Identification Of Membrane And Nuclear Autoantigen Antibodies In The Pathogenesis Of Behcet's Disease (an Autoimmune Disease)

Behcet's disease(BD)is a rare systemic vasculitic disease affecting almost every organ system of the body and particularly characterized by ocular,oral and mucosal ulcerations.In some cases,complications of vasculitis lead to blindness or death.No one knows the exact reason of BD but it may be categorized as an autoimmune disease.The disease occurs frequently in countries along the ancient Silk Road and is especially common in China,Turkey and Japan.The overall incidence rate in developed countries is very low so it could not attract the attention of mainstream scientific research institutions since long time.As a result,the research progress of this disease is relatively slow.So far,the disease is still in a state of no clinical test,no treatment,no basic understanding of the pathogenesis.The current clinical diagnos still relies on doctor's experience to judge,therefore to understand the autoantibodies and autoantigens of the disease and related research is still weak.As autoantibodies are important laboratory diagnostic tools for many other autoimmune diseases.However,as of now,none of BD disease-specific autoantibodies that can be successfully applied to clinical practice in the world.As a matter of subject,we are committed to screen out a number of BD disease candidate autoantigens by establishing a complete set of systems biology methods,combining with traditional biotechnology and modern proteomics.In this study,we have identified four BD disease candidate autoantigens by using Hans Chinese patients' serum.Annexin A1,hnRNP C1/C2,Moesin and Carboxy(C)-terminal amino acid of NUMA.In brief,First,HaCaT and EA.hy926 cells were cultured to extract total RNA and obtained cDNA by Reverse Transcription method(RT-PCR).Then,according to four proteins' gene sequences from NCBI Genbank,we designed the primers for Polymerase Chain Reaction(PCR)and amplified their genes with cDNA.Genes were induced into prokaryotic plasmid pET-28a(+)and transformed the recombinant plasmids into competent E.coli DH5? cells.After PCR verification and sequencing,secondary amplification was performed.Then,extracted plasmids were transformed into competent E.coli BL21.By the induction of 0.1 mM IPTG,proteins were expressed and purified with Ni-NTA resin kit.After that,SDS-PAGE was done to confirm the results of expression and purification.Then expressed proteins were confirmed and validated by applying high throughput technology like Mass spectrometry and other immunological techniques like Western blotting,immune precipitation,immunohistochemistry,indirect immunofluorescence and ELISA.Finally,the data was evaluated by different statistical methods by using up to date softwares.Our findings demonstrate that all above stated autoantigens/autoantibodies are immune targets of BD in Han Chinese patients.