Construction Of 293T Cells Expressing NR1 And Application In The Detection Of Anti NMDA Receptor Autoantibodies

The symptoms of anti-NMDA receptor encephalitis(anti NMDAR encephalitis)is with similar to limbic encephalitis such as cognitive impairment,mental and behavioral abnormalities and so on.But anti NMDAR encephalitis has serious disturbance of consciousness,the mental and behavioral abnormalities and have involved the brain xerotocia central hypoventilation,confusion and other symptoms compared with limbic encephalitis.NMDA receptors are widely distributed in nervous system.Current anti NMDA receptor encephalitis diagnosis is mainly based on the anti NMDA receptor NR1 subunit IgG antibody(+).The positive rate of NR1 subunit IgG antibody in the cerebrospinal fluid of anti NMDA receptor encephalitis patients was 100%.The detection of anti NMDA receptor antibody has a very high sensitivity and specificity in the diagnosis of NMDA encephalitis.The gold standard for laboratory tests for indirect immunofluorescence.At present,the production of the detection kit manufacturers in Europe and the United States,which in China has been widely used and has been clinically tested detection kit is EUROIMMUN company production of antibody detection kit which price is expensive.If the antibody titer were detected,and the detection of serum and CSF specimens,then a single check cost will be increased several times.Objectives: To build a new detection method of anti NMDA receptor autoantibody with lower cost than commercial kits,and compare their detection efficiencies of the samples of anti NMDA receptor encephalitis in our lab.Methods: 1.The coding sequence of GRIN1 was amplified by PCR from a plasmid vector carrying GRIN1,and then the sequence was connected with a FUGW plasmid vector to obtein a FUGW-GRIN1-IRES-EGFP plasmid.2.The recombinant plasmid was transformed to Escherichia coli for amplification,and was collected and identified by PCR.3.The lentivirus expressing NR1 was packaged with a four plasmid system in 293 T cells.4.The cell culture medium containing lentivirus was collected and centrifuged to get the lentiviurs.5.We infected 293 T cells with the virus and detected the expression of NR1.6.We incubated the infected 293 T cells with anti NMDA receptor antibody positive and negative cerebrospinal fluid samples preserved in our laboratory,and performed immunofluorescence to investigate the detection efficiency of this method.Results: 1.The plasmid vector of FUGW-GRIN1-IRES – EGFP was constructed.2.The lentivirus carrying GRIN1 was packeged.3.The 293 T cells expressing NR1 were constructed.4.We detected the cerebrospinal fluid samples of anti NMDA receptor encephalitis patients,and 95% of the results were consistent with commercial kits.Conclusion: 1.We successfully have slow virus particles infected 293 T cells to highly express NMDA receptor NR1 subunit protein by selecting preferably lentiviral vector system.2.We are in agreement with the estimated results of the foreign test kit for the estimation of the antibody titer in the cerebrospinal fluid.Compared with the foreign kit,we can reduce false negative results by observing green fluorescence to distinguish cells that do not express or express small amount of GRIN1 gene.At the same time,using double fluorescence labeling has more advantages in the calculation of the antibody titer.4.The technology has the prospect of application in the laboratory testing according to the present experimental results.