Development Of Novel Fusion Protein Complement Immunosuppressant And Its Application In Rheumatic Arthritis

Objective:The prokaryotic expression vector of recombinant fusion protein p Smart1-CRIg-CD59 was constructed and purified by escherichia coli expression system.Purified fusion protein CRIg-CD59 protein through CD59 immunosuppressive effect and strong complement CRIg complement targeted activity,end up with a highly efficient complement with the activity of local complement targeted immune inhibitor,achieving biological activity in rats with adjuvant induced arthritis,,reduce and even reverse the progress of the disease.Methods:Extracting total RNA from human liver cells HHMa,using reverse transcription-polymerase chain reaction(RT-PCR)and DNA Seamless Cloning technology,the gene sequence which coding people CRIg extracellular IgV period was cloned to p SmartⅠvectors,to obtain p Smart-CRIg;We use the same method to lung cancer cell line A549 in clone CD59 extracellular function segment sequence cloning to p SmartⅠ-CRIg vector,and in the 5’end of CD59 primer design to join(Ser Gly4)3 sequence,flexible would get p Smart-CRIg-CD59 prokaryotic expression plasmid.After selecting the positive colony plasmid for sequencing and verifying the sequence,the CRIg-CD59 protein was expressed and purified by the prokaryotic expression system,and the product was verified by SDS-Page and Western Blot.Hemolytic activity experiments of classical complement pathways and alternative pathways were performed in vitro to verify protein activity,and surface plasmon resonance was used to detect the binding of proteins to C3b and i C3b.Establish adjuvant-induced arthritis models in rats(groups of each model:AIA model group,PBS treatment group,dexamethasone treatment group,CRIg-CD59 treatment group),and regularly perform rat foot and paw swelling,etc.Observe and evaluate the clinical scores and behavioral scores of each group;Elisa method was used to detect local and serum inflammation and chemokine levels.Micro-CT was taken to observe joint erosion and damage,and bone and joint samples were taken for 4%polymerization.Formaldehyde fixation and EDTA2~+decalcification were used to prepare joint sections.HE staining and saffron O-fast green staining were used to observe cartilage damage and synovial hyperplasia.Results:The size of the CRIg extracellular IgV domain sequence cloned from human liver cells HHMa was 795 bp,and the length of the CD59 extracellular functional sequence sequence cloned from the human lung cancer cell line A549 was 231 bp,and finally cloned into CRIg-(Ser Gly4)The 3-CD59 sequence is 1071bp in length,and sequencing results show no mutations.The molecular weight of the target protein after prokaryotic expression and purification was 52.1 kd.In vitro classical and alternative pathway hemolysis experiments show that CRIg-CD59 can reach the maximum complement inhibitory activity at 35μm;surface plasmon resonance detection shows that CRIg-CD59 and C3 cleavage products have a small equilibrium dissociation constant(KD),and the maximum affinity up to 4.96μm.In the rat adjuvant-induced arthritis treatment model,the paw swelling of the rats in the AIA model group and the PBS treatment group was significantly reduced.The swelling of the paw in the dexamethasone treatment group was relieved.Foot swelling was significantly relieved;Elisa results showed that IL-6 and TNFαlevels in the CRIg-CD59 protein treatment group were significantly reduced;Micro CT results showed that joint erosion and destruction symptoms were significantly reduced in the CRIg-CD59 treatment group,and joint section results showed that the CRIg-CD59treatment group Cartilage surface damage and synovial hyperplasia were significantly relieved compared with other groups.Conclusion:The CRIg-CD59 fusion protein constructed in this subject has good complement immunosuppressive activity in vitro,and has significant binding capacity and small dissociation constant with the C3 cleavage product,which enables the CRIg-CD59 protein to be activated by local complement Enriched on the synovium and joints of rat adjuvant-induced arthritis,CRIg-CD59 significantly inhibited cartilage destruction and synovial hyperplasia,and showed good biological activity.This experiment confirmed that CRIg-CD59 has a good therapeutic effect in rat AIA arthritis disease model.