CN110330561A - Source of people targeted complement inhibitor albumen double-mutant mCR2-mDAF and application - Google Patents

Abstract

The invention discloses a kind of complement inhibitor DAF variants, are preparing the application in Parmaceutical for treating disease of autoimmunization system object with the fusion protein of complement receptor 2 variant and the fusion protein.The DAF variant is the molecule modifier obtained after computer modeling, amino acid replacement, the fusion protein of DAF variant and complement receptor 2 variant has higher ligand binding and dissociation rate than its wild type sequence fusion protein, has better ligand binding affinity.Biodistribution is it is demonstrated experimentally that fusion protein provided by the invention enters after rheumatoid arthritis mouse model to have quickly in arthritis position high aggregation and significant anti-stick/anti-inflammatory targeted inhibition effect.In the treatment to MRL/lpr lupus erythematosus mouse, the fusion protein can be obviously improved the survival rate of mouse, the symptoms such as albuminuria, Glomerular score, interstitial inflammation, vasculitis and the crescent/necrosis for the treatment of group mouse be improved significantly.

Description

Source of people targeted complement inhibitor albumen double-mutant mCR2-mDAF and application

Technical field

The invention discloses a kind of fusion proteins, belong to technical field of polypeptide.

Background technique

Complement system is a part of natural immune system, constituent by more than 30 soluble protein molecular compositions Including 30 different kinds of molecules such as complement proper constituent, a variety of regulatory factors and complement receptors.Complement system can be both relatively only by 3 The vertical approach connected each other again is activated, and exempts to play opsonophagocytosis, lytic cell, transmitting inflammation, immunological regulation and removing The various biologicals effect such as epidemic disease compound.Complement activation and its deposition on target structure can also cause cell or group indirectly Knit destruction.Each point in complement pathway generates the complement activation products of mediating tissue damage.It is unsuitable on host tissue Complement activation plays an important role in the pathology of many autoimmunity diseases and inflammatory disease, and is also to cause and such as cardiopulmonary The reason of bio-incompatibility after inflammation and graft rejection related many symptom.Complement inhibition is that these are immune-mediated for treatment Disease and symptom potential treatment mode.

The approach of complement activation has 3, i.e. classical pathway, alternative pathway and mannan binding lectin approach.It participates in mending The ingredient of body Classical pathway includes C1-C9.By its effect in activation, artificially it is divided into three groups, i.e. identification is single Position (Clq, Clr, Cls), activation unit (C4, C2, C3) and film challenging unit (C5-C9) are in the different phase of activation respectively It plays a role in cognitive phase, activation stage and film phase of the attack.This 3 stages are generally in 3 different parts of target cell membrane It carries out.Complement C2, C3, C4, C5 in activation are cracked into 2 or 2 or more segments respectively, are marked with a, b etc. respectively Symbol, such as C3a, C3b, C3c.Wherein C2b, C3b, C4b, C5b are directly or indirectly combined on target cell, in the form of solid phase Molten cell processes are participated in, C3a, C5a are free in liquid phase.Complement may also aggregate into after C5, C6, C7 are activated in activation C567, and special biological function is played together with C3a, C5a.Alternative activation pathway and Classical pathway difference exist It is to have crossed tri- kinds of ingredients of C1, C4, C2 in activation, directly C3 is activated then to complete the chain reaction of each ingredient of C5 to C9, also existed In activated material be not antigen antibody complex but the cell wall constituent-lipopolysaccharides and polysaccharide of bacterium, peptide glycan, phosphorus wall The substances such as acid and the IgA and IgG4 of cohesion.Alternative activation pathway is in bacterial infection early stage, when not yet generating specific antibody, Important anti-infectious function can be played.Mannan binding lectin approach is by mannan-binding lectin in blood plasma The N- amine-galactose or mannose on the Direct Recognition multiple pathogenic microorganisms surface (mannan-binding lectin, MBL), And then successively activate MASP-1, MASP-2, C4, C2, C3, it is formed and the identical C3 of classical pathway and C5 convertase, activating complement Cascade the activated channel of enzymatic reaction.The primary activation object of MBL activated pathway is that mannose group, fucose and N- ammonia are contained in surface The pathogenic microorganism of base galactolipin.Three of the above approach can generate C3 convertase, and C3 molecule is cracked into allergy by C3 convertase Toxin C3a and C3b with opsonic action, C3b molecule can be covalently attached with the amido and hydroxyl on glycoprotein surface, this total Valence effect is mediated by the intramolecular thioester substrate of C3b.Thus, C3b molecule is adsorbable in the intracorporal antimicrobial surface of intrusion, then It is combined with complement receptor 1 (CR1/CD35), iC3b is hydrolyzed to form under the action of the serum H factor and i factor, iC3b is then split Solution is C3d.C3d segment is the minimal segment that cannot be digested again of Complement C_3.It can be with II type complement in conjunction with the microorganism of C3d molecule Receptor (CR2/CD21) combines.

Decay accelerating factor (DAF) i.e. CD55 was separated in 1969 from erythrocyte surface by Hoffman, and by conduct A kind of complement inhibitor becomes the object of scientist's concern.In the classical pathway and alternative pathway activation of complement, C3 is risen Key effect.The activation of C3 needs C3 convertase, and DAF can be competed with C4b in conjunction with C2, can also Competitive assays Factor B and C3b is combined, to prevent the assembling of classical pathway and alternative pathway C3 convertase and accelerate the degradation of C3b and C4b, DAF can also The decay for accelerating C5 convertase, prevents the assembly of C5 convertase.The study found that DAF is the V-type transmembrane protein that GPI anchors, contain 381 amino acid, molecular weight change between 55KD-75KD because of glycosylated difference.35-285 amino acids are 4 spherical The area SCR (short consensus repeat region), is successively rodlike a S/T enrichment region and GPI anchor thereafter.Often A functional areas SCR are made of 60 amino acid.SCR2, SCR3 can convert enzyme effect with two activated pathway of complement, and SCR4 is only Enzyme effect is converted with alternative pathway.3 positively charged lysines (KKK) between SCR2 and SCR3 and positioned at SCR3's Two hydrophobic amino acid-leucines and phenylalanine (LF) are the action sites of C3 convertase, and wherein KKK acts on alternative pathway It is better than classical pathway, and LF has very strong inhibiting effect to two approach.

It is opsonic that the various C3 activation segment that complement activation generates as the ligand of various C3 receptors plays the role of complement. Complement receptor 2 (CR2) is one such complement receptors, and as a kind of transmembrane protein, CR2 passes through in dendritic cells,follicular (FDC), B cell and expression in some T cells and and immune complex combination for above-mentioned cell especially mature B cell Survival and the selection of high-affinity B cell play an important role.CR2 is the member of C3 binding protein family, and by 15-16 A short shared repetition (SCR) structural domain (the characteristic structural unit of C3 binding protein family) composition, C3 binding site are included in 2 In a N-terminal SCR.Different from complement activation inhibitor (DAF, MCP, CR1 and Crry), CR2 is not complement inhibitor, and its Do not combine C3b.The native ligand of CR2 is iC3b, C3dg and C3d, is the C3b in conjunction with the two N-terminal SCR structure domains CR2 Cell combination crack fragment.The generation and its deposition on active cell surface that the cracking of C3 initially causes C3b.C3b segment The generation of multienzyme complex is participated in, which expands complement cascade.On cell surface, especially when containing complement activation When depositing C3b on the host surface (that is, most of host tissues) of regulator, C3b is rapidly converted into the iC3b of inactivation.Even if In the case where the complement regulator combined there is no film, quite high-caliber iC3b is also formed.Then, iC3b is by haemocyanin enzyme Digestion is film binding fragment C3dg and C3d, but this process is relatively slow.Therefore, the C3 activation segment ligand of CR2 once generates Afterwards with regard to relative longevity, and complement activation site is present in high concentration.

The downward of complement activation has been proved for treating several disease indications in animal model and in vitro study It is effective, such as systemic loupus erythematosus and glomerulonephritis, rheumatoid arthritis, cardiovascular shunt and haemodialysis, device Hyperacute rejection, myocardial infarction, reperfusion injury and adult respiratory distress syndrome in official's transplanting.In addition, other inflammation Situation and autoimmunity disease/immune complex disease are also closely related with complement activation, including thermal damage, severe asthma, anaphylaxis Shock, enteritis, rubeola, angioedema, vasculitis, multiple sclerosis, myasthenia gravis, membranoprolifer ative glomerulonephritis and Dry syndrome.

The activation of complement inhibitor targeting complement and disease site are possibly improved to their effect.Because complement is anti-in host It plays an important role in imperial and immune complex catabolism, so the complement inhibitor of orientation can also be reduced especially for a long time The potentially serious side effect of Complement inhibition bring.Recently, it is prepared for being decorated with the modification shape of sialic acid-Lewis x (sLex) The sCR1 of formula, and show that it combines the endothelial cell of expression P and E selection albumen.In the rodent models of inflammatory disease In, it was demonstrated that sCR1sLex is therapeutic agent more more effective than sCR1.In vitro in feasibility study, it was demonstrated that do not target cell with protection It compares, antibody-DAF and antibody-CD59 fusion protein more effectively protect the cell of targeting from the destruction of complement.Pass through coupling Inhibitor and film are inserted into peptide, also have been obtained for the non-specific film targeting of recombination complement inhibitor.

Complement receptor 2 with selectively targeted effect but without Complement inhibition effect is merged with complement inhibitor, It is a kind of new treatment thoughts to play specific inhibitory effect to complement activation.CN101563363B and CN100594037C Complement receptor 2 is reported respectively to be merged with the complement inhibitor F factor, DAF and CD59 for inhibiting the beneficial of different activities Effect.The present invention is quasi- to be carried out further molecule to DAF and complement receptor 2 and changes structure by computer modeling, amino acid replacement, To optimize the space structure of DAF and complement receptor 2 fusion protein, not only improves complement receptor 2 and the targeting of its ligand binding is competing Striving property, it is also desirable to which complement inhibitor is improved to the depression effect of complement activation by the structure that changes of DAF.

Summary of the invention

It is described present invention firstly provides a kind of complement inhibitor DAF variant (mDAF) based on foregoing invention purpose The amino acid sequence of DAF variant is as shown in SEQ ID NO.1.

Secondly, the present invention also provides the polynucleotide molecule for encoding above-mentioned DAF variant, the polynucleotide molecule Sequence is as shown in SEQ ID NO.2.

Third, the present invention provides the fusion protein for containing above-mentioned DAF variant, the fusion protein, which also contains targeting, to be mended The complement targeted molecular in body activation site.

In a preferred technical solution, the complement targeted molecular is complement receptor 2 (CR2).

In one more preferably technical solution, the amino acid sequence of the complement receptor 2 such as SEQ ID NO.3 institute Show, which is the CR2 variant (m CR2) that sequence morphs.

It is further preferable that the complement receptor 2 variant is connect with DAF variant with flexible small peptide GGGSGGGGS.

Particularly preferably, the amino acid sequence of the fusion protein is as shown in SEQ ID NO.5, and in the fusion protein, institute The aminoterminal that complement receptor 2 variant is located at fusion protein is stated, DAF variant is located at the c-terminus of fusion protein.

4th, the present invention provides a kind of polynucleotide molecule for encoding above-mentioned fusion protein, the polynucleotide molecules Sequence as shown in SEQ ID NO.6.

Finally, the present invention provides above-mentioned fusion proteins to prepare the application in Parmaceutical for treating disease of autoimmunization system object.

In a preferred technical solution, the disease includes rheumatoid arthritis and systemic loupus erythematosus.

DAF mutant and complement receptor 2 variant disclosed by the invention are obtained after computer modeling, amino acid replacement The molecule modifier obtained, the fusion protein mCR2-mDAF of the two have higher match than the fusion protein CR2-DAF of its wild type sequence Body combines and dissociation rate, has better ligand binding affinity.The Chinese hamster ovary celI and red blood cell dissolution experiment result of complement-mediated are aobvious Show that the inhibitory effect of mCR2-mDAF ratio CR2-DAF becomes apparent.Inhibit 50%CHO cell dissolution, the concentration of mCR2-mDAF is 10nmol/L, and the concentration of CR2-DAF is 91nmol/L, and efficiency is inhibited to improve 9 times.Biodistribution is it is demonstrated experimentally that this hair The fusion protein of bright offer can be quickly in arthritis position high aggregation after entering rheumatoid arthritis mouse model, it was demonstrated that this hair Bright mCR2-mDAF has special targeting to C3d, and mCR2-mDAF ratio CR2-DAF has better improvement degree, and has Apparent dose-dependence, it was demonstrated that mCR2-mDAF have it is excellent it is anti-stick/anti-inflammatory targeted inhibition effect, to the inflammation of body Disease reaction has good therapeutic effect.MCR2-mDAF disclosed by the invention in the treatment of MRL/lpr lupus erythematosus mouse, It can be obviously improved the survival rate of mouse, entire therapeutic process can protect MRL/lpr lupus erythematosus mouse, mCR2- completely MDAF treatment group survival rate is 100%.And albuminuria, Glomerular score, interstitial inflammation, the blood vessel of mCR2-mDAF treatment group The symptoms such as scorching and crescent/necrosis be improved significantly, show mCR2-mDAF provided by the invention in preparation autoimmune disease There is excellent application prospect in sick therapeutic agent.

Detailed description of the invention

Fig. 1 .mCR2-mDAF sequence variations schematic diagram；

Fig. 2 .CR2-DAF sequence diagram；

The 12%SDS-PAGE qualification result of Fig. 3 .mCR2-mDAF；

The Western Blot qualification result of Fig. 4 .mCR2-mDAF；

Biodistribution experimental result of Fig. 5 .mCR2-mDAF in RA mouse；

Fig. 6 .mCR2-mDAF treats RA mouse test results；

The survival rate comparative diagram of Fig. 7 .mCR2-mDAF treatment MRL/lpr mouse；

The albuminuria that Fig. 8 .mCR2-mDAF treats MRL/lpr mouse changes comparative diagram.

Specific embodiment

The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and It is apparent.But examples are merely exemplary for these, does not constitute any restrictions to protection scope of the present invention.

Computer modeling

One, CR2 combined scheme

Using the Builder program in II software package of Insight, small peptide is shown in the method from the beginning built.Utilize molecule The method of docking (docking) is formed by the structure of compound after simulating CR2 in conjunction with C3d, through molecular mechanics and Molecule Motion Mechanics optimization obtains the Stable conformation of CR2/C3d compound.The binding site of the two is analyzed, determines that CR2 and C3d is tied The core sequence of conjunction, and obtain the mode to interact between amino acid when the two combines and energy.Based on the analysis results, it replaces Certain amino acid of CR2, mould is built again, obtains the Stable conformation of CR2 mutant peptide-C3d compound.Analyze CR2 mutant peptide and C3d Between the mode that interacts and energy, affinity size when comparing CR2 mutation front and back in conjunction with C3d.

The simulation of CR2 Yu C3d compound tentatively have been carried out, and has devised following mutant, has improved its knot with C3d It closes:

1,Thr86-----Ser；2,Phe130-----Tyr；3,Glu133-----Gln；4,Asp92-----Glu；5, Thr25-----Ser (CR2 mutation after amino acid sequence as shown in SEQ ID NO.3, polynucleotide sequence such as SEQ ID Shown in NO.4, mutational site is as shown in Figure 1, the CR2 wild type sequence before mutation is as shown in Figure 2).

Two, DAF combined scheme

On the basis of CR2 combined scheme, based on the considerations of mCR2-mDAF fusion protein entirety conformation, guarantee DAF with Under the premise of its action site (C3b and C4b) is specifically bound, the molecular mechanics and molecular dynamics combined to it optimizes, right The amino acid of the binding sequence two sides of core is mutated, in a manner of obtaining and have more excellent interaction and bigger affinity Mutant, the mutational site of the DAF mutant finally obtained are as follows:

1.Thr 68-----Ser；2.Asn71-----Ser；3.Asn 220-----Asp；4.Asn 236-----Ser (for the amino acid sequence after DAF mutation as shown in SEQ ID NO.1, polynucleotide sequence is mutated position as shown in SEQ ID NO.2 Point is as shown in Figure 1, the CR2 wild type sequence before mutation is as shown in Figure 2).

The preparation of embodiment 1.mCR2-mDAF, mCR2-DAF, CR2-DAF fusion protein

1. material expression carrier selects pEE14.1 (Lonza biologics)；Chinese hamster ovary celI is used for protein expression, culture Liquid is the DMEM containing 10% fetal calf serum, is purchased from Invitrogen company.The anti-DAF mAb 1H4 and 1A10 of mouse, mouse are anti-human CR2mAb 171, anti-sheep red blood cell IgM and all secondary antibodies are purchased from Sigma company.

2. method

The sero-fast preparation of 2.1 rabbit-anti Chinese hamster ovary celI films and people DAF according to document Harlow, E., and Lane, D.Antibodies:a laboratory manual.Cold Spring Harbor Laboratory.Cold Spring Harbor, New York, the method that USA.1988:726. is introduced obtain.

The buildings of 2.2 expression recombinants and protein expression cDNA structural gene by encode 4 end N- SCR units of CR2 with The sequence of encoding D AF extracellular region is connected.The mutational site of mCR2 mutant includes: 1, Thr86-----Ser；2, Phe130-----Tyr；3,Glu133-----Gln；4,Asp92-----Glu；5,Thr25-----Ser.Complement inhibitor DAF Mutational site includes: 1.Thr 68-----Ser；2.Asn71-----Ser；3.Asn 220-----Asp；4.Asn 236-----Ser.CR2 with DAF wild type sequence before mutation is as shown in Fig. 2, the two is connected with soft small peptide GGGSGGGGS).

Then mCR2, CR2 are connect with complement inhibitor mDAF or DAF respectively, mCR2, CR2 (mCR2- at the end N- MDAF, mCR2-DAF, CR2-DAF) catenation sequence be GGGSGGGGS.Gene frame round pcr synthesizes (mCR2-mDAF fusion The amino acid sequence of albumen is as shown in SEQ ID NO.5, and polynucleotide sequence is as shown in SEQ ID NO.6).All cloning process (referring to CN104940953A) is carried out on pEE14.1 carrier.PEE14.1 efficient expression vector contains special glutamine (GS) gene expression system.GS enzyme is responsible for that glutamy and amine is used to synthesize glutamine as substrate.Some mammal cell lines Without GS gene, so cannot survive in the environment of not adding glutamine.For these cell lines, the GS gene of transfection It can be used as a kind of selection markers, the cell grown in no Glutamin medium can be filtered out.Methionine imines (Methionine sulphoximine, MSX) can inhibit the activity of GS enzyme, and certain cell lines (CHO-K1) can generate endogenous Glutamine, can containing MSX without being grown in Glutamin medium, it is this can inhibit GS enzymatic activity added with enough MSX Culture medium provide screening pressure, to filter out required cell strain.MCR2-mDAF, mCR2-DAF, CR2-DAF fusion Gene connects after EcoRI, SmalI double digestion after PCR method synthesizes with pEE14.1 carrier, recombinant vector PEE14.1-mCR2-mDAF and pEE14.1-mCR2-DAF, pEE14.1-CR2-DAF transfection CHO-K1 cell of FuGENE 6, After transfection 24 hours, selective d MEM culture medium (without glutamine) is changed, and be added MSX to final concentration of 50 μM, about 2 perimeters Positive colony out, the normal Chinese hamster ovary celI without transfected plasmids gradually fall off from bottle wall, and cell cracking is dead.It is 3 plants of selection thin Born of the same parents collect cell conditioned medium respectively with having blood serum medium and serum free medium culture 3 days, are partially concentrated 5 times through PEG20000 It is spare, remaining -80 DEG C preservations.With the expression (mCR2-mDAF of ELISA method detection mCR2-mDAF, mCR2-DAF, CR2-DAF For amino acid sequence as shown in SEQ ID NO.5 and shown in Fig. 1, CR2-DAF amino acid sequence is as shown in Figure 2).Recombinant protein is to divide Form is secreted to express in Chinese hamster ovary celI.It is higher than using the expression quantity of the Chinese hamster ovary celI stably expressing cell line of serum free medium culture and is used The cell of culture medium culture containing serum.

The purifying of 2.3 recombinant proteins purifies the albumen in cell culture supernatant using affinity chromatography.According to Operation manual, chromatographic column is by forming the affine column coupling for resisting DAF mAb (1H4) and HiTrap NHS to activate.Recombination will be contained The pH value of albumen culture supernatant is adjusted to 8.0, crosses column with the rate of 0.5mL/min.Then pillar is washed with 6~8 times of volume PBS, weight Histone is with 0.1mol/L glycine (pH2.4) elution of 2~3 times of column volumes.Fusion protein is collected into 1mol/L Tris to delay In fliud flushing (pH8.0), and dialyse in PBS.

2.4SDS-PAGE and Western blot identification

The albumen of purifying carries out in the SDS-PAGE glue containing 100g/L.Gel coomassie brilliant blue staining.Western Recombinant protein is detected with mouse anti-DAF mAb 1H4 in blot experiment.

3. result

Recombinant protein (mCR2-mDAF, mCR2-DAF, CR2- are separated to from the Chinese hamster ovary celI culture supernatant of stable transfection DAF) there is the target patch of expected size as the result is shown in 100~200 μ g/L, SDS-PAGE (Fig. 3) and Western blot (Fig. 4) Section.

Embodiment 2.CR2 fusion protein and C3 aglucon interaction dynamics are analyzed

The dynamic analysis surface of the C3dg (C3dg- biotin) of CR2 fusion protein and biotin labeling interaction Plasmon resonance (SPR) detection system is detected (BIAcore3000 instrument).It is average according to each flow cell The amount of 50mg/L, by people C3dg- biotin (Guthridge, J.M., et al.Structural studies in 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): it 5931-5941.) is arrived with the speed injection of 2 μ L/min BIAcore streptavidin sensor core on piece acts on 20min, and buffer is that 0.5 × PBS (pH7.4) (contains 0.5g/L Tween20).The spr signal that is obtained from the C3dg of capture generate BIAcore reacton (range be 250 to 500).Fusion protein group is not added as control.25 DEG C, with the flow velocity of 25 μ L/min, with 0.5 × PBST (0.5g/L Tween20 after) washing, its affinity is assessed by detection CR2 fusion protein concentration (15.6~500nmol/L).

Dynamic analysis data show that be most suitable for engagement reaction model with spherical detection parameters at 1: 1.SPR testing result is aobvious Show, mCR2-mDAF ratio mCR2-DAF has higher combination and dissociation rate (table 1).And the affinity ratio CR2-DAF of mCR2-DAF It is high.

Kinetic parameter of 1 recombination fusion protein of table in conjunction with C3dg- biotin

The experiment of 3. complement lysis of embodiment

To measure the inhibitory activity to complement, the Chinese hamster ovary celI of 60%~80% fusion is separated with ethylenediamine tetra-acetic acid, is used DMEM is washed 2 times, is then resuspended in DMEM, make its final concentration of 10⁶A cell/mL.100mL/L rabbit is added in cell suspension Anti- Chinese hamster ovary celI film antiserum, 4 DEG C of effect 30min, makes cell sensitization.Then antiserum is abandoned, cell is resuspended in diluted with DMEM In NHS, final volume is 50 μ L or 100 μ L.37 DEG C of effect 60min finally measure Cells viability with placenta orchid dyeing exclusive method (living cells and dead cell count).Recombination fusion protein is charged first in NHS after being diluted with DEME, is added to Chinese hamster ovary celI Suspension.Final concentration be subject to 100g/L NHS can lead to about 90% antibody sensitized control Chinese hamster ovary celI dissolution.Complement-mediated The sheep red blood cell (EAs) of red blood cell dissolution inhibition experiment antibody sensitized is measured.Hemolytic test is buffered in gelatin veronal Liquid (GVB⁺⁺) in carry out, final volume be 300 μ L, include 2.5 × 10⁷EAs, NHS are according to 1: 300 dilution.Reaction mixture exists 37 DEG C of incubation 60min are eventually adding the 300 μ L solution of EDTA-PBS containing 10mmol/L and terminate reaction.Centrifugation, takes supernatant, Quantitative detection is carried out to the ferroheme in supernatant with optical spectrum imagers under 413nm wavelength.

Fusion protein complement inhibitor Activity determination: the Chinese hamster ovary celI and red blood cell dissolution experiment of complement-mediated the results show that CR2-DAF is more obvious than non-targeted sDAF inhibitory effect, and the inhibitory effect of mCR2-DAF ratio CR2-DAF becomes apparent, mCR2- The inhibitory effect highest of mDAF.Inhibit 50%CHO cell dissolution, the concentration of mCR2-mDAF is 10nmol/L, and mCR2-DAF's is dense Degree is 15nmol/L, and the concentration of CR2-DAF is 91nmol/L, and non-targeted sDAF needs 415nmol/L (table 2).In addition, in cell In dissolution inhibition experiment, sDAF is stronger than Chinese hamster ovary celI to the protective effect of red blood cell.

The complement inhibitor concentration that table 2. can inhibit 50% cell to dissolve

Embodiment 4.mCR2-mDAF fusion protein RA experiment in vivo:

1. biodistribution is tested

MCR2-mDAF, CR2-DAF and DAF are marked respectively using Iodogen method¹²⁵I is being coated with 200 μ g In the EP pipe of Iodogen, the ScFv of 150 μ l of 50mmLo/L PBS (pH7.4), BSA containing the 1mg dissolution of existing preparation is sequentially added 100μl(100μg)、Na¹²⁵15 μ l (185MBq) of I solution is interrupted gentle agitation at room temperature and marks pipe 15min.SEP-PAK C18 Column uses methanol, distilled water and 0.1% trifluoroacetic acid (TFA) each 5mL successively to wash respectively makes its activation；Mixture upper prop is marked, 0.1%TFA elution；60% acetonitrile solution elution, 1.5mL eluent before collecting.After freeze-dried, with BSA containing 1mg/mL PBS solution dilution, packing, -80 DEG C of freezer storages are spare.0.1mL collagen II is subcutaneously injected in male DBA/1J mouse tail root With complete Freund's adjuvant (being purchased from Sigma Co., USA), reinforce within the 21st day once establishing rheumatoid arthritis (CIA) mouse mould (model foundation is referring to the foundation of C57BL/6 mouse CIA model and its preliminary screening of monitoring system, liberation army medical journal for type In June, 2004 the 6th phase 472-474 of volume 29).Test and group technology are as follows: 1. control group tail vein injection¹²⁵I--DAF (2ug), the respectively sacrificed by decapitation for 24 hours, after 48h collect blood, take out histoorgan, weigh and measure its radioactivity (μ Ci), As a result it is scaled ID%/g tissue.2. arthritis group tail vein injection¹²⁵I--DAF fusion protein (2ug), respectively for 24 hours, 48h, After 72h, 96h, takes and ibid handle, detect.3. mCR2-mDAF arthritis treatment group, tail vein injection are primary¹²⁵I-mCR2- MDAF fusion protein (0.25ug) is taken afterwards for 24 hours and ibid handles, detects.4. CR2-DAF arthritis treatment group, tail vein injection Once¹²⁵I-CR2-DAF fusion protein (0.25ug) is taken afterwards for 24 hours and ibid handles, detects.As a result as shown in figure 5, showing this hair Bright mCR2-mDAF fusion protein can be in the arthritis position high aggregation (legend of the sequence from top to bottom of marginal data in Fig. 5 It is corresponded with the sub- column figure of the sequence from left to right in grouping column figure each in Fig. 5).

The treatment of rheumatoid arthritis 2. (CIA) mouse model

Using rheumatoid arthritis (CIA) mouse model (ibid), is tested since the 21st day, be grouped as follows: 1. injecting 50 μ l PBS control groups (N=15).2. CR2-DAF low dose therapy group (N=13), CR2-DAF of injection merges egg daily White (0.25) mg.3. CR2-DAF high-dose therapy group (N=14) injects CR2-DAF fusion protein (0.25) mg twice daily. 4. mCR2-mDAF low dose therapy group (N=14) injects mCR2-mDAF fusion protein (0.25) mg daily.⑤mCR2- MDAF high-dose therapy group (N=16) injects mCR2-mDAF fusion protein (0.25) mg twice daily.Beginning in 23rd day is clinical Scoring scores to joint of animal inflammation degree by following standard: 0 point, no arthritis；1 point, light inflammation and claw are rubescent；2 points, Severe erythema and swelling influence claw function；It is 3 points, claw or dysarthrasis, stiff, lose function.Every mouse four limbs highest Total score is 12 points.As a result as shown in fig. 6, since the 23rd day, the arthritis of mCR2-mDAF fusion protein Liang Ge treatment group is serious Degree is significantly lower than PBS group.Wherein the scoring of the 5. group (2 injection groups) be 4. group (1 injection group) 1/2 hereinafter, being 3. group (CR2-DAF double injection group) 1/4 hereinafter, be 4. group (CR2-DAF one injection group) 1/5 hereinafter, being 1. the 1/7 or less of group (PBS control group).

Above the results show mCR2-mDAF fusion protein of the invention has special targeting to C3d, has more Resist well and stick/anti-inflammatory targeted inhibition effect, there is good therapeutic effect to the inflammatory reaction of body, be significantly higher than CR2- The therapeutic effect of DAF.

Therapeutic effect of the embodiment 5.mCR2-mDAF and CR2-DAF in MRL/lpr lupus erythematosus mouse

1. the raising of survival rate

MRL/lpr mouse from 16 weeks to 24 week mCR2-mDAF treatment group (n=26) than CR2-DAF treatment group (n=26) and The comparison of PBS control group (n=24) mouse survival rate

MRL/lpr lupus erythematosus mouse model is that Murphy and Roths were established in 1979 earliest, by multiple Strains of Mouse It is formed by the complicated hybrid process after 12 generations, the 75% of the murine genes of the model comes from from LG/J, 12.6% AKR/J, 12.1% come from C57BL/6 Strains of Mouse from C3H/Di and 0.3%.MRL/lpr mouse contains and cell spontaneity , there is lymphocytosis gene, leads to T cell hyperplasia, lymph nodes of body as a whole in the related Fas gene recessive mutation of programmed death Enlargement and erosive arthritis, anti-DNA, anti-Sm, anti-Su, anti-nucleosides P antibody, high titre ANA, hypergammaglobulinemia And rheumatoid factor.The mouse was fallen ill earliest in 8 weeks, and autoantibody can be detected in serum at this time.It is observed that at 12 weeks Lymphnoditis.12-16 weeks, MRL/lpr mouse started to generate a large amount of autoantibodies including anti-dsDNA antibody.Nearly 16 weeks Multiple organ is involved when age, and the stabilization renal function occurred characterized by serious albuminuria is degenerated.16-24 week old mouse is proliferated Property the glomerulonephritis that mediates of immune complex, vasculitis, and eventually lead to renal failure and dead, the death rate can achieve 50%.

16 weeks MRL/lpr mouse for having already appeared renal failure symptom are randomly divided into three groups, first group (n=26) by the present embodiment Receive 0.2mg mCR2-mDAF weekly from the 16-24 weeks, second group (n=26) receives weekly 0.2mg from the 16-24 weeks CR2-DAF, third group (n=24) are control group, receive the PBS of equivalent weekly from the 16-24 weeks.Three groups of administration route is equal For tail vein injection.MCR2-mDAF is evaluated according to administration group and the survival rate of control group, CR2-DAF is to MRL/lpr lupus erythematosus The protective rate of mouse.

Experimental result is as shown in fig. 7, receive the mouse of mCR2-mDAF, CR2-DAF treatment, due in complement activation pathway C3d be targeted the CR2-DAF of C3d and effectively inhibit, therefore, the survival rate of MRL/lpr lupus erythematosus mouse significantly improves, The entire therapeutic process of mCR2-mDAF treatment group can protect MRL/lpr lupus erythematosus mouse, survival rate 100%, CR2- completely DAF group is able to maintain at the 24th week to be compared in 70% or more survival rate with control group, from the mouse of 19 Zhou Qi treatment groups Survival rate significantly improves.

2. renal function improvement

Mouse is placed in metabolic cage and studies mCR2-mDAF, CR2-DAF is to MRL/lpr lupus erythematosus mouse urinary albumin The influence of secretion.Collect the twenty-four-hour urine liquid of a mouse every two weeks since 16 weeks.To prevent bacterial growth, in collecting pipe Ampicillin, gentamicin and chloramphenicol is added.It is drawn using the Mouse albumin sample of known concentration by ELISA method Standard curve, and determine the urinary albumin excretion situation of experiment mice, and contain using the creatinine in biochemical instruments measurement mouse urine Amount.Last evaluation result is indicated with 24 hours urinary albumins of every experiment mice (mg) and creatinine (mg) ratio.Urinate white egg The white higher expression renal function of creatinine ratio is damaged.As shown in figure 8, MRL/lpr mouse mCR2-mDAF treatment group (n= 20) than CR2-DAF treatment group (n=20) and the comparison of PBS control group (n=20) albuminuria situation at the 22nd week and 24 weeks, with Control group is compared, and the Proteinuria level for the treatment of group is substantially reduced (P < 0.01), and mCR2-mDAF treatment group (n=20) compares CR2-DAF Treatment group (n=20) Proteinuria level more reduces (P < 0.01).Prove that mCR2-DAF provided by the invention more can significantly change Kind renal impairment symptom.

3. the inflammatory reaction of kidney mitigates

After experiment, longitudinally dissection is two halves to excision mouse kidney, and wherein half carries out immunofluorescence analysis, the other half 10% neutral formalin is fixed, solid paraffin embedded section, with hematoxylin eosin staining method and periodic acid Schiff stain method to paraffin The renal tissue slice of processing is dyed, using blind respectively to being sliced the glomerulus inflammation observed, hyperplasia, crescent certainly It is formed, downright bad symptom scores, while also scoring the variation of renal interstitial.It scores and is divided into 0,1,2,3,4 Pyatyis, 0 To be not damaged, 4 be serious damage.Perivascular inflammatory exudation evaluation uses semiquantitative way, by two independent observers' blind The blood vessel for being sliced 10 or more to every is evaluated.Inflammation is scored at 0-3, and 0 is no inflammation, and 1 is the blood vessel lower than 50% It is surrounded by 3 confluent monolayer cells, 2, which are 3-6 layers for the blood vessel greater than 50%, surrounds, and 3 show for most serious, and the cell more than 6 layers is surround.It comments The results are shown in Table 3 for valence.

Table 3.MRL/lpr mouse week treatment Hou24Zhou treatment group and PBS control group kidney damage situation from 16 weeks to 23 Comparison

Grouping	Glomerular score	Interstitial inflammation	Vasculitis	Crescent/necrosis
					PBS control group (n=14)	12.8±4.2	3.6±0.6	100%	65%
CR2-DAF treatment group (n=16)	8.7±3.1	3.1±0.5	75%	50%
					MCR2-DAF treatment group (n=14)	5.4±2.6	2.0±0.4	60%	20%
MCR2-mDAF treatment group (n=16)	3.5±1.7	1.6±0.3	30%	10%

Compared with control group, mCR2-mDAF ratio CR2-DAF Glomerular score, interstitial inflammation, vasculitis and crescent/ The more obvious reduction (P < 0.05) such as necrosis.

Sequence table

<110>Beijing Kang Pumeite innovates medical sci-tech Co., Ltd

<120>source of people targeted complement inhibitor albumen double-mutant mCR2- mDAF and application

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 250

<212> PRT

<213> Homo sapiens

<400> 1

Asp Cys Gly Leu Pro Pro Asp Val Pro Asn Ala Gln Pro Ala Leu Glu

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Gly Arg Thr Ser Phe Pro Glu Asp Thr Val Ile Thr Tyr Lys Cys Glu

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Glu Ser Phe Val Lys Ile Pro Gly Glu Lys Asp Ser Val Ile Cys Leu

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Lys Gly Ser Gln Trp Ser Asp Ile Glu Glu Phe Cys Asn Arg Ser Cys

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Glu Val Pro Ser Arg Leu Ser Ser Ala Ser Leu Lys Gln Pro Tyr Ile

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Thr Gln Asn Tyr Phe Pro Val Gly Thr Val Val Glu Tyr Glu Cys Arg

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Pro Gly Tyr Arg Arg Glu Pro Ser Leu Ser Pro Lys Leu Thr Cys Leu

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Gln Asn Leu Lys Trp Ser Thr Ala Val Glu Phe Cys Lys Lys Lys Ser

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Cys Pro Asn Pro Gly Glu Ile Arg Asn Gly Gln Ile Asp Val Pro Gly

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Gly Ile Leu Phe Gly Ala Thr Ile Ser Phe Ser Cys Asn Thr Gly Tyr

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Lys Leu Phe Gly Ser Thr Ser Ser Phe Cys Leu Ile Ser Gly Ser Ser

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Val Gln Trp Ser Asp Pro Leu Pro Glu Cys Arg Glu Ile Tyr Cys Pro

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Ala Pro Pro Gln Ile Asp Asn Gly Ile Ile Gln Gly Glu Arg Asp His

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Tyr Gly Tyr Arg Gln Ser Val Thr Tyr Ala Cys Asp Lys Gly Phe Thr

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Met Ile Gly Glu His Ser Ile Tyr Cys Thr Val Ser Asn Asp Glu Gly

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Glu Trp Ser Gly Pro Pro Pro Glu Cys Arg

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<212> DNA

<213> Homo sapiens

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gactgtggcc ttcccccaga tgtacctaat gcccagccag ctttggaagg ccgtacaagt 60

tttcccgagg atactgtaat aacgtacaaa tgtgaagaaa gctttgtgaa aattcctggc 120

gagaaggact cagtgatctg ccttaagggc agtcaatggt cagatattga agagttctgc 180

aatcgtagct gcgaggtgcc aagcaggcta agttctgcat ccctcaaaca gccttatatc 240

actcagaatt attttccagt cggtactgtt gtggaatatg agtgccgtcc aggttacaga 300

agagaacctt ctctatcacc aaaactaact tgccttcaga atttaaaatg gtccacagca 360

gtcgaatttt gtaaaaagaa atcatgccct aatccgggag aaatacgaaa tggtcagatt 420

gatgtaccag gtggcatatt atttggtgca accatctcct tctcatgtaa cacagggtac 480

aaattatttg gctcgacttc tagtttttgt cttatttcag gcagctctgt ccagtggagt 540

gacccgttgc cagagtgcag agaaatttat tgtccagcac caccacaaat tgacaatgga 600

ataattcaag gggaacgtga ccattatgga tatagacagt ctgtaacgta tgcatgtgat 660

aaaggattca ccatgattgg agagcactct atttattgta ctgtgagtaa tgatgaagga 720

gagtggagtg gcccaccacc tgaatgcaga 750

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<213> Homo sapiens

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Ile Ser Cys Gly Ser Pro Pro Pro Ile Leu Asn Gly Arg Ile Ser Tyr

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Tyr Ser Thr Pro Ile Ala Val Gly Ser Val Ile Arg Tyr Ser Cys Ser

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Gly Thr Phe Arg Leu Ile Gly Glu Lys Ser Leu Leu Cys Ile Thr Lys

35 40 45

Asp Lys Val Asp Gly Thr Trp Asp Lys Pro Ala Pro Lys Cys Glu Tyr

50 55 60

Phe Asn Lys Tyr Ser Ser Cys Pro Glu Pro Ile Val Pro Gly Gly Tyr

65 70 75 80

Lys Ile Arg Gly Ser Ser Pro Tyr Arg His Gly Glu Ser Val Thr Phe

85 90 95

Ala Cys Lys Thr Asn Phe Ser Met Asn Gly Asn Lys Ser Val Trp Cys

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Gln Ala Asn Asn Met Trp Gly Pro Thr Arg Leu Pro Thr Cys Val Ser

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Val Tyr Pro Leu Gln Cys Pro Ala Leu Pro Met Ile His Asn Gly His

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His Thr Ser Glu Asn Val Gly Ser Ile Ala Pro Gly Leu Ser Val Thr

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Tyr Ser Cys Glu Ser Gly Tyr Leu Leu Val Gly Glu Lys Ile Ile Asn

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Cys Leu Ser Ser Gly Lys Trp Ser Ala Val Pro Pro Thr Cys Glu Glu

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Ala Arg Cys Lys Ser Leu Gly Arg Phe Pro Asn Gly Lys Val Lys Glu

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Pro Pro Ile Leu Arg Val Gly Val Thr Ala Asn Phe Phe Cys Asp Glu

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Gly Tyr Arg Leu Gln Gly Pro Pro Ser Ser Arg Cys Val Ile Ala Gly

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Gln Gly Val Ala Trp Thr Lys Met Pro Val Cys

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<213> Homo sapiens

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atttcttgtg gctctcctcc gcctatccta aatggccgga ttagttatta ttctaccccc 60

attgctgttg gttccgtgat aaggtacagt tgttcaggta ccttccgcct cattggagaa 120

aaaagtctat tatgcataac taaagacaaa gtggatggaa cctgggataa acctgctcct 180

aaatgtgaat atttcaataa atattcttct tgccctgagc ccatagtacc aggaggatac 240

aaaattagag gctcttcacc ctacagacat ggtgaatctg tgacatttgc ctgtaaaacc 300

aacttctcca tgaacggaaa caagtctgtt tggtgtcaag caaataatat gtgggggccg 360

acacgactac caacctgtgt aagtgtttac cctctccagt gtccagcact tcctatgatc 420

cacaatggac atcacacaag tgagaatgtt ggctccattg ctccaggatt gtctgtgact 480

tacagctgtg aatctggtta cttgcttgtt ggagaaaaga tcattaactg tttgtcttcg 540

ggaaaatgga gtgctgtccc ccccacatgt gaagaggcac gctgtaaatc tctaggacga 600

tttcccaatg ggaaggtaaa ggagcctcca attctccggg ttggtgtaac tgcaaacttt 660

ttctgtgatg aagggtatcg actgcaaggc ccaccttcta gtcggtgtgt aattgctgga 720

cagggagttg cttggaccaa aatgccagta tgt 753

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Ile Ser Cys Gly Ser Pro Pro Pro Ile Leu Asn Gly Arg Ile Ser Tyr

1 5 10 15

Tyr Ser Thr Pro Ile Ala Val Gly Ser Val Ile Arg Tyr Ser Cys Ser

20 25 30

Gly Thr Phe Arg Leu Ile Gly Glu Lys Ser Leu Leu Cys Ile Thr Lys

35 40 45

Asp Lys Val Asp Gly Thr Trp Asp Lys Pro Ala Pro Lys Cys Glu Tyr

50 55 60

Phe Asn Lys Tyr Ser Ser Cys Pro Glu Pro Ile Val Pro Gly Gly Tyr

65 70 75 80

Lys Ile Arg Gly Ser Ser Pro Tyr Arg His Gly Glu Ser Val Thr Phe

85 90 95

Ala Cys Lys Thr Asn Phe Ser Met Asn Gly Asn Lys Ser Val Trp Cys

100 105 110

Gln Ala Asn Asn Met Trp Gly Pro Thr Arg Leu Pro Thr Cys Val Ser

115 120 125

Val Tyr Pro Leu Gln Cys Pro Ala Leu Pro Met Ile His Asn Gly His

130 135 140

His Thr Ser Glu Asn Val Gly Ser Ile Ala Pro Gly Leu Ser Val Thr

145 150 155 160

Tyr Ser Cys Glu Ser Gly Tyr Leu Leu Val Gly Glu Lys Ile Ile Asn

165 170 175

Cys Leu Ser Ser Gly Lys Trp Ser Ala Val Pro Pro Thr Cys Glu Glu

180 185 190

Ala Arg Cys Lys Ser Leu Gly Arg Phe Pro Asn Gly Lys Val Lys Glu

195 200 205

Pro Pro Ile Leu Arg Val Gly Val Thr Ala Asn Phe Phe Cys Asp Glu

210 215 220

Gly Tyr Arg Leu Gln Gly Pro Pro Ser Ser Arg Cys Val Ile Ala Gly

225 230 235 240

Gln Gly Val Ala Trp Thr Lys Met Pro Val Cys Gly Gly Gly Ser Gly

245 250 255

Gly Gly Gly Ser Asp Cys Gly Leu Pro Pro Asp Val Pro Asn Ala Gln

260 265 270

Pro Ala Leu Glu Gly Arg Thr Ser Phe Pro Glu Asp Thr Val Ile Thr

275 280 285

Tyr Lys Cys Glu Glu Ser Phe Val Lys Ile Pro Gly Glu Lys Asp Ser

290 295 300

Val Ile Cys Leu Lys Gly Ser Gln Trp Ser Asp Ile Glu Glu Phe Cys

305 310 315 320

Asn Arg Ser Cys Glu Val Pro Ser Arg Leu Ser Ser Ala Ser Leu Lys

325 330 335

Gln Pro Tyr Ile Thr Gln Asn Tyr Phe Pro Val Gly Thr Val Val Glu

340 345 350

Tyr Glu Cys Arg Pro Gly Tyr Arg Arg Glu Pro Ser Leu Ser Pro Lys

355 360 365

Leu Thr Cys Leu Gln Asn Leu Lys Trp Ser Thr Ala Val Glu Phe Cys

370 375 380

Lys Lys Lys Ser Cys Pro Asn Pro Gly Glu Ile Arg Asn Gly Gln Ile

385 390 395 400

Asp Val Pro Gly Gly Ile Leu Phe Gly Ala Thr Ile Ser Phe Ser Cys

405 410 415

Asn Thr Gly Tyr Lys Leu Phe Gly Ser Thr Ser Ser Phe Cys Leu Ile

420 425 430

Ser Gly Ser Ser Val Gln Trp Ser Asp Pro Leu Pro Glu Cys Arg Glu

435 440 445

Ile Tyr Cys Pro Ala Pro Pro Gln Ile Asp Asn Gly Ile Ile Gln Gly

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Glu Arg Asp His Tyr Gly Tyr Arg Gln Ser Val Thr Tyr Ala Cys Asp

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Lys Gly Phe Thr Met Ile Gly Glu His Ser Ile Tyr Cys Thr Val Ser

485 490 495

Asn Asp Glu Gly Glu Trp Ser Gly Pro Pro Pro Glu Cys Arg

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<210> 6

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<212> DNA

<213> Homo sapiens

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atttcttgtg gctctcctcc gcctatccta aatggccgga ttagttatta ttctaccccc 60

attgctgttg gttccgtgat aaggtacagt tgttcaggta ccttccgcct cattggagaa 120

aaaagtctat tatgcataac taaagacaaa gtggatggaa cctgggataa acctgctcct 180

aaatgtgaat atttcaataa atattcttct tgccctgagc ccatagtacc aggaggatac 240

aaaattagag gctcttcacc ctacagacat ggtgaatctg tgacatttgc ctgtaaaacc 300

aacttctcca tgaacggaaa caagtctgtt tggtgtcaag caaataatat gtgggggccg 360

acacgactac caacctgtgt aagtgtttac cctctccagt gtccagcact tcctatgatc 420

cacaatggac atcacacaag tgagaatgtt ggctccattg ctccaggatt gtctgtgact 480

tacagctgtg aatctggtta cttgcttgtt ggagaaaaga tcattaactg tttgtcttcg 540

ggaaaatgga gtgctgtccc ccccacatgt gaagaggcac gctgtaaatc tctaggacga 600

tttcccaatg ggaaggtaaa ggagcctcca attctccggg ttggtgtaac tgcaaacttt 660

ttctgtgatg aagggtatcg actgcaaggc ccaccttcta gtcggtgtgt aattgctgga 720

cagggagttg cttggaccaa aatgccagta tgtggaggtg ggtcgggtgg cggcggatcc 780

gactgtggcc ttcccccaga tgtacctaat gcccagccag ctttggaagg ccgtacaagt 840

tttcccgagg atactgtaat aacgtacaaa tgtgaagaaa gctttgtgaa aattcctggc 900

gagaaggact cagtgatctg ccttaagggc agtcaatggt cagatattga agagttctgc 960

aatcgtagct gcgaggtgcc aagcaggcta agttctgcat ccctcaaaca gccttatatc 1020

actcagaatt attttccagt cggtactgtt gtggaatatg agtgccgtcc aggttacaga 1080

agagaacctt ctctatcacc aaaactaact tgccttcaga atttaaaatg gtccacagca 1140

gtcgaatttt gtaaaaagaa atcatgccct aatccgggag aaatacgaaa tggtcagatt 1200

gatgtaccag gtggcatatt atttggtgca accatctcct tctcatgtaa cacagggtac 1260

aaattatttg gctcgacttc tagtttttgt cttatttcag gcagctctgt ccagtggagt 1320

gacccgttgc cagagtgcag agaaatttat tgtccagcac caccacaaat tgacaatgga 1380

ataattcaag gggaacgtga ccattatgga tatagacagt ctgtaacgta tgcatgtgat 1440

aaaggattca ccatgattgg agagcactct atttattgta ctgtgagtaa tgatgaagga 1500

gagtggagtg gcccaccacc tgaatgcaga 1530

Claims (10)

1. a kind of complement inhibitor DAF variant, which is characterized in that the amino acid sequence of the DAF variant such as SEQ ID Shown in NO.1.

2. a kind of polynucleotide molecule of DAF variant described in coding claim 1, which is characterized in that the polynucleotides point The sequence of son is as shown in SEQ ID NO.2.

3. a kind of fusion protein containing DAF variant described in claim 1, which is characterized in that the fusion protein also contains The complement targeted molecular in targeting complement activation site.

4. fusion protein according to claim 3, which is characterized in that the complement targeted molecular is complement receptor 2.

5. fusion protein according to claim 4, which is characterized in that the amino acid sequence of the complement receptor 2 such as SEQ Shown in ID NO.3.

6. fusion protein according to claim 5, which is characterized in that the complement receptor 2 and DAF molecule are with flexible small peptide GGGSGGGGS connection.

7. fusion protein according to claim 6, which is characterized in that the amino acid sequence of the fusion protein such as SEQ ID Shown in NO.5.

8. a kind of polynucleotide molecule of fusion protein described in coding claim 7, which is characterized in that the polynucleotide molecule Sequence as shown in SEQ ID NO.6.

9. any fusion protein of claim 3-7 is preparing the application in Parmaceutical for treating disease of autoimmunization system object.

10. application according to claim 9, the disease includes rheumatoid arthritis and systemic loupus erythematosus.