMO patients is seriously damaged or blind,and about50%of relapsing NMO patients are unable to walk independentlywithin5years of onset. Immunosuppressive drugs are regarded as the besttreatment for neuromyelitis optica, whereas immunomodulatory treatments arepresently recommended for early treatment of multiple sclerosis. In2004,Lennon and colleagues discovered a highly specific serum autoantibodymarker, NMO-IgG,was found in the sera of NMO, which binds toaquaporin-4(AQP4),the most abundant water channel in the CNS. Thedetection of AQP4autoantibodies may play an important role in thedifferential diagnosis of NMO and MS, which has been enven regarded as animportant index to support the diagnosis in the Wingerchuk Reviseddiagnostic criteria for neuromyelitis optica, but the assays for the detection ofAQP4antiantibodies are still to be solved.The purpose of this study is to construct human aquaporin-4(AQP4)recombinant plasmid and to detect its stable expressions in Human EmbryonicKidney Epithelial (HEK293). This assay can identify the NMO and MS preliminarily, make the treatment more targeted and individualized, andfacilitate our further exploration for the possible pathogenesis of NMO. It alsoprovides an experimental basis for the disease activity, prediction of relapseand prognosis.Methods:1Acquisition of human AQP4gene1.1Design and synthesis of Primer: We designed primers respectivelyreferring to the full length human AQP4gene sequence published inGenebank.Upstream primer contained NheⅠrestriction enzyme sites anddownstream primer contained XhoⅠrestriction sites. The internal referencebeta actin (β-actin) as positive control.1.2Extraction of total RNA from brain tissue: The total RNA wasextracted with a TRizol-based protoco from adult brain tissue, obtained fromDepartment of Neurosurgery in Second Hospital of Hebei MedicalUniversity.The extracted product was subjected to agarose gel electrophoresisand measure its purity and concentration.1.3AQP4gene amplified by RT-PCR: We reverse-transcribed RNA intocDNA using the random primers, then used it as the template for PCR toamplify AQP4gene using random primers. PCR products were identified andpurified by agarose gel electrophoresis.2Construction of recombinant plasmids: AQP4isoforms A and pEGFP-N1were digested respectively overnight by restriction endonucleases Nhe Ⅰand Xho Ⅰ, then identify and purify the products. The digested pEGFP-N1and AQP4were ligated with T4ligase.Then the ligation product wastransformed into competent cells-E. coli DH5α, in kanamycin sulfateresistant LB agar overnight, with empty plasmid as the control. Selectedmonoclonal tomorrow and extracted the recombinant plasmid after expandingculture.3Identification of recombinant plasmid3.1Identification by restriction enzyme: The recombinant plasmid andempty plasmid were digested by both NheⅠand XhoⅠ or either, the obtained products were analyzed by agarose gel electrophoresis to detectwhether the recombinant plasmid can be cut out the complete AQP4gene afterboth enzyme digestion.3.2Identification by sequencing: The recombinant plasmid was sent toShanghai SANGON for sequencing. The sequencing outcome was contrastedwith sequence of human AQP4from GeneBank.4Cell transfection, identification and selection of stable cells4.1Transfection of HEK293cells: The day before transfection, theappropriate numbers of cells were seeded on24-well plates.When cultured to70%to80%cell fusion, HEK293cells were transfected with a vector carryinghuman AQP4gene or a control vector without human AQP4by Lipofectamine2000.4.2Detect the expression of green fluorescent protein (GFP): Observe theexpression of GFP of recombinant plasmid group, empty plasmid group andnon-transfection group respectively after48h under an inverted fluorescencemicroscope.4.3Screening of transfected cells: Observe the transient transfectionefficiency of transfected cells after24h, meanwhile800mg/L G418wereadded into the medium. Two weeks later, selected the stable cells and observedthe stable transfection efficiency. We combined with the application of flowcytometry to improve the stable transfection rate.5Identification of AQP4in HEK293cellsDetect the AQP4in HEK293cells by RT-PCR, Western Blot, and indirectimmunofluorescence respectively in cell and protein level. Fixed cells werefurther observed under confocal microscopy to determine whether the fusionprotein of stable cells have membrane localization effects.Results:1Human AQP4gene amplified by RT-PCR: Agarose gel electrophoresisof RT-PCR product shows clear specific amplification bands, consistent withthe expectations.2Identification of recombinant plasmid: The both enzyme digestion products of recombinant plasmid were analyzed by agarose gel electrophoresis.It can be observed a specific band at the place of theoretically expected value,while singe enzyme digestion products show no specific band. Thecoincidence rate of sequencing results and the theoretical sequence was morethan99%.3Identification and screening of transfected cells: Expression of GFP wasfound in the recombinant plasmid group and the fluorescence were mainlylocated on the cell membrane, while the empty plasmid group showed GFPexpression and fluorescence were mainly located in the wrapped slurry. Therewas no GFP expression in HEK293cells without transfection. Antibioticscombined with flow cytometry makes stable transfection rate reaches above90%.4Identification of AQP4in HEK293cells: Detect AQP4by RT-PCR:RT-PCR products were analyzed by agarose gel electrophoresis, a specificband was seen at972bp. Detect AQP4by Western Blot: The extracted proteinfrom cells transfected with recombinant plasmid was analyzed by WB, aspecific band was visible in the position of61KD, and the cells transfectedwith empty plasmid showed no specific bands. Detect AQP4by indirectimmunofluorescence: Cells transfected with recombinant plasmid afterantigen-antibody reaction were obseverd under an inverted fluorescencemicroscope. When exciting light was blue, cell surface showed greenfluorescence, when green, cell surface showed the red fluorescence in the cellmembrane,and the two fluorescence coincided well. Cellular localization offusion protein (AQP4-GFP): Observe the GFP under a confocal microscope,itfills the entire cytoplasm in the cells transfected with empty plasmid, nosignificant local positioning,while localized on the cell membranesignificantly in cells transfected with recombinant plasmid.Conclusion:Recombinant plasmid pEGFP-N1-AQP4is constructed and clonedsuccessfully, and expressed stably in HKE293cells. It provided a highsensitive and specific detection method for AQP4antibody in the serum of NMO patients and facilitated our exploration for the possible pathogenesis ofNMO. At the same time, it provided a significativ experimental basis for thedisease activity, prediction of relapse and prognosis.