Human target complement inhibitor protein double mutant mcr2-mdaf and its application
A technology of inhibitors and complement receptors, applied in the field of fusion proteins, which can solve problems such as slow process
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Embodiment 1
[0037] Preparation of embodiment 1.mCR2-mDAF, mCR2-DAF, CR2-DAF fusion protein
[0038] 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.
[0039] 2. Method
[0040] 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.
[0041] 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 of DAF. The mutation sites of mCR2 mutant include: ...
Embodiment 2
[0048] Example 2. Kinetic analysis of interaction between CR2 fusion protein and C3 ligand
[0049] 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
[0053] Embodiment 3. Complement lysis experiment
[0054] 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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