The Construction Of CRMP5-293T Cell Models And Its Significance In Demyelinating Diseases Of The Central Nervous System

BackgroundDemyelinating diseases of the central nervous system(CNS)include neromyelitis optica spectrum disorders(NMOSDs),multiple sclerosis(MS),acute disseminated encephalomyelitis(ADEM),longitudinally extensive transverse myelitis(LETM),optic neuritis(ON)and so on.At present,the most common diseases in clinical are neromyelitis optica spectrum disorders and multiple sclerosis.There are so many differences in there pathogenesis,pathological changes and treatment.Since some foreign scholars discovered the aquaporin 4(aquaporin 4,AQP4)antibody in 2004,and this antibody has been recognized as the most sensitive and specific immunomarker of NMOSDs.Although the discovery of this antibody had a great significance in distinguish nmosds of MS,there are10-40%NMOSDs patients can not discover the antibody in their serum.The etiology of NMOSDs patients with negative AQP4 antibodies is not clear at present.Some scholars speculated that other antibodies may be involved in the pathological process of NMOSDs,and CRMP 5 antibody may be one of them.Section 1:The construction of CRMP 5-293T cell models ObjectivesTo construct the CRMP5 plasmids separately,and transfect them into different HEK293T cells.So that we can obtain CRMP5-293T cell models for the following CRMP5antibody dectection.Methods1.The construction of CRMP 5 plasmids1.1.Design and synthesis of primersAccording to the human CRMP5 gene sequences published in the Genebank,we used primer-design software Premier 5.0 to design the forward and reverse primers for the gene of CRMP5 separately.All primers were synthesized in a gene company.1.2.Obtainment of the CRMP5 and CRMP5 geneWe extracted the whole RNA from human somatic cells using Trizol method and detected its purity and integrity.And then we made use of the above CRMP5 primers and the whole human RNA in two different RT-PCR reactions,so that we obtained the CRMP5-cDNA.PCR products were identified and purified by agarose gel electrophoresis.1.3.Enzyme digestion and plasmid constructionDigested and construction of CRMP5 plasmids:PCR products and pEnter vector were digested overnight by restriction endonucleases ASC I and Not I,then the digested CRMP5 and vector were ligated with T7 ligase.Then the ligation product were transformed into competent cells with JM09,in kanamycine sulfate resistant LB agar overnight.The empty vector plasmid was as the control.Selected monoclonal tomorrow and extracted the expression plasmid after expanding culture.1.4.Identification of the CRMP5 and CRMP5 plasmidsExpression plasmid products extracted by double digestion were analyzed by agarose gel electrophoresis to detect the integrity of CRMP5 gene.Identification by sequencing:The expression plasmid was sent to biosune gene company for sequencing.The sequencing outcome was contrasted with sequence of human CRMP5 from GeneBank.2.Cell culture and transfectionResuscitated and cultured the HEK 293T cells.24 hours before the transfection,passage cultured of the 293T cells and when the cell density reached 70-80%,we trasfected the CRMP5 and CRMP5 plasmids into different 293T cells separately using the transfection reagents Lipofectamine 3000.3.Identification of stable cell linesWe used western blotting and indirect immunofluorescent assay to identify the expression of CRMP5 and CRMP5 in 293T cells.The successful CRMP5-293T and CRMP5-293T cell models would be used in the following antibody detection.Results1.Gel electrophoresis identification of the PCR productsHuman CRMP5 gene amplified by RT-PCR:the product of RT-PCR in Agarose gel electrophoresis shows clear specific amplification bands(1695bps),consistent with the expectations.2.Identification of the CRMP5 and CRMP5 plasmids2.1.Gel electrophoresis identification of the plasmids digestionThe gel electrophoretogram showed electrophoretic bands consistent with the theoretical value and confirmed that the CRMP5 plasmid was constructed successfully.2.2.Identification of gene sequencing analysisBLAST sequence alignment showed that the CRMP5 gene was 100%in accordance with the original sequences.3.CRMP5 expression in the cellsWe found Western blotting presented specific bands near 65 kDa.By the IIFA,CRMP5 positive antibody binding cell on the surface and intracellular showed red fluorescence excitation,and was superimposed with DAPI stained blue fluorescence.ConclusionThe CRMP5-293T cell model was constructed successfully by cloning the CRMP5gene and constructing the expression plasmid to be transfected into 293T cells.The cell model will provide a reliable experimental method for the detection of CRMP5 antibody.Section 2:Detection of CRMP5 antibody in serum from patients with demyelinating diseases of the CNSObjectivesTo verify the expression of CRMP5 in the brain,and detect the CRMP5 antibody in the sera of patients by the CRMP5-293T cell model constructed in Part One,then explore the role of CRMP5 antibody in demyelinating diseases of the central nervous system.Methods1.Cases and samples collectionThe study protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Guangzhou Medical University.Written informed consent was provided by all participants.We collected 62 patients diagnosed of demyelinating diseases of the CNS in the Second Affiliated Hospital of Guangzhou Medical University from September 2016 to September 2017.All patients had been detected for AQP4 antibody in their serum.Among them,16 cases were NMOSDs,13 cases were MS,15 cases were OIND,18 cases were IND,The control group included 20 patients diagnosed of ischemic stroke.And all patients had been drawn venous blood in the morning in fasting state before using steroid or immunosuppressor.2.The detection of CRMP5 antibody in surumWe used indirect immunofluorescence assays to detect CRMP5 antibody in patients'serum basing on CRMP5-293T cell models separately.The second antibody was CY3labeled mouse anti human IgG.3.Detection of CRMP5 Antibody in Commercial Western Blotting KitIt can be seen that the target bands of appeared on the blotting strip with the theoretical value,which agree with the CBA positive CRMP5 antibody patients.It is confirmed the successful establishment of the cell model and the positive reaction of serum CRMP5.4.Statistical analysisAll results had been analyzed by the SPSS 16.0 software.Since the results of our study were categorical data,we used X~2test to compare the positive rates among different groups,There were no significant differences among NMOSDs and control groups(p>0.05).Results CRMP5 antibody positive rates in different demyelinating diseasesCRMP5 antibodies were present in 1/13(7.69%)of MS patients,1/16(6.25%)of NMOSDs patients,0/15(0%)of OIND patients,0/18(0%)of OND patients,and 0/20(0%)of healthy controls.There were no significant differences among MS and control groups(p=0.316).ConclusionWe have success to constructe a method that based on cell transfection to detect CRMP5antibody in patient serum,which is helpful for clinical diagnosis of CRMP5 antibody associated diseases.