Human target complement inhibitor protein mcr2-daf and its application
A technology of complement and complement receptor, which is applied in the field of fusion proteins, can solve the problems of slow process, etc., and achieve the effects of improved inhibition efficiency, good ligand binding ability, and excellent anti-adhesion/anti-inflammatory targeted inhibition effect
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Embodiment 1
[0033] Example 1. Preparation of CR2 mutant peptide-DAF fusion protein
[0034] 1 Materials The expression vector was pEE14.1 (Lonza biologicals); CHO cells were used for protein expression, and the culture medium was DMEM containing 10% fetal bovine serum, which was purchased from Invitrogen. Mouse anti-DAF mAb 1H4 and 1A10, mouse anti-human CR2 mAb 171, anti-goat erythrocyte IgM and all secondary antibodies were purchased from Sigma.
[0035] 2 methods
[0036] 2.1 The preparation of rabbit anti-CHO cell membrane and human DAF antiserum was described in Harlow, E., and Lane, D. Antibodies: a laboratory manual. Cold Spring Harbor Laboratory. Cold Spring Harbor, New York, USA. 1988: 726. method to obtain.
[0037] 2.2 Construction of expression recombinant and protein expression The cDNA structural gene is composed of 4 N-terminal SCR units encoding CR2 connected with the sequence encoding the extracellular region encoding DAF. The complement inhibitor sequence is 1-250 bas...
Embodiment 2
[0044] Example 2. Kinetic analysis of interaction between CR2 fusion protein and C3 ligand
[0045] Kinetic analysis of the interaction between CR2 fusion protein and biotin-labeled C3dg (C3dg-biotin) was detected by surface plasmon resonance (SPR) detection system (BIAcore3000 instrument). Human C3dg-biotin (Guthridge, J.M., et al. Structural studies solution of the recombinant N-terminal pair of short consensus / complement repeat domains of complement receptor type 2 (CR2 / CD21) and interactions with its ligand C3dg.Biochemistry.2001,40(20):5931–5941.) were injected onto the BIAcore streptavidin sensor chip at a speed of 2μL / min for 20min, and the buffer was 0.5× PBS (pH7.4) (containing 0.5g / L Tween20). SPR signals acquired from captured C3dg yielded BIAcore response units (range 250 to 500). The group without fusion protein was used as the control. After washing with 0.5×PBST (0.5g / LTween20) at a flow rate of 25μL / min at 25°C, the affinity of the CR2 fusion protein was ev...
Embodiment 3
[0049] Embodiment 3. Complement lysis experiment
[0050] To measure the inhibitory activity on complement, 60%-80% confluent CHO cells were separated with ethylenediaminetetraacetic acid, washed twice with DMEM, and then resuspended in DMEM to make the final concentration of 10 6 cells / mL. Add 100mL / L rabbit anti-CHO cell membrane antiserum to the cell suspension and react at 4°C for 30min to sensitize the cells. Then the antiserum was discarded, and the cells were resuspended in NHS diluted with DMEM to a final volume of 50 μL or 100 μL. After 60 minutes at 37°C, the cell viability was measured by placenta blue staining and exclusion method (both live and dead cells were counted). The recombinant fusion protein was diluted with DEME and added to NHS first, and then added to CHO cell suspension. The final concentration was based on the control CHO cells lysed by 100 g / L NHS which can cause about 90% antibody sensitization. Complement-mediated inhibition of erythrocyte lys...
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