CN105542006A - Preparation method and use of anti-EPOR fusion protein antibody - Google Patents

Abstract

The invention discloses a preparation method and use of an anti-EPOR fusion protein antibody, and relates to the field of gene engineering. The anti-EPOR fusion protein antibody provided by the invention can inhibit or block an EPO-EPOR pathway, thereby inhibiting proliferation and metastasis of tumor cells; the anti-EPOR fusion protein polyclonal antibody provided by the invention is used for preparation of drugs for control or treatment of tumor.

Description

The preparation method of anti-EPOR fusion rotein antibody and purposes

Technical field

The present invention relates to genetically engineered field, particularly relate to preparation method and the purposes of anti-EPOR fusion rotein antibody.

Background technology

Erythropoietin (erythropoietin, EPO) be a kind of glycoprotein hormones, belong to cytokine superfamily members as a kind of hemopoieticgrowth factor, he and erythropoietin receptor (erythropoietinreceptor, EPOR) combine, play an important role in erythrocytic production.

The biological effect of erythropoietin (erythropoietin, EPO) is regulated by its special cell surface receptor-erythropoietin receptor (erythropoietinreceptor, EPOR).Erythropoietin receptor (erythropoietinreceptor, EPOR) be a kind of mad magic albumen of volume containing 508 amino acid deformity, molecular weight is 66kDa, be made up of outer ligand binding domain, membrane-spanning domain and born of the same parents' internal area three parts of born of the same parents, research in recent years shows that EPO and acceptor thereof are present in the tumor cell line of many non-red systems equally, the expression of EPO and EPOR is found in multiple human tumor, comprises mammary cancer, renal cell carcinoma, prostate cancer, colorectal carcinoma, incidence squamous cell cancer etc.

Summary of the invention

Object of the present invention is exactly to overcome above-mentioned prior art Problems existing, a kind of erythropoietin receptor antibody is provided, the impact of erythropoietin-erythropoietin receptor pathway activity can be suppressed, thus block the tumour of erythropoietin-erythropoietin receptor expression.

For realizing object of the present invention, first aspect present invention provides a kind of antibody of anti-EPOR fusion rotein, and it can suppress or block EPO-EPOR path, is obtained by the fusion protein immunization animal of the aminoacid sequence had as shown in SEQIDNO.3.

Wherein, described fusion rotein is by Histidine signal peptide and be positioned at the 25th of human body EPOR albumen and form to 254 amino acids residues.

Particularly, described EPOR fusion rotein is obtained by eukaryotic expression.

Especially, described EPOR fusion rotein obtains by being transfected into the expression of human body renal epithelial cell.

Preferably, described human body renal epithelial cell is HEK-293 cell.

For realizing object of the present invention, second aspect present invention provides a kind of gene of anti-EPOR fusion rotein, and the fusion rotein of its coding described in first aspect, comprises the aminoacid sequence as shown in SEQIDNO.3.

For realizing object of the present invention, third aspect present invention provides a kind of recombinant vectors, carries out, in effective connection procedure, having the gene described in second aspect at it.

Wherein, described recombinant vectors utilizes eukaryotic expression vector pcDNA3.1 (+)-myc-His-Cmammalianexpressionvector to obtain.

For realizing object of the present invention, fourth aspect present invention provides a kind of host cell, and it contains the recombinant vectors described in the third aspect.

For realizing object of the present invention, fifth aspect present invention provides a kind of compound, comprises the antibody of the anti-EPOR fusion rotein described in first aspect, is applied to the preparation suppressing or block EPO-EPOR path medicine.

For realizing object of the present invention, sixth aspect present invention provides a kind of using the application of the antibody of the anti-EPOR described in first aspect as preparation tumor.

For realizing object of the present invention, seventh aspect present invention provides a kind of antibody preparing anti-EPOR fusion rotein, is prepared by following steps:

Outer to signal peptide and EPOR section peptide to be connected in carrier for expression of eukaryon successively by the method for PCR and transfection is expressed to host cell, to obtain EPOR fusion rotein;

EPOR fusion rotein described in purifying, and carry out Mass Spectrometric Identification;

By EPOR fusion protein immunization animal correct for qualification, obtain the antiserum(antisera) with anti-EPOR fusion rotein;

Antiserum(antisera) described in purifying, obtains the antibody of anti-EPOR fusion rotein.

Wherein, described carrier for expression of eukaryon is pcDNA3.1 (+)-myc-His-Cmammalianexpressionvector.

Wherein, described host cell is human body renal epithelial cell HEK293 cell.

Beneficial effect of the present invention is embodied in:

1, the antibody of anti-EPOR fusion rotein provided by the invention can effectively suppress EPO-EPOR path, thus the tumour that blocking-up EPO-EPOR pathway activity causes is expressed, the generation of the cell of the polyclonal antibody suppression in various degree that the application provides obviously can be found out, therefore the polyclonal antibody that provides of the application and there is the application that the gene of expressing described polyclonal antibody may be used for preparation tumor in the kidney tumor cell system of the UT-7 cell and classics that rely on EPO growth.

2, the present invention utilizes human body renal epithelial cell as host cell, and the polyclonal antibody purity of the anti-EPOR fusion rotein prepared is high, and strong with the binding ability of antigen, height of tiring, when extension rate reaches 256000, still can detect and obtain potent antibodies.

Accompanying drawing explanation

When considered in conjunction with the accompanying drawings, by referring to detailed description below, the present invention can be understood more complete, better and easily learn wherein many adjoint advantages.Accompanying drawing described herein is used to provide a further understanding of the present invention, forms a part of the present invention, and schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention, as figure, wherein:

Fig. 1 is the mass spectrum order-checking qualification result figure of the recombinant epo R fusion rotein after the purifying described in embodiment 3, represents that albumen size is 35-36kDa in figure;

Fig. 2 is the colour developing figure of the serum titer detected result described in embodiment 4, and figure A represents that three exempt from rear antiserum titre detection colour developing figure; Figure B represents that four exempt from rear antiserum titre detection colour developing figure; After figure C represents whole bloodletting, antiserum titre detects colour developing figure; Figure D puts rear antiserum(antisera) antibody purification bioactivity colour developing figure eventually, and in figure, I, II represents twice repetition;

Fig. 3 is the qualification result figure of the anti-EPOR fusion rotein antibody described in embodiment 5;

Fig. 4 is the result figure of the anti-EPOR fusion rotein antibody suppression EPO-EPOR pathway activity described in embodiment 6; Wherein figure E is pathway activity inhibition figure, the figure F of anti-EPOR fusion rotein antibody to different clone is the active inhibition figure of different underpass action time, scheme G is different antibodies concentration underpass activity inhibition figure;

Fig. 5 is the result figure of the impact that the anti-EPOR antibodies on tumor cell described in embodiment 7 grows; Wherein, to scheme H be anti-EPOR antibody on UT-7 cell proliferation affect result figure, figure I be anti-EPOR antibody caki-1 kidney cancer cell is increased affect result figure.

Embodiment

Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.

Embodiment 1: the structure of recombinant epo R expression plasmid

1, expression signal peptide Osteonectin and Histidine 6-His label prepare

Normal people's genomic dna is adopted to be template, by standard PCR amplification expression signal peptide Osteonectin sequence, wherein, upstream primer used is with HindIII restriction enzyme site, base sequence is agctAagcttgcctgccgcctgcctgcct, and as shown in SEQIDNO.4, downstream primer is with BamHI restriction enzyme site and 6 His (Histidine) sequences, base sequence is agctggatccgtgatggtgatggtgatgcagctgaggggctgccaagg, as shown in SEQIDNO.5.

Wherein, the aminoacid sequence of described expression signal peptide is as shown in SEQIDNO.1.

According to the aminoacid sequence amplification EPOR gene fragment of HUMANEPOR, and reclaim after the agarose gel electrophoresis of 1% concentration, obtain the EPOR gene fragment after increasing.EPOR gene fragment is adopted after HindIII and BamHI endonuclease digestion with eukaryotic expression vector pcDNA3.1 (+)-myc-His-Cmammalianexpressionvector and is connected, obtain recombinant plasmid pcDNA3.1 (+)-myc-His-Cmammalianexpressionvector-Osteonectin-6His, and by recombinant plasmid transformed bacillus coli DH 5 alpha, obtain recombinant bacterium DH5 α/Osteonectin-6His.Carry out DNA sequencing through PCR screening positive clone, after checking, obtain building correct recombinant plasmid DH5 α/Osteonectin-6His.

2, the outer section peptide recombinant plasmid preparation of people EPOR is expressed

Adopt normal people's genomic dna to be template, by the outer section peptide sequence of standard PCR amplification people EPOR, upstream primer used is with BamHI restriction enzyme site, and sequence is agctggatccgcgcccccgcctaacctc, as shown in SEQIDNO.6.Downstream primer is with EcoRI restriction enzyme site, and sequence is agctgaattctcacgtcaggatgagggg, as shown in SEQIDNO.7, obtains the outer section peptide sequence of people EPOR.

The outer section peptide sequence of the people EPOR obtained increasing reclaims after the agarose gel electrophoresis of 1% concentration, obtains the gene fragment expressing the outer section peptide of people EPOR.By the gene fragment of acquisition and recombinant plasmid pcDNA3.1 (+)-myc-His-Cmammalianexpressionvector-Osteonectin-6His, connect after adopting BamHI and EcoRI endonuclease digestion, obtain recombinant plasmid pcDNA3.1 (+)-myc-His-Cmammalianexpressionvector-Osteonectin-6His-EPOR, by its transformation of E. coli DH5 α, obtain recombinant bacterium DH5 α/Osteonectin-6His-EPOR, DNA sequencing is carried out through PCR screening positive clone, checking obtains building correct recombinant plasmid DH5 α/Osteonectin-6His-EPOR.

Positive DH5 α/Osteonectin-6His-EPOR is seeded to the liquid nutrient medium that 1LLB/Amp is prepared by 1% peptone, 0.5% yeast extract, 1%NaCl, in 37 DEG C, shaking culture is spent the night under 180rpm condition.Second angel spends the large extraction reagent kit of intracellular toxin plasmid and extracts plasmid, obtains recombinant epo R expression plasmid.

Embodiment 2: the expression of recombinant epo R fusion rotein

1, the preparation of HEK293 cell

HEK-293 cell that is good for growth conditions, degree of converging more than 95% is inoculated in 6 orifice plates with the inoculum size of 1*10^5, by the DMEM culture medium culturing containing 10% foetal calf serum, repeatedly put upside down mixing to be placed on 37 DEG C, to cultivate 24 hours in 5%CO2 incubator, obtain can be used for transient transfection, cell attachment completely, density reach 80% HEK293 cell.

Wherein, HEK293 cell comes from the biological product collecting center of ATCC-USS.

2, the transient transfection of HEK-293 cell and expression

Recombinant epo R expression plasmid embodiment 1 obtained is transfected into cultivate to be had in 6 orifice plates of HEK293 cell, fully slowly adds in cell factory after mixing.Cell factory is placed in 37 DEG C, 5%CO ₂cultivate in incubator.After 4 hours, add 266mlCellBoost5, continue after mixing to cultivate 3-4 days, afterwards, the purifying collected supernatant liquor and be used for target protein under 7000rpm condition centrifugal 20min minute.

Embodiment 3: the purifying of recombinant epo R fusion rotein and qualification

Centrifugal for the cell culture supernatant 3000rpm of results 20min is removed precipitation.Dilute with the BindingBuffer of equivalent, the histidine-tagged protein purification kit adopting Beaver company to provide carries out the purifying of target protein, obtains the recombinant epo R fusion rotein after purifying, as shown in Figure 1.

As seen from Figure 1, the recombinant epo R fusion protein molecule amount after purifying is about 35-36KDa.

The Maldi-TOF-TOF that the recombinant epo R fusion rotein obtained adopts Sangon Biotech (Shanghai) Co., Ltd. to provide is carried out mass spectrum order-checking, and it is consistent that the rear recombinant epo R fusion rotein of checking and normal human express the outer section peptide sequence of EPOR.

Embodiment 4: the acquisition of anti-EPOR fusion rotein antibody and purifying

1, immune-treated

Take the EPOR albumen 3mg that embodiment 3 obtains, to 4 monthly ages, the Healthy female new zealand white rabbit of 2.1kg carries out immunity, just exempt from dosage be 0.3mg/ only, two to exempt from, three to exempt from, four exempt from dosage be 0.15mg/ only.Just exempting from antigen is that proteantigen and equal-volume Freund's complete adjuvant mix emulsification, and two to exempt from, three to exempt from, four to exempt from antigen be that proteantigen and equal-volume Freund's incomplete adjuvant mix emulsification, process is set to 2 repetitions, and be designated as A rabbit and B rabbit, wherein, immunologic process is:

One exempts from: the 1st day, and immunity antigen is Freund's complete adjuvant+proteantigen;

Two exempt from: the 21st day, and immunity antigen is Freund's incomplete adjuvant+proteantigen;

Three exempt from: the 35th day, and immunity antigen is Freund's incomplete adjuvant+proteantigen;

Three exempt from rear blood sampling: the 42nd day, and ear vein blood sampling 1ml, ELISA detect antiserum titre;

Four exempt from: the 49th day, and immunity antigen is Freund's incomplete adjuvant+proteantigen;

Four exempt from rear blood sampling: the 56th day, and ear vein blood sampling 1ml, ELISA detect antiserum titre;

Whole bloodletting: the 57th day, ELISA detects antiserum titre and reaches requirement, and carotid artery adopts whole blood.

Freund's complete adjuvant and Freund's incomplete adjuvant all purchased from Sigma.

2, serum titer detects

2.1. indirect ELISA detects

Exempt from three, four to exempt from, antiserum(antisera) after whole bloodletting and whole bloodletting purifying utilizes ELISA method to detect serum titer, concrete grammar is:

By antigen with 0.05mol/l carbonate (PH=9.6) by 0.2ug/ hole wrapper sheet, 100ul/ hole, 4 DEG C of overnight incubation, take out rear 0.05%Tween-20 (PBST) and wash three times, 3 minutes/time, then add 5% skim-milk 100ul confining liquid to every hole, close 60 minutes for 37 DEG C, taking-up 0.05%Tween-20 (PBST) washs three times again, 3 minutes/time.By the serum of rabbit respectively according to 1:1000 dilution, then doubling dilution, hatches 1 hour for 37 DEG C, and taking-up 0.05%Tween-20 (PBST) washs three times, 3 minutes/time.With horseradish enzyme labelling goat anti-rabbit igg (H+L), 1:8000 dilutes, and hatches 45min for 37 DEG C.Taking-up 0.05%Tween-20 (PBST) washs five times, 3 minutes/time.Add substrate solution (TMB) 100ul/ hole, reaction 15min, finally adds 100ul2mol/L sulfuric acid termination reaction.Under 450nm wavelength, OD value is measured by microplate reader (China of section ST-360).

2.2 detected results and analysis

Record three to exempt from rear antiserum titre detected result and see Fig. 2 A and table 1, record four to exempt from rear antiserum titre detected result and see Fig. 2 B and table 2, after recording whole bloodletting, antiserum titre detected result is shown in Fig. 2 C and table 3, records eventually to put rear antiserum(antisera) antibody purification bioactivity and the results are shown in Figure 2D and table 4.

Table 1

Note: according to inspecting standard, serum titer value >=2.0 × feminine gender value then represents significant extension rate;

K represents 1000 multiples.

Fig. 2 A according to detected result can find out, the combination that the antibody of coloured moiety and antigen can be good, and associative list 1 is known, and when extension rate reaches 128K, A rabbit and B rabbit still can detect potent antibodies.

Table 2

Note: according to inspecting standard, serum titer value >=2.0 × feminine gender value then represents significant extension rate;

K represents 1000 multiples.

Fig. 2 B according to detected result can find out, the combination that the antibody of coloured moiety and antigen can be good, and associative list 2 is known, and when extension rate reaches 256K, A rabbit and B rabbit still can detect potent antibodies.

Table 3

Note: according to inspecting standard, serum titer value >=2.0 × feminine gender value then represents significant extension rate;

K represents 1000 multiples.

Fig. 2 C according to detected result can find out, the combination that the antibody of coloured moiety and antigen can be good, and associative list 3 is known, and when extension rate reaches 512K, A rabbit and B rabbit still can detect potent antibodies.

Table 4

Note: according to inspecting standard, serum titer value >=2.0 × feminine gender value then represents significant extension rate;

K represents 1000 multiples.

Fig. 2 D according to detected result can find out, the combination that the antibody of coloured moiety and antigen can be good, and associative list 4 is known, and when extension rate reaches 512K, A rabbit and B rabbit still can detect potent antibodies.

3, immunoaffinity purification

Serum (containing total antibody) is attached on albumin A microballon.Antibody and albumin A pearl are mixed and made into thin homogenate, be add about lml microballon in the solution of 10ml in total amount, incubated at room 1h, shake mixing gently, microballon is washed 2 times with the 0.2mol/L Sodium Tetraborate (pH9.0) of 10 times of volumes, each with the centrifugal 30s of centrifugal 2rain or 10000g of 3000g, with 0.2mol/L Sodium Tetraborate (pH9.0) the resuspended microballon of 10 times of volumes, add enough dimethyl pimelate (solid) in microballon homogenate, final concentration is made to be 20mmol/L, incubated at room 30min, and mix gently, microballon 1 time is washed with termination reaction with 0.2mo/L thanomin (pH8.0).Then be resuspended in 0.2mol/L thanomin, incubated at room 2h, mix gently.Microballon is with after PBS washing, be resuspended in PBS, add Thiomersalate to preserve, microbead sample before coupling and after coupling is added after boiling in Laemmli sample buffer, check coupling effect, get respectively and be equivalent to 1ul and 9ul2 increment product electrophoresis in 10%SDS-polyacrylamide gel, and with coomassie brilliant blue staining, when coupling effect is good, show the heavy chain band of a 55kDa in microbead sample before coupling, and the sample after coupling is without this zone, proceeds in chromatography column by the microballon of antibody bag quilt, use PBS rinsing vessel, collect residual microballon.If possible, only use in conjunction with the antibody microballon matrix needed for antigen whole in goods, post washed by the damping fluid identical with preparing antigen with 20 times of column volumes, EPOR antigen liquid is added on post, antigenic solution is made to flow through chromatography column by the speed of every milliliter of about lml/h of column volume, wash post with the binding buffer liquid (PBS) of 20 times of column volumes, wash post with the pre-elution buffer of 20 times of column volumes.Use pre-elution buffer, adopt stepwise elution method, pass through chromatography column with the elution buffer of 0.5 times of column volume continuously, be in charge of and collect each component, detect the antigenic content of often pipe, each pipe high for concentration is merged.To the protein eluate dialysis obtained to change damping fluid, final acquisition concentration is the antibody of 2.0mg/ml, about 5ml.

Embodiment 5: the qualification of anti-EPOR fusion rotein antibody

Negative PBS control and rabbit igg contrast and positive EpoR are set and detect antibody (purchased from santacruzsc-5624) contrast, adopt conventional westernblot method to utilize the associativity of EPOR antibody and EPOR albumen to resist EPOR fusion rotein antibody to identify, qualification result as shown in Figure 3, by obviously finding out in figure that antibody prepared by the application and EPOR albumen have good associativity.

Embodiment 6: anti-EPOR fusion rotein antibody suppression EPO-EPOR pathway activity

1, anti-EPOR fusion rotein antibody is on the impact of EPO-EPOR pathway activity

UT-7 cell, kidney tumor cell 786-0, caki-1 of relying on EPO growth are spread 6 orifice plates, serum free medium overnight starvation is changed after cell grows to degree of converging 80%, often organizing, cell per well adds PBS20uL respectively, rabbit igg contrasts 100ug, EPOR antibody 100ug, cultivate 2 hours, add bovine serum FBS10uL to every hole again, cultivate 30 minutes, finally extract the total protein of each cell, adopt western to detect each group of EPO-EPOR pathway activity, detected result as shown in Figure 4 E.

Can obviously be found out by Fig. 4 E, after applying anti-EPOR antibody, in UT-7,786-0, caki-1 clone, EPO-EPOR pathway activity all has and declines in various degree.

2, under different action time anti-EPOR fusion rotein antibody on the impact of EPO-EPOR pathway activity

The UT-7 cell relying on EPO growth is spread 6 orifice plates, serum free medium overnight starvation is changed after cell grows to degree of converging 80%, every hole adds bovine serum FBS10ul, cultivate 30min, again to often organizing cell per well respectively at 0min, 120min, 180min, 210min, 220min, 225min, 230, 235, 240min adds EPOR antibody 250ug/mL (the final corresponding reaction times 0, 5, 10, 20, 30, 60, 120, 240min), finally extract the total protein of each cell, western is adopted to detect each group of EPO-EPOR pathway activity, detected result as illustrated in figure 4f.

EPOR vent effect is suppressed with regard to Absorbable organic halogens after antibody effect 30min.

Obviously can be found out after antibody effect 30min, there is stabilization checking EPOR vent effect by Fig. 4 F.

3, under different pharmaceutical concentration anti-EPOR fusion rotein antibody on the impact of EPO-EPOR pathway activity

The UT-7 cell relying on EPO growth is spread 6 orifice plates, serum free medium overnight starvation is changed after cell grows to 10^5/mL, EPOR antibody 500ug/mL is added respectively again to often organizing cell per well, 250ug/mL, 125ug/mL, rabbit igg contrast (Sheng Gong company) 500ug/mL, 250ug/mL, 125ug/mL, cultivates 2 hours, then adds bovine serum FBS10uL to every hole, cultivate 30 minutes, finally extract the total protein of each cell, adopt western to detect each group of EPO-EPOR pathway activity, detected result as shown in Figure 4 G.

Can obviously find out by figure, EPOR antibody suppression EPO-EPOR pathway activity and concentration are proportionate.

Embodiment 7: the impact of anti-EPOR antibodies on tumor cell growth

Anti-EPOR antibody, rabbit igg and PBS solution are joined respectively people's megakaryoblast type leukemia cell's UT-7 cell and kidney cancer cell caki-1, observation and comparison its on the impact of cell enlargement, concrete operation step is as follows:

1. cell cultures

UT-7 and caki-1 cell is spread 6 orifice plates (each clone 3 plate, every plate 5 hole, every hole 5*10^4 cell), at serum free medium overnight starvation, make cell cycle synchronization, change 5%FBS culture medium culturing next day, and add different pharmaceutical culturing cell (every 24h dosing is once) by following condition:

Plate 1 (UT-7cell): every hole adds anti-EPOR antibody 250ug/mL

Plate 2 (UT-7cell): every hole adds rabbit igg contrast 250ug/mL

Plate 3 (UT-7cell): every hole adds PBS and contrasts 20uL

Plate 1 (caki-1cell): every hole adds anti-EPOR antibody 250ug/mL

Plate 2 (caki-1cell): every hole adds rabbit igg contrast 250ug/mL

Plate 3 (caki-1cell): every hole adds PBS and contrasts 20uL.

2, result is observed

After 24h, 48h, 72h, 96h, 120h, carrying out cell counting respectively, (UT-7 cell is suspension cell, connects tally counting after collected by centrifugation is resuspended; Caki-1 is attached cell, the resuspended counting of collecting cell after digestion), repeat 3 times, get proliferation rate mean number and draw cell proliferation growth curve.Obtain the proliferation rate of UT-7 cell proliferation rate as shown in Figure 5 and caki-1 cell.

As can be seen from Fig. 5 H, the cell proliferation rate adding anti-EPOR antibody to people's megakaryoblast type leukemia cell is significantly lower than adding rabbit igg and adding the cell proliferation rate of PBS.As can be seen from Fig. 5 I, the cell proliferation rate adding anti-EPOR antibody in kidney cancer cell is significantly lower than adding rabbit igg and adding the cell proliferation rate of PBS.Illustrate that the anti-EPOR fusion rotein that the application obtains significantly can suppress or block EPO-EPOR path, thus the propagation of inhibition tumor cell and transfer.

Although above-mentioned to invention has been detailed description; but be not limited thereto; those skilled in the art can principle according to the present invention modify, and therefore, all various amendments carried out according to principle of the present invention all should be understood to fall into protection scope of the present invention.

Claims (9)

1. an anti-EPOR fusion rotein antibody, is characterized in that, for suppressing or blocking EPO-EPOR path, is obtained by the fusion protein immunization animal of the aminoacid sequence had as shown in SEQIDNO.3.

2. an antibody as claimed in claim 1, is characterized in that, described EPOR fusion rotein is obtained by eukaryotic expression.

3. express a gene for EPOR fusion rotein, it is characterized in that, comprise the aminoacid sequence as shown in SEQIDNO.3, for encoding said fusion protein.

4. a recombinant vectors, is characterized in that, carries out, in effective connection procedure, having gene as claimed in claim 3 at it.

5. a host cell, is characterized in that, containing recombinant vectors according to claim 5.

6. a compound, comprises the antibody of anti-EPOR fusion rotein according to claim 1, for suppressing or block the preparation of EPO-EPOR path medicine.

7. an antibody as claimed in claim 1 is as the application preparing tumor.

8. prepare a method for antibody as claimed in claim 1, it is characterized in that, comprising:

Outer to signal peptide and EPOR section peptide to be connected in carrier for expression of eukaryon successively by the method for PCR and transfection is expressed to host cell, to obtain EPOR fusion rotein;

EPOR fusion rotein described in purifying, and carry out Mass Spectrometric Identification;

By EPOR fusion protein immunization animal correct for qualification, obtain the antiserum(antisera) with anti-EPOR fusion rotein;

Antiserum(antisera) described in purifying, obtains anti-EPOR fusion rotein antibody.

9. method as claimed in claim 9, it is characterized in that, described host cell is human body renal epithelial cell HEK-293 cell.