The Expression Of Human Aquaporin-4 And Its Application In The Diagnosis Of Neuromyelitis Optica

Background Neuromyelitis optica(neuromyelitis optica, NMO) is a severe demyelinating disorder in the central nervous system which predominantly affects the optic nerves and spinal cord. Generally, it occurs suddenly and develops rapidly. Moreover, it’s a serious risk of the health of human. There has been a large controversy whether NMO is an independent disease or a subtype of mutiple sclerosis(mutiple sclerosis, MS) since the concept of NMO was first described in 1894. Later, Lennon et al found that the antibody against aquaporin-4(AQP4)was the special and pathogenic antibody in the patients of NMO, which validated that NMO was a humoral immune mediated autoimmune disease different from MS. There are many differences in the treatment and the prognosis between NMO and MS. If we detect the antibody against AQP4 in the serum of the patients who is highly suspected NMO, it will be a great help in the aspect of differential diagnosis, treatment and prognosis between NMO and MS. So far there hasn’t been a highly sensitive and specifical kit for detecting the anti-AQP4 antibody in our country. Based on the status at present, it’s very necessary to establish a method to detect the autoantibody named anti-AQP4 antibody in the serum of the Chinese. It’s very clear that the main epitope of anti-AQP4 antibody binding is the extracellular of its target protein AQP4 now. Therefore, clone and express the extracellular of AQP4 may provide raw material used for detecting the antibody against AQP4. In addition, we expressed the full length of AQP4 by eukaryoti expression system, and compared the differences between the protein of AQP4 extracellular and the full length of AQP4 in the detection of the AQP4 antibody. Aim1. To establish an ELISA method to detect the anti-AQP4 antibody in the serum through constructing a prokaryotic expression plasmid p ET-32a(+)-AQP4 extracellular to express the protein of AQP4 extracellular.2. The recombinant plasmid p CMV6-AC-GFP-AQP4 was transfected into HEK293 cells and the full length of AQP4 was expressed in the cells, to establish a CBA method to detect the AQP4 antibody in the NMO patients. Methods1. Analyze the structure of human AQP4 by using bioinformatics software TMHMM 2.0,and synthesize the oligonucleotide sequences of human AQP4 extracellular according to the E. coli preferred codons.2. Construct a prokaryotic expression plasmids p ET-32a(+)-AQP4 extracellular through the technology of molecular cloning.3. The recombinant expression plasmids constructed were transformed to E. coli BL21(DE3) for expression under induction of IPTG.Then analyze the fusion proteins expressed by SDS-PAGE electrophoresis.4. The host bacteria was cultured and induced to express target proteins in large scale. The expressed fusion proteins were purified by nickel ion affinity hromatography. The proteins purified were identified by SDS-PAGE and western blot.5. The target proteins were coated in the microplate of ELISA. We evaluatedthe biological activity of the proteins expressed using the positive serums which were screened by indirect immunofluorescence method that was thought to be the gold standard in the worldwide.6. The recombinant plasmid p CMV6-AC-GFP-AQP4 was transfected into HEK293 cells by the technology of liposome transfection,and p CMV6-AC-GFP was transfected at the same time as a control. The cells were fixed and then used to detect the AQP4 antibody in the serums of NMO patients by indirect immunoflorescence. Results1. Restriction analysis and sequencing proved recombinant plasmid was constructed correctly.2. The results of SDS-PAGE showed that the proteins induced by the prokaryotic expression plasmids p ET-32a(+)-AQP4 extracellular can express in the supernatant in a soluble form. The relative molecular mass of the fusion proteins expressed is 26 000 dalton approximately.3. The proteins induced could reach high purity after one step affinity chromatography.4. The fusion proteins showed specific binding to mouse anti-His monoclone antibody.5. The proteins of AQP4 extracellular can’t bind to the anti-AQP4 antibody specially.6. The green fluorescent is filled with the whole cells which were transfected only GPF, but the green fluorescent is only located on the membrane of cells which were transfected GFP-AQP4.7. The cells which were transfected the GFP-AQP4 can specially bind to the AQP4 antibody in the serums of NMO patients. Conclusions1. We have constructed recombinant expression plasmid successfully and expressed recombinant proteins in E. coli BL21, which laid a foundation of further study about the extracellular of human aquaporin4 in the diagnosis of neuromyelitis optica.2. Once we express the the extracellular of human aquaporin4 only, they can’t bind to AQP4 antibody specially. Maybe this phenomenon is related to the three-dimensional structure that was destroyed and the OAP structures which were formed by itself whether or not.