CN102757497B - Anti-PEDF monoclonal antibody, and preparation method and application thereof - Google Patents
Anti-PEDF monoclonal antibody, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an anti-PEDF monoclonal antibody, and a preparation method and application thereof. The preparation method comprises the following steps: immunizing a BALB/c mouse with PEDF protein, fusing splenocyte and myeloma cell of the immune mouse, screening and cloning to obtain a hybridoma strain capable of secreting the anti-PEDF monoclonal antibody, and finally acquiring the required monoclonal antibody. The monoclonal antibody disclosed by the invention can be used for preparing medicines for treating PEDF-increase wound healing diseases.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of anti-PEDF monoclonal antibody and its preparation method and application.
Background technology
Diabetes microvascular complication (diabetic microvascular complication) is that diabetic subject is disabled, lethal major cause.Increasing research shows suppressed due to angiogenesis (angiogensis), and obstacle appears in diabetic subject's local wound healing (wound healing), and research estimates that diabetic foot (diabetic foot) appears in 15% diabetic subject.There is no at present specific treatment means both at home and abroad, cause every year 72,000 patient's amputation.The cause of disease that diabetic foot occurs is except wound, bacterium infect repeatedly, and wound healing impaired (impaired wound healing) is one of them important factor.In specific situation, as the generation of diabetes, vasculogenesis is obstructed, and wound healing process is broken or postpones, and chronic injury (chronic wound) grows up, and finally may cause ulcer, amputation.
Angiogenesis, from the online a large amount of processes that generate new vessel of already present capillary vessel.Angiogenesis has the active effect of recovering blood confession and promoting wound healing under wound and peripheral circulation occlusion condition.The formation of new vessel is subject to the balance regulation of angiogenesis stimulating factor and angiogenesis supressor, stimulating factor comprises vascular endothelial growth factor (vascular endothelial grow factor, VEGF), fibroblast growth factor (fibroblast growth factor, FGF), transforming growth factor (transforming growth factor, TGF), Thr6 PDGF BB (platelet-derived growth factor, PDGF) etc., supressor comprises angiostatin angiostatin, Endostatin endostatin, pigment epidermal derived factors (pigment epithelium-derived factor, PEDF) etc.
PEDF is the stronger angiogenic inhibitor of activity of finding at present, has the effect of the neonate tumour blood vessel of inhibition simultaneously, and its Cytological Basis is the endothelial cell apoptosis activating by induction.Research report PEDF significantly raises in I type, type ii diabetes and metabolic syndrome Blood of Patients recently; Our preliminary experiment found that the insulin resistant mice serum PEDF of high fat induction is apparently higher than normal diet control group.The PEDF that diabetes raise may induce capillary endothelium apoptosis, suppresses diabetes peripheral blood vessel new life, hinders diabetic foot wound healing process thereby mediate it, and blocking-up PEDF has the potential using value of control diabetic foot wound healing obstacle.
Summary of the invention
The object of the invention is to according to the deficiencies in the prior art, a kind of wound healing potential drug albumen slowly of diabetes and the metabolic syndrome etc. that can become preparation treatment PEDF rising is provided.
Aforesaid method of the present invention is achieved by the following technical programs:
A preparation method for anti-PEDF monoclonal antibody, is by pedf protein immunity BALB/c mouse, then merges with splenocyte and the myeloma cell of immunized mice, obtains the hybridoma cell strains of the anti-PEDF monoclonal antibody of secretion through screening and cloning, obtains required monoclonal antibody.
The medicine of the wound healing disease of diabetes and metabolic syndrome etc. that the anti-PEDF monoclonal antibody of the present invention can raise for the preparation for the treatment of PEDF.
Compared with prior art, the present invention has following beneficial effect:
Diabetes microvascular complication (diabetic microvascular complication) is that diabetic subject is disabled, lethal major cause.Increasing research shows suppressed due to angiogenesis (angiogensis), there is obstacle in diabetic subject's local wound healing (wound healing), research estimates that diabetic foot ulcer (diabetic foot ulcers) appears in 15% diabetic subject, there is no at present specific treatment means both at home and abroad, cause every year 72,000 patient's amputation.This research proposes PEDF monoclonal antibody first can be for the preparation of the medicine of the wound healing disease of diabetes and the metabolic syndrome etc. for the treatment of PEDF rising.
Brief description of the drawings
Fig. 1 is the present invention pedf protein SDS-PAGE result figure that recombinates;
Fig. 2 is the non-sex change gel electrophoresis of PEDF monoclonal antibody figure after purifying;
Fig. 3 is that the immunizing potency of different extent of dilution PEDF monoclonal antibodies detects (western blotting);
Fig. 4 is that PEDF monoclonal antibody detects endogenous pedf protein (western blotting);
Fig. 5 is the wound healing rate statistics of injection pedf protein and contrast GST;
Fig. 6 is the wound healing situation of injection pedf protein and contrast GST;
Fig. 7 is the wound healing rate statistics of diabetic groups and PEDF monoclonal antibody group;
Fig. 8 is the wound healing situation of diabetic groups and PEDF monoclonal antibody group.
Embodiment
Further explain the present invention below in conjunction with embodiment, but embodiment does not limit in any form to the present invention.Method in following examples, equipment, material, if not specified, be method, equipment and the material of this area routine.
Embodiment 1 preparation of PEDF monoclonal antibody and qualification
One, PEDF monoclonal antibody preparation
1. PEDF recombinant protein preparation
Select pET30a (+) (purchased from Novagen) as carrier, according to people source PEDF gene order design primer, the cDNA of pcr amplification PEDF gene, select EcoRI and HindIII restriction enzyme (purchased from Takara) enzyme to cut the PEDF cDNA after purifying, connect with the pET30a (+) cutting through same enzyme, obtain connecting product pET30a (+)-PEDF.By pET30a (+)-PEDF plasmid transformation escherichia coli BL-21 DE3, after IPTG induction, collect bacterium, resuspended with Binding buffer, ultrasonic centrifugal rear acquisition soluble recombinant protein.Again by albumen and 4 DEG C of abundant combinations of Ni-NTA resin (purchased from Novagen), upper prop afterwards, wash away foreign protein with Washing buffer, finally with Elution buffer, albumen is eluted, identify as immunogenic (see figure 1) through SDS-PAGE again, the final pedf protein concentration 3.3mg/ml obtaining, purity >98%, albumen yield >15mg/L.
2. animal immune
Adopt 5 6-8 female BALB/C mouse inbred lines in all ages, and by standard program booster immunization, totally 4 immunity, tire from starting for the second time ELSA detection, in standard state, the 4th time final immune, and select two mouse of best immunne response by the result of ELISA.
3. hybridoma merges screening
The splenocyte of two mouse of best immunne response and myeloma cell are carried out to cytogamy, the hybridoma obtaining obtains the clone of high-titer by propagation and ELISA screening, by limiting dilution assay, 2-5 positive parent cell carried out subclone and identify subclass, 6 cell strains of final acquisition, each cell strain has 2 subclones.
4. the production of monoclonal antibody and purifying
1) ELISA method and Western blot detect immunizing potency and the specificity (in table 1) of cells and supernatant, select to be further used for the cell strain 2D9F10 of immune mouse.
Table 1
ELISA?results?of?supernatant?samples：
| Cell lines | 1:10 | 1:30 | 1:90 | 1:270 | 1:810 | 1:2430 | Negative（Medium） | Titer | Isotype |
| 2D9F10 | 2937 | 2918 | 3029 | 2878 | 2485 | 2512 | 0.127 | >1:2430 | IgG1， K |
| 2D9F12 | 2631 | 2617 | 2610 | 2465 | 2596 | 2288 | 0.127 | >1:2430 | IgG1， K |
| 5B11D2 | 2871 | 2723 | 2704 | 2608 | 2350 | 2147 | 0.127 | >1:2430 | IgG1， K |
| 5B11F1 | 2990 | 2875 | 2563 | 2350 | 2306 | 2105 | 0.127 | >1:2430 | IgG1， K |
| 5C2E9 | 3052 | 3139 | 3033 | 2771 | 2670 | 2395 | 0.127 | >1:2430 | IgG1， K |
| 5C2D12 | 2997 | 3052 | 3094 | 2997 | 2876 | 2713 | 0.127 | >1:2430 | IgG1， K |
Starting?dilution：1:10
The?titer?is?the?highest?dilution?with?P/N（positive/negative）>=2.1。
2) preparation of Syngenic mice monoclonal antibody ascites
Cultivate hybridoma, 5 BALB/C mice of immunity altogether, every mouse peritoneal injection 1 × 10
6individual mouse hybridoma cell 2D9F10.After 7 days, observe the mouse ascites condition of production, as belly obviously expands, can extract ascites.After ascites collection, the centrifugal 10min of 13000rpm, except degrease and precipitation, collects supernatant liquor, is ascites monoclonal antibody.
3) purifying: affinity purification ascites, obtains monoclonal antibody protein.
Steady to baseline with binding buffer balance protein G, by upper prop after binding buffer liquid balance for ascites monoclonal antibody, collect stream and wear liquid, then stream is worn to liquid upper prop again, be washed till baseline with Washing buffer steady.Add Eluting Buffer wash-out, collect elution peak, be the ascites monoclonal antibody of purifying.
Two, monoclonal antibody CHARACTERISTICS IDENTIFICATION
1, the molecular mass of antibody qualification:
Adopt non-sex change glue to measure monoclonal antibody, result as shown in Figure 2.
2, the mensuration of antibody titer
ELISA result is as shown in table 2, and western blot result as shown in Figure 3.
Table 2
ELISA?results?for?the?purified?monoclonal?antibody(A450nm)
| Antibody dilution | Purified antibody |
| 1000 | 2.817 |
| 3000 | 2.866 |
| 9000 | 2.721 |
| 27000 | 2.592 |
| 81000 | 2.041 |
| negative | 0.114 |
| titer | >81000 |
Starting?dilution?1:1000(1.0μg/ml)。
3, the mensuration of antibodies specific
PEDF monoclonal antibody western blot method detects cell and different tissues sample, sees Fig. 4.
The application of embodiment 2 PEDF monoclonal antibodies
One, the foundation of diabetes animal model
Get 40 8 weeks C57BL/6J mouse (Zhongshan University's animal center provides), be divided into normal group and diabetic groups, every group 20, carrying out respectively normal diet and high lipid food (Guangdong Province's animal center provides) feeds 4 weeks, rear diabetic groups continuous small dose abdominal injection U-9889 (Streptozotocin, STZ) (dosage is 70mg/kg) 7 days, by fasting plasma glucose, insulin tolerance (insulin tolerance test, ITT), glucose tolerance (glucose tolerance test, GTT) experiment detects and determines diabetes model modeling success.
Two, the result for the treatment of of the foundation of periphery trauma model and PEDF monoclonal antibody
The mouse of above-mentioned normal group is divided into 2 groups at random, GST group and PEDF group.After mouse back is disinfected with depilatory cream depilation, make the circular mark of two diameter 6mm of biopsy punch tool, excision holostrome skin.The mouse of normal group is injected respectively GST and GST-PEDF (2.5 mg/kg) every day, take pictures every day, carry out assess wound healing rate by measuring wound area, from the 3rd day, the wound healing rate of two groups has obvious significant difference, Continuous Observation 7 days finds afterwards, and the speed of wound healing of injection PEDF group has slowed down 37.5% than injection GST group, shows the PEDF healing rate (seeing Fig. 5-6) of wound of can slowing down that raises.
Equally the mouse of diabetic groups is also divided into 2 groups at random, diabetic groups and PEDF antibody group.Use the same method, make the circular mark of two diameter 6mm of biopsy punch tool, excision holostrome skin.Diabetic groups mouse, inject respectively PBS and PEDF monoclonal antibody (0.34mg/kg) every day, take pictures every day and carry out assess wound healing rate by measuring wound area, from the 5th day, the existing obvious significant difference of wound healing rate of two groups, Continuous Observation is found for 10 days afterwards, the speed of wound healing of injection PEDF antibody group has improved 38.1% than injection PBS group, show the healing rate that the PEDF monoclonal antibody of above-mentioned preparation can accelerated in wounds, for PEDF monoclonal antibody for the preparation for the treatment of PEDF raise diabetes and metabolic syndrome etc. wound healing disease drug provision according to (seeing Fig. 7-8).
Claims (1)
1. the application in the medicine of the diabetes that anti-PEDF monoclonal antibody raises at preparation treatment PEDF; The preparation method of described anti-PEDF monoclonal antibody is by pedf protein immunity BALB/c mouse, then merges with splenocyte and the myeloma cell of immunized mice, obtains the hybridoma cell strains of the anti-PEDF monoclonal antibody of secretion through screening and cloning, obtains required monoclonal antibody.
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