Fusion protein, preparation method therefor and application thereof in preparing ophthalmic disease treatment, anti-inflammation and anti-tumor medicament

Abstract

The invention discloses a fusion protein, a preparation method thereof and application thereof in preparing ophthalmic disease treatment, anti-inflammation and anti-tumor medicament, and belongs to the field of biopharmaceutical technology. The present invention uses a flexible (F) or rigid (R) linker to fuse two polypeptides to respectively obtain two bifunctional fusion proteins, namely two multi-functional fusion protein macromolecules obtained by linking antiangiogenesis polypeptides HM-3, interleukin 4 and immunoglobulin Fc fragments via an amino acid linker, which can improve drug efficacy, prolong half-life and enhance stability, has the characteristics of strong effect, low toxicity and the like, and can be used for the prevention and treatment of solid tumors and various types of inflammations and neovascular ophthalmic diseases. The fusion protein is expressed in a eukaryotic cell by a genetic engineering method and purified by affinity chromatography or the like.

Claims

1. A fusion protein comprising: an antiangiogenesis polypeptide HM-3 sequence, an interleukin 4 (IL-4) peptide sequence and an Fc fragment sequence of a IgG1 antibody, wherein the HM-3 sequence, the interleukin 4 (IL-4) peptide sequence and the Fc fragment sequence of the IgG1 antibody are linked by linkers, and wherein the amino acid sequence of the fusion protein is SEQ ID NO: 1.

2. A fusion protein comprising: an antiangiogenesis polypeptide HM-3 sequence, an interleukin 4 (IL-4) peptide sequence and an Fc fragment sequence of a IgG1 antibody, wherein the HM-3 sequence, the interleukin 4 (IL-4) peptide sequence and the Fc fragment sequence of the IgG1 antibody are linked by linkers, and wherein the amino acid sequence of the fusion protein is SEQ ID NO: 2.

3. A DNA encoding the fusion protein according to claim 1, wherein the DNA comprises the SEQ ID NO: 3.

4. A DNA encoding the fusion protein according to claim 2, wherein the DNA comprises the SEQ ID NO: 4.

5. A method of treating rheumatoid arthritis, eye vascular diseases and/or retinopathy, comprising preparing a medicament comprising the fusion protein according to claim 1, and administering the medicament to a subject in need thereof at a pharmaceutically acceptable dosage.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a structural schematic diagram of a protein corresponding to SEQ ID NO: 1 according to the present invention;

(2) FIG. 2 is a structural schematic diagram of a protein corresponding to SEQ ID NO: 2 according to the present invention;

(3) FIG. 3 is an electrophoretogram of a fragment obtained by gel extraction of Fc-IL4DM-HM3-1 and expression vector pcDNA3.4/MCS(+) according to the present invention;

(4) FIG. 4 is a diagram showing the results of PCR verification of bacterial liquid according to the present invention;

(5) FIG. 5 is a diagram showing the results of capture of fusion protein according to the present invention;

(6) FIG. 6 is a diagram showing the fine purification of fusion protein according to the present invention;

(7) FIG. 7 is a diagram showing the results of analysis of a fusion protein sample by an SDS-PAGE method according to the present invention;

(8) FIG. 8 is a diagram showing the results of analysis of a fusion protein sample by HPLC according to the present invention.

DETAILED DESCRIPTION

(9) The invention is further described below in conjunction with specific examples.

Example 1

(10) (I) Acquisition of Fusion Protein Gene and Construction of Expression Vector

(11) The antiangiogenesis polypeptide HM-3 sequence is shown in SEQ ID NO: 5, the interleukin 4 sequence is shown in SEQ ID NO: 6, and the human immunoglobulin IgG1-Fc region (SEQ ID NO: 7) is linked to IL4DM-HM3 protein via different linker peptides Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser (SEQ ID NO: 9) flexible (F) linker and AlaGluAlaAlaAlaLysGluAlaAlaAlaLysGluAlaAlaAlaLysGluAlaAlaAlaLysAla (SEQ ID NO: 10) rigid (R) linker to design two novel Fc fusion proteins Fc-IL4DM-HM3, in which the amino acid sequence of the protein I constructed by the flexible (F) linker is shown in SEQ ID NO: 1, and the amino acid sequence of the protein II constructed by the rigid (R) linker is shown in SEQ ID NO: 2. According to the codon preference in CHO cell, the coding sequences of two novel Fc fusion proteins Fc-IL4DM-HM3 are optimized, in which NheI cleavage sites, Kozak sequences, and signal peptides are introduced at the 3′ end, and XhoI cleavage sites are introduced at the 5′ end, thereby obtaining the following DNA sequences SEQ ID NO: 3 and SEQ ID NO: 4 by a whole gene synthesis method.

(12) The DNA sequences of the above 2 fusion proteins Fc-IL4DM-HM3 were synthesized by a commissioned biotechnology company, ligated to a pUC57 vector to form a cloning vector, and stored in E. coli DH5a to form a clone strain. The two fusion proteins all used pcDNA3.4/MCS(+) as the expression vectors, and the vector construction processes are completely identical. Therefore, Fc-IL4DM-HM3-1 is taken as an example, and the experimental procedures were as follows.

(13) 1. Under sterile conditions, the Fc-IL4DM-HM3-1 clone strain sent by the biotechnology company was picked up from the surface of the bacteria penetrans and inoculated into two tubes containing 5 mL of Amp-resistant LB medium at 37° C., 120 rpm under shaking overnight.

(14) 2. After the culture of the bacteria solution in the two tubes, 2.5 mL of sterile 60% glycerol was added in one tube, mixed well, and then charged into sterile centrifuge tubes, with 1 mL per tube, to prepare glycerin tubes, which were frozen and stored at −80° C. The bacteria solution in the other tube was centrifuged at 12,000 rpm for 1 min to collect thalli, and a cloning vector of Fc-IL4DM-HM3-1 was extracted by using a conventional commercial plasmid miniprep kit.

(15) 3. The restriction endonuclease NheI/XhoI was used to perform double enzyme digestion of the Fc-IL4DM-HM3-1 cloning vector and the expression vector pcDNA3.4/MCS(+), and the inserted fragment Fc-IL4DM-HM3-1 having cohesive ends and the expression vector pcDNA3.4/MCS(+) were separated by horizontal nucleic acid electrophoresis and recovered by using a commercial DNA gel extraction kit. The DNA fragment recovery results are shown in FIG. 3.

(16) 4. Using the T4 ligase, the ligation between the inserted fragment Fc-IL4DM-HM3-1 and the expression vector pcDNA3.4/MCS(+) obtained by gel extraction were carried out at 16° C. according to the molar ratio of inserted fragment to vector of 1:5 for 16 h.

(17) 5. 20 uL of DNA ligation was added to 100 uL of freshly thawed E. coli TOP10 competent cells, mixed gently, and placed in an ice bath for 30 min. After being heat-shocked at 42° C. for 45 s, the mixture was quickly placed in an ice bath for 2 to 3 minutes. 900 uL of non-resistant LB medium was added to the mixture, and cultured at 37° C. for 1 h under shaking. The mixture was centrifuged at 4500 rpm for 1 min at 4° C., 900 uL of supernatant was discarded under sterile conditions, the remaining bacterial solution and the precipitated thalli were mixed evenly via gentle blowing-suction, all of which was aspirated by a pipette, and coated to an Amp-resistant LB solid plate and statically cultured at 37° C. for 12 h.

(18) 6. 20 single colonies were picked up and inoculated in a tube containing 5 mL of Amp-resistant LB medium, and cultured at 37° C., 120 rpm under shaking overnight.

(19) 7. Among the strains inoculated in the previous step, each of normally growing strains was stored in 3 glycerol tubes. At the same time, each strain was tested by bacterial PCR verification (see FIG. 4), positive clones were screened, and the preserved glycerol tubes were sent to the biotechnology company for sequencing verification. The correct expression vector was finally obtained.

(20) 8. The glycerol tubes preserving the strain having correct sequencing was taken out, inoculated into a 250 mL shake flask containing 30 mL of Amp-resistant LB medium, incubated at 37° C., 120 rpm overnight, stored in 20 glycerol tubes, which were stored at −80° C. At this point, the construction of the Fc-IL4DM-HM3-1 expression vector was finished.

(21) (II) Expression of Fusion Protein

(22) Transient transfection is one of the ways to introduce DNA into eukaryotic cells. In transient transfection, recombinant DNA is introduced into highly infectious cell lines to obtain transient but high levels of expression of target gene. Enough proteins can be obtained for experiments in a short period of time, saving cell screening time in stable transfection.

(23) The Expi293 Expression System is used to express two novel fusion proteins Fc-IL4DM-HM3. Since the expression processes of the fusion proteins are completely identical, the Fc-IL4DM-HM3-1 is used as an example. The experimental procedures are as follows.

(24) 1. Plasmid Preparation.

(25) A glycerol tube preserving strain with Fc-IL4DM-HM3-1 expression vector was taken from a refrigerator at −80° C., inoculated into a 2 L shake flask containing 500 mL of Amp-resistant LB medium, and cultured at 37° C., 160 rpm under shaking overnight.

(26) After the completion of the culture, the mixture was centrifuged at 5000 g for 5 min to collect thalli, and the plasmid was extracted using a commercial EndoFree Plasmid Maxi Kit. The plasmid concentration was controlled to be 1 mg/mL or above (if it is lower than this concentration, concentration is required), and then sterilized by filtration using a sterile 0.22 μm pore size filter to complete plasmid preparation.

(27) 2. Early-Stage Preparation of Transient Transfection of Cells

(28) The 293F cells used for transfection were passaged at a cell density of 0.4*10.sup.6 cells/mL for every four days from the day of thawing, and at least three passages were performed, followed by the transient transfection. During the passage, the passage volume was expanded as needed based on the volume of the final transfection medium.

(29) 3. Transient Transfection (Taking 30 mL Transfection Volume as an Example, Multiply as Needed)

(30) (1) One day before the experiment, 6*10.sup.7 live cells were inoculated into 30 mL Expi293 Expression Medium, and cultured at 37° C., 8% CO.sub.2, 125 rpm under shaking.

(31) (2) On the day of the experiment, the cells cultured on the previous day were counted firstly, the cell density should be 3-5*10.sup.6 cells/mL, and the viability was greater than 95%.

(32) (3) 7.5*10.sup.7 cells were aspirated into a new 125 mL Erlenmeyer flask and the preheated Expi293 Expression Medium was added to 25.5 mL.

(33) (4) Preparation of plasmid-transfection reagent mixture

(34) a. 30 μg of plasmid DNA was re-dissolved in 1.5 mL of Opti-MEM I Reduced Serum Medium and mixed gently.

(35) b. 81 μL of ExpiFectamine 293 Reagent was added to Opti-MEM I Reduced Serum Medium to a volume of 1.5 mL. The mixture was gently mixed and incubated for 5 min at room temperature (long incubation period affects conversion efficiency).

(36) c. The above two solutions were mixed gently, and incubated for 20-30 min at room temperature to complete the preparation of the plasmid-transfection reagent mixture.

(37) (5) 3 mL of plasmid-transfection reagent mixture was added to the cell culture liquid of step 3 to 28.5 mL in total.

(38) (6) The mixture was cultured at 37° C., 8% CO.sub.2, 125 rpm under shaking for 20 h.

(39) (7) 150 μL of ExpiFectamine 293 Transfection Enhancer 1 and 1.5 mL of ExpiFectamine 293 Transfection Enhancer 2 were added. At this point, the total volume was 30 mL.

(40) (8) The mixture was cultured at 37° C., 8% CO.sub.2, 125 rpm under shaking. The culture was terminated after 6 days and protein purification was carried out.

(41) (III) Purification of Fusion Protein

(42) Protein A is a cell-wall protein isolated from Staphylococcus aureus, which binds to mammalian IgG mainly through the Fc fragment and has very high specificity and binding ability, and is widely used for purification of IgG antibodies and IgG-Fc fusion proteins. The two novel fusion proteins Fc-IL4DM-HM3-1 have IgG-Fc fragments and thus the purification processes are completely identical. Therefore, Fc-IL4DM-HM3-1 produced by transient transfection at 1.6 L scale is used as an example. The experimental procedures are as follows.

(43) 1. Sample pretreatment: 1.6 L of transiently transfected cell culture liquid after culture termination was centrifuged at 7500 rpm for 20 min at 4° C., and the supernatant obtained was about 1.46 L for the next protein A capture.

(44) 2. Affinity capture of target protein (see FIG. 5)

(45) The column information is as follows

(46) TABLE-US-00001 Packing Mabselect SuRe Column XK50/20 Column height (cm) 10 Cross-sectional area 19.62 of column (cm.sup.2) Packing volume (mL) 196.2

(47) a. The sterilization was first performed with 500 mL of 0.2 M NaOH at a flow rate of 10 mL/min.

(48) b. The column was equilibrated with 20 mM PB, and 0.15 M NaCl, pH 7.0, the volume was about 1000 mL, and the flow rate was 20 mL/min.

(49) c. Loading: the sample was pre-adjusted to a neutral pH, and the flow rate was 20 mL/min.

(50) d. The column was washed with 20 mM PB and 0.15 M NaCl, pH 7.0, the volume was about 800 mL, and the flow rate was 20 mL/min.

(51) e. The target protein was eluted with 50 mM citric acid-sodium citrate, and 0.15 M NaCL, pH 3.0, collection was started at onset 20 mAu and stopped at post-peak 20 mAu; and the flow rate was flow rate 20 mL/min.

(52) f. The column was finally washed with 500 mL of 0.2 M NaOH, rinsed with water to neutral, and the column was stored with 20% ethanol.

(53) 3. Further separation and purification by gel chromatography (FIG. 6)

(54) Column Parameters:

(55) TABLE-US-00002 Packing Superdex200 Column XK50/60 Column height (cm) 58 Cross-sectional area 19.62 of column (cm.sup.2) Packing volume (mL) 1138 Flow rate ml/min Loading 1-10% loading volume

(56) a. The sterilization was performed with 300 mL of 0.5 M NaOH at a flow rate of 10 mL/min, and followed by rinsed with ultrapure water to about neutral.

(57) b. The column was equilibrated with a PBS buffer, pH 7.4, the equilibrium volume was about 1500 mL, and the flow rate was 10 mL/min.

(58) c. Loading: the sample was a protein A eluent, and the loading volume was 40 mL.

(59) d. The sample was collected, peak 3 was the target protein peak, for the collection of peak 3, collection was started at onset 10 mAu peak and stopped at post-peak 10 mAu.

(60) e. Finally, the column was stored with 0.1 M NaOH and the flow rate was 10 mL/min.

(61) 4. Ultrafiltration concentration of sample: the samples of peak 3 were combined and subjected to ultrafiltration concentration. A 10 kDa ultrafiltration membrane was selected, and the samples were concentrated to a target protein concentration of more than 5 mg/mL, and then the samples were charged and stored in a refrigerator at −80° C. The initial concentration was about 0.29 mg/mL, and the samples were finally concentrated to 27 mL, and the concentration was about 5.53 mg/mL; the sample were charged and cryopreserved. At the same time, samples were subjected to release detection by SDS-PAGE and HPLC (FIG. 7 and FIG. 8), and then used for druggability evaluation studies.

Example 2

(62) Inhibitory Effect of Fusion Protein on Proliferation of Various Tumor Cells

(63) The MTT assay was used to detect the inhibitory effect of the integrin blocker fusion protein obtained in Example 1 on the proliferation of various tumor cells, including melanoma cell B16F10, gastric cancer cell MGC-803, lung cancer cell A549, liver cancer cell Hep-G2, breast cancer cell MDA-MB-231, colon cancer cell HCT-116, human glioma U87, and cervical cancer cell Hela.

(64) The tumor cells were cultured in a 5% CO.sub.2 incubator at 37° C. to a density of 90% or more, and collected by trypsinization. The cells were resuspended in the culture liquid and counted under a microscope. The cell concentration was adjusted to 3.0×10.sup.4 cells/mL. The cell suspension was inoculated into a 96-well plate, 100 μL per well, and cultured overnight in a 5% CO.sub.2 incubator at 37° C. The fusion proteins I, II, and the positive drug Taxol were diluted with the culture liquid to respective predetermined concentrations. After the cells were fully adhered, each dilution was added to a 96-well plate, 100 μL per well, respectively. The integrin blocker fusion proteins I and II were added as an administration group, Taxol was used as a positive control group, and the culture liquid without any drug was used as a blank control group, and incubated in an 5% CO.sub.2 incubator at 37° C. for 48 hours. 20 μL of 5 mg/mL MTT was added to each well of a 96-well plate, and incubation was continued for 4 hours. The medium was aspirated and 100 μL of DMSO per well were added to dissolve. The absorbance was measured at 570 nm with a microplate reader with a reference wavelength of 630 nm, and the proliferation inhibition (PI) was calculated. The formula is as follows:

(65) $PI\left(\%\right)=1-\frac{N_{\mathrm{test}}}{N_{\mathrm{control}}}\times 100\%$

(66) where N.sub.test is the OD value of the test group and N.sub.control is the OD value of the blank control group.

(67) Data Statistics:

(68) The test was repeated 5 times independently. The results obtained from the test were calculated as mean±SD, and statistical t-test was performed. P<0.05 was considered as significant difference, and P<0.01 was considered as an extremely significant difference. The experimental results are shown in Tables 1-8.

(69) TABLE-US-00003 TABLE 1 Inhibitory effect of fusion protein I and II on proliferation of melanoma cell B16F10 Group (n = 5) Dose (μg/mL) A570 nm/A630 nm PI (%) Protein I 1 1.1081 ± 0.0159 11.75% 2 1.0012 ± 0.0786 20.26% 4 0.8611 ± 0.0643* 31.42% 8 0.6974 ± 0.0421** 44.46% 16 0.5234 ± 0.0769** 58.31% 32 0.4331 ± 0.0591** 65.51% 64 0.3032 ± 0.0279** 75.85% 128 0.1954 ± 0.0499** 84.44% 256 0.0834 ± 0.0334** 93.36% Protein II 1 1.1154 ± 0.0382 11.17% 2 1.0259 ± 0.0232 18.29% 4 0.8991 ± 0.0725* 28.39% 8 0.7251 ± 0.0429** 42.25% 16 0.6411 ± 0.0659** 48.94% 32 0.5034 ± 0.0279** 59.91% 64 0.3865 ± 0.0189** 69.22% 128 0.2939 ± 0.0319** 76.59% 256 0.1749 ± 0.0209** 85.07% Taxol 5 0.6011 ± 0.0144** 52.13% control — 1.2556 ± 0.0411  0.00% *P < 0.05, **P < 0.01 vs control.

(70) The results showed that fusion protein I and protein II could effectively inhibit melanoma cell B16F10, and the inhibition rate reached 40% or more at a concentration of 8 μg/mL.

(71) TABLE-US-00004 TABLE 2 Inhibitory effect of protein I and protein II on proliferation of gastric cancer cell MGC-803 Group (n = 5) Dose (μg/mL) A570 nm/A630 nm PI (%) Protein I 1 0.8701 ± 0.0456  8.92% 2 0.8301 ± 0.0343 13.11% 4 0.7531 ± 0.0236* 21.17% 8 0.6859 ± 0.0395 28.20% 16 0.5692 ± 0.0222* 40.42% 32 0.4365 ± 0.0239** 54.31% 64 0.3068 ± 0.0398** 67.88% 128 0.1696 ± 0.0431** 82.25% 256 0.0945 ± 0.0249** 90.11% Protein II 1 0.8821 ± 0.0306  7.66% 2 0.7852 ± 0.0125 17.81% 4 0.7049 ± 0.0323* 26.21% 8 0.7421 ± 0.0460 22.32% 16 0.6342 ± 0.0302* 33.61% 32 0.4523 ± 0.0271** 52.65% 64 0.3796 ± 0.0133** 60.26% 128 0.2276 ± 0.0511** 76.18% 256 0.1112 ± 0.0115** 88.36% Taxol 5 0.4146 ± 0.0186** 56.60% control — 0.9553 ± 0.0113  0.00% *P < 0.05 **P < 0.01 vs control.

(72) The results showed that fusion protein I and protein II could effectively inhibit gastric cancer cell MGC-803, and the inhibition rate reached 50% or more at a concentration of 32 μg/mL.

(73) TABLE-US-00005 TABLE 3 Inhibitory effect of protein I and protein II on proliferation of lung cancer cell A549 Group (n = 5) Dose (μg/mL) A570 nm/A630 nm PI (%) Protein I 1 0.6466 ± 0.0503  4.14% 2 0.5947 ± 0.0173 11.83% 4 0.5603 ± 0.0416 16.93% 8 0.5225 ± 0.0293* 22.54% 16 0.4866 ± 0.0299* 27.86% 32 0.4395 ± 0.0124** 34.84% 64 0.3837 ± 0.0396** 43.11% 128 0.3263 ± 0.0218** 51.62% 256 0.2609 ± 0.0265** 61.32% Protein II 1 0.6215 ± 0.0196  7.86% 2 0.6051 ± 0.0125 10.29% 4 0.5833 ± 0.0339 13.52% 8 0.5516 ± 0.0313* 18.22% 16 0.5067 ± 0.0241* 24.88% 32 0.4698 ± 0.0178** 30.35% 64 0.4259 ± 0.0336** 36.86% 128 0.3509 ± 0.0116** 47.98% 256 0.2775 ± 0.0267** 58.86% Taxol 5 0.3226 ± 0.0309** 52.17% control — 0.6745 ± 0.0231  0.00% *P < 0.05 **P < 0.01 vs control.

(74) The results showed that fusion protein I and protein II could effectively inhibit lung cancer cell A549, and the inhibition rate reached 45% or more at a concentration of 128 μg/mL.

(75) TABLE-US-00006 TABLE 4 Inhibitory effect of protein I and protein II on proliferation of liver cancer cell Hep-G2 Group (n = 5) Dose (μg/mL) A570 nm/A630 nm PI (%) Protein I 1 0.9884 ± 0.0424  3.77% 2 0.9666 ± 0.0276  5.89% 4 0.9169 ± 0.0253 10.73% 8 0.8793 ± 0.0133* 14.39% 16 0.7989 ± 0.0305* 22.22% 32 0.7564 ± 0.0114* 26.36% 64 0.6915 ± 0.0242** 32.67% 128 0.6558 ± 0.0189** 36.15% 256 0.6024 ± 0.0134** 41.35% Protein II 1 0.9816 ± 0.0382  4.43% 2 0.9555 ± 0.0197  6.97% 4 0.9133 ± 0.0384 11.08% 8 0.8856 ± 0.0115* 13.78% 16 0.8076 ± 0.0411* 21.37% 32 0.7622 ± 0.0175* 25.79% 64 0.7116 ± 0.0369** 30.72% 128 0.6809 ± 0.0216** 33.71% 256 0.6108 ± 0.0468** 40.53% Taxol 5 0.4257 ± 0.0099** 58.55% control — 1.0271 ± 0.0531  0.00% *P < 0.05 **P < 0.01 vs control.

(76) The results showed that fusion protein I and protein II had certain inhibitory effects on liver cancer cell Hep-G2, and the inhibition rate increased along with the increase of concentration.

(77) TABLE-US-00007 TABLE 5 Inhibitory effect of protein I and protein II on proliferation of breast cancer cell MDA-MB-231 Group (n = 5) Dose (μg/mL) A570 nm/A630 nm PI (%) Protein I 1 0.8133 ± 0.0262  5.77% 2 0.7992 ± 0.0250  7.40% 4 0.7798 ± 0.0409  9.65% 8 0.7371 ± 0.0378* 14.60% 16 0.6706 ± 0.0185* 22.30% 32 0.6109 ± 0.0130* 29.22% 64 0.5499 ± 0.0186** 36.29% 128 0.4982 ± 0.0326** 42.28% 256 0.4410 ± 0.0171** 48.91% Protein II 1 0.8213 ± 0.0202  4.84% 2 0.8008 ± 0.0199  7.22% 4 0.7805 ± 0.0430  9.57% 8 0.7219 ± 0.0333* 16.36% 16 0.6779 ± 0.0130* 21.46% 32 0.6186 ± 0.0160* 28.33% 64 0.5555 ± 0.0331** 35.70% 128 0.5011 ± 0.0275** 41.94% 256 0.4476 ± 0.0282** 48.06% Taxol 5 0.4071 ± 0.0301** 52.83% control — 0.8631 ± 0.0409  0.00% *P < 0.05 **P < 0.01 vs control.

(78) The results showed that fusion protein I and protein II could effectively inhibit breast cancer cell MDA-MB-231, and the inhibition rate reached 40% or more at a concentration of 128 μg/mL.

(79) TABLE-US-00008 TABLE 6 Inhibitory effect of protein I and protein II on proliferation of colon cancer cell HCT-116 Group (n = 5) Dose (μg/mL) A570 nm/A630 nm PI (%) Protein I 1 0.6640 ± 0.0246  2.40% 2 0.6412 ± 0.0181  5.75% 4 0.6089 ± 0.0131 10.50% 8 0.5503 ± 0.0319* 19.11% 16 0.5285 ± 0.0222* 22.31% 32 0.4529 ± 0.0190** 33.43% 64 0.3726 ± 0.0190** 45.23% 128 0.2826 ± 0.0151** 58.46% 256 0.2071 ± 0.0271** 69.56% Protein II 1 0.6610 ± 0.0280  2.84% 2 0.6440 ± 0.0143  5.34% 4 0.6112 ± 0.0495 10.16% 8 0.5615 ± 0.0125* 17.46% 16 0.5416 ± 0.0375* 20.39% 32 0.4889 ± 0.0109** 28.13% 64 0.4000 ± 0.0020** 41.20% 128 0.3126 ± 0.0255** 54.05% 256 0.2324 ± 0.0283** 65.84% Taxol 5 0.2098 ± 0.0164** 69.16% control — 0.6803 ± 0.0441  0.00% *P < 0.05 **P < 0.01 vs control.

(80) The results showed that fusion protein I and protein II could effectively inhibit colon cancer cell HCT-116, and the inhibition rate reached 40% or more at a concentration of 64 μg/mL.

(81) TABLE-US-00009 TABLE 7 Inhibitory effect of protein I and protein II on proliferation of human glioma U87 Group (n = 5) Dose (μg/mL) A570 nm/A630 nm PI (%) Protein I 1 0.7370 ± 0.0190  4.52% 2 0.6916 ± 0.0275 10.40% 4 0.6209 ± 0.0314* 19.56% 8 0.5377 ± 0.0212** 30.34% 16 0.4788 ± 0.0366** 37.97% 32 0.3520 ± 0.0223** 54.40% 64 0.2330 ± 0.0112** 69.81% 128 0.1346 ± 0.0283** 82.56% 256 0.0820 ± 0.0230** 89.38% Protein II 1 0.7480 ± 0.0299  3.10% 2 0.7042 ± 0.0166  8.77% 4 0.6359 ± 0.0330* 17.62% 8 0.5512 ± 0.0577** 28.59% 16 0.4996 ± 0.0333** 35.28% 32 0.3688 ± 0.0171** 52.22% 64 0.2640 ± 0.0395** 65.80% 128 0.1564 ± 0.0206** 79.74% 256 0.0984 ± 0.0222** 87.25% Taxol 5 0.1825 ± 0.0163** 76.38% control — 0.7719 ± 0.0188  0.00% *P < 0.05 **P < 0.01 vs control.

(82) The results showed that fusion protein I and protein II could significantly inhibit human glioma U87, and the inhibition rate reached 50% or more at a concentration of 32 μg/mL.

(83) TABLE-US-00010 TABLE 8 Inhibitory effect of protein I and protein II on proliferation of cervical cancer cell Hela Group (n = 5) Dose (μg/mL) A570 nm/A630 nm PI (%) Protein I 1 1.1729 ± 0.0183  8.28% 2 1.0658 ± 0.0295 16.66% 4 0.9739 ± 0.0238* 23.84% 8 0.9526 ± 0.0166* 25.51% 16 0.8248 ± 0.0105* 35.50% 32 0.7109 ± 0.0119** 44.41% 64 0.5643 ± 0.0265** 55.87% 128 0.3629 ± 0.0215** 71.62% 256 0.1461 ± 0.0350** 88.58% Protein II 1 1.1066 ± 0.0282 13.47% 2 1.0001 ± 0.0162 21.72% 4 0.9368 ± 0.0835* 26.74% 8 0.9812 ± 0.0179 23.27% 16 0.8276 ± 0.0395* 35.28% 32 0.7018 ± 0.0133** 45.12% 64 0.5845 ± 0.0499** 54.29% 128 0.3792 ± 0.0213** 70.35% 256 0.1456 ± 0.0845** 88.61% Taxol 5 0.2078 ± 0.0162** 83.75% control — 1.2788 ± 0.0987  0.00% *P < 0.05 **P < 0.01 vs control.

(84) The results showed that fusion protein I and protein II could significantly inhibit cervical cancer cell Hela, and the inhibition rate reached about 55% at a concentration of 64 μg/mL.

(85) Taken together, the inhibitory effects of fusion protein I and protein II integrin blockers on proliferation of various tumor cells are shown in Tables 1-8. The fusion proteins can effectively inhibit proliferation of gastric cancer, lung cancer, liver cancer, breast cancer, melanoma, colon cancer, glioma, and cervical cancer. Among them, the inhibition rate of melanoma, gastric cancer and human glioma reached 50% or more at a concentration of 32 μg/mL; the inhibition rate of colon cancer cells reached 40% or more, and the inhibition rate of cervical cancer cells reached 50% or more at a concentration of 64 μg/mL; higher concentrations were required to achieve effective inhibition on lung cancer, liver cancer, and breast cancer cells.

Example 3

(86) Detection of Inhibitory Effect of Fusion Proteins I and II on Migration of Human Umbilical Vein Endothelial Cells by Three-Dimensional Transwell Assay

(87) Human umbilical vein endothelial cells (HUVECs) were cultured with endothelial cell culture fluid containing 5% fetal bovine serum and 1×ECGS in a 5% CO.sub.2 incubator at 37° C., to a confluence of 90% or more, and then inhibitory effect of fusion protein I and protein II on migration of endothelial cells were detected by transwell assay, in which only endothelial cells HUVEC of passage 2 to 8 were used, and the specific operation was as follows.

(88) (1) 10 mg/mL Matrigel (a basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells) diluted with a DMEM medium (Dulbecco's Modified Eagle Medium) at a ratio of 1:4, coated on a transwell chamber membrane, and air-dried at room temperature;

(89) (2) HUVEC cells cultured to logarithmic phase were digested with 0.2% EDTA, collected, washed twice with PBS, followed by resuspended in an endothelial cell culture liquid containing 0.1% BSA, and counted under a microscope, and the cell concentration was adjusted to 1×10.sup.5 cells/mL;

(90) (3) Test solutions for each group were formulated and diluted to 100 μL with a cell culture liquid containing 0.1% BSA;

(91) Groups were divided as follows:

(92) Blank control group: a cell culture liquid containing no drug;

(93) Endostar group: 5 mg/mL Endostar solution diluted to a predetermined concentration with a cell culture liquid containing no drug;

(94) Fusion protein group: the fusion protein diluted to 10 μg/mL with a cell culture liquid containing no drug;

(95) (4) The cells were inoculated into a transwell chamber, 100 μL per well, and test solutions for each group were added to the chamber. To a 24-well plate, 0.6 mL of endothelial cell culture liquid containing 5% fetal bovine serum and 1×ECGS was added to stimulate cell migration, and incubated at 5% CO.sub.2 at 37° C. for 24 h;

(96) (5) The culture liquid in the well was discarded. The cells were fixed with 90% alcohol at room temperature for 30 min, stained with 0.1% crystal violet for 10 min at room temperature, and rinsed with water. Un-migrated cells in the upper layer were gently wiped off by a cotton swab. The observation was carried out under a microscope and four fields of view were selected for taking photos and counting. The migration inhibition (MI) was calculated according to the formula:

(97) $MI\left(\%\right)=1-\frac{N_{\mathrm{test}}}{N_{\mathrm{control}}}\times 100\%$

(98) where N.sub.test is the migration cell number in the test group, and N.sub.control is the migration cell number in the blank control group.

(99) Data Statistics:

(100) The test was repeated 3 times independently. The results obtained from the test were calculated as mean±SD, and statistical t-test was performed. P<0.05 was considered as a significant difference, and P<0.01 was considered as extremely significant difference. The experimental results are shown in Table 9.

(101) TABLE-US-00011 TABLE 9 Migration inhibition of HUVEC by protein I and protein II Group (n = 5) Dose (μg/mL) Migration cell number MI (%) Protein I 0.25 2040.0 ± 180.21 18.17% 0.5   1738 ± 366.83* 30.28% 1  933.0 ± 150.58** 62.58% 2  731.0 ± 236.53** 70.68% 4  786.0 ± 45.50** 68.47% 8 1385.0 ± 176.11* 44.44% Protein II 0.25 2122.0 ± 75.18 14.88% 0.5 1215.0 ± 221.60** 51.26% 1  684.0 ± 28.58** 72.56% 2  578.0 ± 275.42** 76.82% 4  874.0 ± 141.50** 64.94% 8 1758.0 ± 180.03* 29.48% Sunitinib 8.0 .Math. 10.sup.−6  441.0 ± 150.58** 82.31% control — 2493.0 ± 85.12 *P < 0.05 **P < 0.01 vs control.

(102) It can be seen from the experimental results that under the action of fusion proteins I and II, the number of migrated endothelial cells was significantly reduced compared with that of the negative control, and the migration inhibition of HUVEC was significant at a concentration of 2 μg/mL. The inhibition rate was 70% or more, and the inhibition rate on cell migration was extremely significant compared with that of the negative control (P<0.01). Between 0.5 μg/mL and 4 μg/mL, the best inhibitory effect was achieved.

Example 4

(103) Effect of Fusion Proteins I and II on Proliferation of Mouse Spleen Lymphocytes

(104) The spleen of a mouse was taken out under sterile conditions, washed 3 times with an empty 1640 medium, ground in 5 mL syringe core, filtered through a 200-mesh sieve, and prepared into a single cell suspension, the single cell suspension was centrifuged (1000 rpm, 5 min), and the supernatant was discarded. Tris-NH.sub.4Cl was used to break the red blood cells, which were allowed to stand in an ice water bath for 4 min and centrifuged (1000 rpm, 5 min), the supernatant was discarded, and the cells were washed twice with sterile PBS. Finally, cells were suspended in a 10% fetal calf serum RPMI 1640 culture liquid (5 mL), counted, adjusted to a cell concentration of 5×10.sup.6 cells/mL, and cultured in a 96-well culture plate.

(105) The experiment comprises a blank control group, a concanavalin A (ConA) group, a dexamethasone (Dex) group (0.02 mg/mL), and protein I and protein II used as experimental groups. After each group was added with spleen lymphocyte suspension, 100 μL per well, the blank control group was added with 100 μL of empty 1640 culture liquid, ConA group was added with ConA (final concentration of 5 μg/mL), and Dex group was added with Dex, and protein I and protein II were added with ConA (final concentration of 5 μg/mL) on the basis of addition of different concentrations of extracts. The cells were statically cultured in a cell incubator at 37° C. for 48 h. After the completion of the culture, 20 μL of MTT was added to each well, and the culture was continued for 4 h. Finally, all the solutions in each well were discarded, 100 μL of DMSO was added to each well, and the mixture was shaken and detected by a microplate reader for OD value at 570 nm. 5 parallels were preformed for per well. The experimental results are shown in Table 10.

(106) TABLE-US-00012 TABLE 10 Effect of protein I and protein II on the proliferation of mouse spleen lymphocytes Group (n = 5) Dose (μg/mL) A570 nm/A630 nm PI (%) Protein I 8 0.5520 ± 0.0182  6.27% 32 0.4902 ± 0.0122* 16.76% 128 0.3741 ± 0.0911** 36.47% Protein II 8 0.5633 ± 0.0237  4.35% 32 0.4963 ± 0.0886* 15.72% 128 0.3965 ± 0.0122** 32.67% ConA — 0.6201 ± 0.0349 — Dex 20 0.3468 ± 0.1144** 41.11% control — 0.5889 ± 0.0528 *P < 0.05 **P < 0.01 vs control.

(107) The results showed that fusion protein I and protein II could inhibit mouse spleen lymphocytes to some extent compared with the ConA group.

Example 5

(108) Effect of Fusion Proteins I and II on IL-1β Production by Mouse Peritoneal Macrophages

(109) (1) IL-1β production: mice were intraperitoneally injected with 1 mL of broth medium containing 6% starch, and three days later, the mouse peritoneal macrophages were aseptically taken, washed twice with a 1640 medium, adjusted to the cell concentration of 2×10.sup.6 cells/mL, inoculated into a 24-well culture plate, 1 mL/well, incubated for 3 h in a cell culture incubator, and shaken once every 30 min, so that the cells were allowed to adhere sufficiently. Then, the cells were washed twice with a culture liquid to remove un-adhered cells. The blank group was added with PBS, the positive group was added with the positive drug dexamethasone Dex, and the control groups were the low, medium and high concentrations of fusion protein I and protein II. After administration, the culture was continued for 48 h, the cells were centrifuged at 1000 r/min for 15 min. The supernatant was taken as a sample for testing activity of IL-1β.

(110) (2) Determination of IL-1β content: the detection was performed by using mouse IL-1β enzyme-linked immunosorbent assay kit from R&D company. According to the instructions of the kit, the procedures were as follows: the reaction well for the test samples and standards with different concentration were sealed with sealing tapes, respectively, incubation was performed at 37° C. for 90 min; the plate was washed four times; a biotinylated antibody working solution (100 μL/well) was added, the reaction well was sealed with sealing tapes, and incubation was performed at 37° C. for 60 min; the plate was washed four times; an enzyme conjugate working solution (100 μL/well) was added, the reaction well was sealed with sealing tapes, incubation was performed at 37° C. for 30 min; the plate was washed four times; a developer (100 μL/well) was added, incubation was performed at 37° C. away from light for 10 to 20 min, a stop solution (100 μL/well) was added and mixed, and the OD450 value was measured. The experimental results are shown in Table 11.

(111) TABLE-US-00013 TABLE 11 Effect of protein I and II on IL-β production by mouse peritoneal macrophages Group (n = 5) Dose (μg/mL) IL-1β (pg/ml) PI (%) Protein I 8 722.85 ± 9.95 16.04% 32 495.30 ± 14.58* 42.47% 128 322.56 ± 13.52** 62.53% Protein II 8 755.56 ± 17.82 12.24% 32 610.06 ± 19.97* 29.14% 128 468.96 ± 12.59** 45.53% Model group 20 860.96 ± 5.53* Dex 20 322.46 ± 20.56** 61.38% control —   8.79 ± 2.26* *P < 0.05, **P < 0.01 vs control.

(112) The results showed that fusion protein I and II could significantly inhibit IL-113 production by mouse peritoneal macrophages.

Example 6

(113) Effect of Fusion Proteins I and II on Subacute Inflammation of Tampon Granuloma in Rat

(114) 40 parts of degreasing cotton, 30 mg for each part, were accurately weighed with an analytical balance and kneaded into balls having substantially the same shape and size, which were autoclaved at 1.5 kpa for 30 min and dried at 50° C. for further use.

(115) 40 male SD rats were randomly divided into 4 groups with 10 rats for each group. The groups were a model group, a dexamethasone positive group (10 mg/kg), and fusion protein I and protein II experimental groups at an effective dose of 64 mg/kg, respectively. Rats were anesthetized with sodium pentobarbital (40 mg/kg) via intraperitoneal injection before administration. The abdominal coat was cut off, and the skin of the middle of the lower abdomen was cut under sterile conditions. The incision was about 1 cm long and the subcutaneous tissue was expanded with a vascular clamp. A sterile dry tampon was subcutaneously implanted into one side of the groin, the incision was sutured, and an appropriate amount of amoxicillin was spread at the incision to prevent infection. After the surgery is finished, the groups were administered once by injection every 5 days from the day of surgery. On day 7, the rats were sacrificed by cervical dislocation at the 24th hour after administration, the inguinal skin was cut, the tampon was taken out together with the surrounding granulation tissue and the surrounding tissue was removed. After drying for additional 48 h in an oven at 60° C., the weight was accurately weighed. The granuloma weight was calculated: granuloma weight (mg/100 g body weight)=net weight of granulation (mg)/rat body weight (100 g). The experimental results are shown in Table 12.

(116) TABLE-US-00014 TABLE 12 Effect of protein I and protein II on subacute inflammation of tampon granuloma in rat Granuloma Weight (mg)/Weight Group (n = 10) gain (g) (100 g) PI (%) Protein I (64 mg/kg)  32.09 ± 10.55* 0.37 ± 0.09* 19.57% Protein II (64 mg/kg)  33.89 ± 9.79** 0.38 ± 0.02** 17.39% Dex (10 mg/kg) −32.98 ± 7.68** 0.22 ± 0.06** 52.17% control  26.62 ± 5.59* 0.46 ± 0.12* *P < 0.05, **P < 0.01 vs control.

(117) The experimental results showed that fusion protein I and II could significantly inhibit tampon granuloma in rat at an effective dose of 64 mg/kg, compared with the blank model group. Although the positive drug had a higher inhibition rate, the weight loss of the rat was obvious, and the side effects were larger. In contrast, the fusion protein was relatively safe.

Example 7

(118) Effect of Fusion Proteins I and II on Peritoneal Capillary Permeability in Mice

(119) 80 Kunming mice were randomly divided into 8 groups with 10 mice for each group, which were a blank model group, a dexamethasone positive group (10 mg/kg), and fusion protein I, protein II experimental groups at high, medium and low doses (128, 32, 8 mg/kg), respectively. The drug was administered once by injection every 5 days, and the blank model group was given an equal volume of physiological saline for 5 days and fed normally. On the 5th day after the administration, a 5 g/L of Evans blue physiological saline solution was injected into the tail vein at 10 kg/mL, followed by intraperitoneal injection (10 kg/mL) HAc solution (6 mL/L) to induce inflammation. After 20 min, the mice were sacrificed by cervical dislocation. 5 mL of physiological saline was intraperitoneally injected, the abdomen was gently rubbed for 2 min, the abdominal cavity was cut, a peritoneal washing solution was collected and centrifuged at 4000 rpm for 10 min, 1 mL of supernatant was taken, and 3 mL of physiological saline was added to obtain a 4 mL of dilution. The absorbance OD value of the dilution was measured by an ultraviolet spectrophotometer at a wavelength of 590 nm, and the amount of pigment exudation was expressed by the OD590 nm value, and peritoneal capillary permeability in mice was examined. The experimental results are shown in Table 13.

(120) TABLE-US-00015 TABLE 13 Effect of protein I and protein II on peritoneal capillary permeability in mice Dose Exudation rate Group (n = 10) (mg/kg) (OD590) PI (%) Protein I 8 0.59 ± 0.02 10.61% 32 0.46 ± 0.02* 30.30% 128 0.35 ± 0.05** 46.97% Protein II 8 0.55 ± 0.07 16.67% 32 0.42 ± 0.09* 36.36% 128 0.31 ± 0.06** 53.03% Dex 10 0.27 ± 0.03** 59.09% control — 0.66 ± 0.04* *P < 0.05, **P < 0.01 vs control.

(121) The results showed that fusion protein I and II could significantly inhibit the increase of peritoneal capillary permeability in mice induced by glacial acetic acid. The higher the dose, the stronger the effect.

Example 8

(122) Effect of Fusion Proteins I and II on Xylene-Induced Ear Swelling in Mice

(123) 80 Kunming mice were divided into 8 groups with 10 mice for each group and numbered. A physiological saline group was used as a blank control group, an aspirin group (200 mg/kg) was used as a positive control group, and fusion proteins I and II at high, medium and low doses (128, 32, 8 mg/kg) was used as the experimental groups. Mice were administered once by injection every 5 days. The blank control group was given an equal volume of physiological saline. On the fifth day after the administration, 0.05 mL of xylene was applied to both sides of the right ears of the mice to induce inflammation, and the left ears were not applied and were normal ears. After 2 h, the mice were sacrificed by dislocation, and the ears were cut along the auricle. Ear pieces were taken with a puncher and weighed, and the swelling degree and swelling rate were calculated. Swelling degree=right ear piece weight-left ear piece weight, swelling rate=(swelling degree/left ear piece weight)×100%. The experimental results are shown in Table 14.

(124) TABLE-US-00016 TABLE 14 Effect of protein I and protein II on xylene-induced ear swelling in mice Group Dose Swelling (n = 10) (mg/kg) degree (mg) PI (%) Protein I 8 6.18 ± 0.20  7.21% 32 5.21 ± 0.31* 21.77% 128 4.19 ± 0.28** 37.09% Protein II 8 5.92 ± 0.15 11.11% 32 5.01 ± 0.84* 24.77% 128 3.89 ± 0.39** 41.59% Aspirin 200 3.12 ± 0.61** 53.15% control — 6.56 ± 0.47* *P < 0.05, **P < 0.01 vs control.

(125) The experimental results showed that high doses of fusion proteins I and II could significantly inhibit xylene-induced ear swelling in mice, and the inhibitory effect could be enhanced with the increase of dose.

Example 9

(126) Effect of Fusion Proteins I and II on Acute Inflammation of Toe Swelling in Rat Induced by Carrageenan

(127) 80 SD rats were randomly divided into 8 groups with 10 mice for each group. The groups were a blank model group, a dexamethasone positive group (5 mg/kg) and fusion protein I and protein II experimental groups at high, medium and low doses (128, 32, 8 mg/kg), respectively. The drug was administered once by injection every 5 days, and the model group was given an equal volume of physiological saline for 3 days and fed normally. On the third day after the administration, 0.1 mL of 1% carrageenan was injected subcutaneously into the right hind toes of the rats to induce inflammation. The foot volume was measured at 1 h, 3 h, 5 h, and 7 h after inflammation. The swelling degree of the foot was calculated according to the following formula: the swelling degree of the foot (mL)=the volume of the foot after inflammation−the volume before inflammation. The number of milliliters of spilled liquid was recorded (method: the protruding point of the right joint was circled with a ballpoint pen and used as a measurement mark, and the right hind feet of the rats were sequentially placed in the volume measuring device, so that the hind limbs were exposed outside the cylinder, and the depth of the immersion was limited to the overlap of the circle and the liquid level. After the foot entered the liquid, the liquid level was raised, and the volume of the spilled liquid was the volume of the right hind foot of the rat, and the normal volume of the right hind foot of each mouse is sequentially determined). The experimental results are shown in Table 15.

(128) TABLE-US-00017 TABLE 15 Effect of protein I and protein II on acute inflammation of toe swelling in rat induced by carrageenan Swelling degree(mL) Group (n = 10) Dose (mg/kg) 1 h 3 h 5 h 7 h Protein I 8 0.27 ± 0.13 0.41 ± 0.15 0.44 ± 0.15 0.35 ± 0.09* 32 0.29 ± 0.07* 0.36 ± 0.21 0.37 ± 0.14* 0.34 ± 0.11* 128 0.28 ± 0.15* 0.32 ± 0.08** 0.34 ± 0.21* 0.32 ± 0.18** Protein II 8 0.26 ± 0.13 0.42 ± 0.12* 0.42 ± 0.13 0.38 ± 0.08* 32 0.25 ± 0.21 0.30 ± 0.14* 0.35 ± 0.09* 0.32 ± 0.19 128 0.27 ± 0.05* 0.32 ± 0.15* 0.33 ± 0.10** 0.31 ± 0.16** Dex 10 0.21 ± 0.11** 0.26 ± 0.10** 0.26 ± 0.11** 0.25 ± 0.09* control — 0.26 ± 0.22 0.44 ± 0.17 0.54 ± 0.06 0.39 ± 0.21 *P < 0.05, **P < 0.01 vs control.

(129) The experimental results showed that the toes of the rats in each group were rapidly swollen after modeling, and the peak of swelling was reached at about 3-5 h, which began to subside at 7 h. The fusion proteins I and II at high dose could significantly inhibit toe swelling in rat induced by carrageenan, and the inhibitory effect was not significant at low dose.

Example 10

(130) Effect of Fusion Proteins I and II on Chronic Inflammation of Adjuvant Arthritis in Rat

(131) Model Establishment:

(132) 80 SPF SD rats were randomly divided into 8 groups. Rats in each group were lightly anesthetized with ether. Then, 0.1 mL of complete Freund's adjuvant containing inactivated Mycobacterium tuberculosis was injected subcutaneously into the left hind foot of the rats. Primary arthritis occurred in the left hind foot of the rat, and at about 13 days post-modeling, secondary arthritis occurred in the right hind foot. A blank control group was injected with an equal volume of physiological saline. The drug was administered 13 days after modeling. The methotrexate group was administered once by injection every 5 days for 15 days, 4 times in total; the fusion protein I and protein II at high, medium and low doses (128 mg/kg, 32 mg/kg, 8 mg/kg) were administered by injection once every 5 days for 15 days.

(133) Efficacy Evaluation:

(134) 1. Primary and Secondary Toe Swelling Degree

(135) Using a foot volume measuring method, a marker was made with a fat-soluble marker at the left and right posterior ankle joints of each rat, and the left and right hind feet of the animal were respectively immersed in the volume measuring device. The immersion depth was bounded by the marker, and the reading value at the scale pipette of the device was the initial volume of the animal's left and right hind feet.

(136) The day of modeling was considered as the 0th day and recorded as d0. The volume of the left hind foot (modeling foot) was measured from the first day dl after modeling every 2 days. When the swelling occurred (i.e., secondary arthritis occurred) at the contralateral non-inflammatory foot (right hind foot), the administration was started. The volume of the left and right hind feet was measured once every 2 days, and the degree of primary and secondary toe swelling was determined, which was calculated as follows:
Primary toe swelling (mL)=left hind foot volume on the day of measurement−initial volume of left hind foot
Secondary toe swelling (mL)=right hind foot volume on the day of measurement−initial volume of right hind foot

(137) 2. Clinical Score

(138) Systemic score: the systemic score was taken every 2 days after the onset of secondary inflammation.

(139) Hind foot: no swelling=0 score, one hind foot swelling=1 score, two hind feet swelling=2 scores;

(140) Forefoot: no swelling=0 score, one forefoot swelling=1 score, two forefeet swelling=2 scores;

(141) Ears: no redness and nodules=0 score, redness or nodules in one ear=1 score, redness and nodules in both ears=2 scores;

(142) Nose: no swelling=0 score, obvious swelling=1 score;

(143) Tail: no nodules=0 score, nodules=1 score; full score was 8 scores.

(144) Arthritis index score: the arthritis index score was performed every 2 days after the onset of secondary inflammation.

(145) Normal=0 score; erythema and mild swelling in the ankle joint=1 score; erythema and mild swelling from the ankle to the metatarsophalangeal joint or metacarpal joint=2 scores; erythema and moderate swelling from the ankle to the metatarsophalangeal joint or metacarpal joint=3 scores; erythema and severe swelling from the ankle to the metatarsophalangeal joint or metacarpal joint=4 scores; each foot had a full score of 4 scores, and each rat had a maximum score of 16 scores.

(146) 3. Weight Gain

(147) The initial body weight of each group of rats was weighed before modeling. The body weight was measured every 2 days from dl after modeling, from which the initial body weight was subtracted to obtain the weight gain of each group of rats. The experimental results are shown in Table 16.

(148) TABLE-US-00018 TABLE 16 Effect of protein I and protein II on chronic inflammation of adjuvant arthritis in rat Group Dose Foot swelling degree (mL) Clinical score Weight gain (n = 10) (mg/kg) Left Right Whole body Arthritis index (g) Protein I 8 1.94 ± 0.31  1.97 ± 0.08  2.09 ± 0.17*  6.79 ± 0.52  44.14 ± 21.57  32 1.67 ± 0.04** 1.56 ± 0.22* 1.84 ± 0.43  5.75 ± 0.47*  48.61 ± 16.92* 128 1.41 ± 0.17**  1.35 ± 0.08** 1.54 ± 0.08** 4.36 ± 0.45** 51.87 ± 16.34* Protein II 8 1.88 ± 0.08  1.89 ± 0.55  2.14 ± 0.25*  6.97 ± 0.57  45.29 ± 11.82  32 1.59 ± 0.23*  1.63 ± 0.33* 1.89 ± 0.37  5.68 ± 0.44*  47.91 ± 19.30* 128 1.35 ± 0.17** 1.37 ± 0.77* 1.57 ± 0.18** 4.23 ± 0.69**  50.88 ± 15.37** Methotrexate 1 1.22 ± 0.21**  1.25 ± 0.13** 1.34 ± 0.19** 2.96 ± 0.57**  47.35 ± 17.40** control — 2.14 ± 0.09  2.12 ± 0.18  2.59 ± 0.28  7.54 ± 0.36  29.50 ± 20.32  *P < 0.05, **P < 0.01 vs control.

(149) The experimental results showed that after modeling, the left hind foot in each group was swollen rapidly (primary inflammation), and on the 13th day, the hind foot (non-contralateral inflammatory foot) began to be red and swollen (i.e., secondary inflammation occurred). The arthritis index and systemic score began to increase, reaching the highest value on the 19th day, and the swelling degree and score of each group were gradually decreased along with administration. The primary toe swelling degree was used to reflect the therapeutic effect of each treatment group on primary arthritis. The high and medium doses of each administration group could treat primary arthritis to a certain extent compared with the model group. The positive drug methotrexate had the best effect, and the fusion protein I and protein II were effective in high dose groups, with extremely significant differences (**P<0.01). The secondary toe swelling degree was used to reflect the therapeutic effect of each treatment group on secondary arthritis.

Example 11

(150) Inhibitory Effect of Fusion Proteins I and II on Proliferation of Human Retinal Vascular Endothelial Cell (HRCEC)

(151) The activity of the integrin blocker polypeptide to inhibit proliferation of human retinal vascular endothelial cells was examined by MTT assay. HRCEC cells were cultured in a 5% CO.sub.2 incubator at 37° C. to a density of 90% or more, and then collected by trypsinization. The cells were resuspended in the culture liquid and counted under a microscope. The cell concentration was adjusted to 3.0×10.sup.4 cells/mL. The cell suspension was inoculated into a 96-well plate, 100 μL per well, and cultured overnight in a 5% CO.sub.2 incubator at 37° C. The polypeptide I, the polypeptide II, the polypeptide III, and the Avastin® (bevacizumab) were diluted with the culture liquid to respective predetermined concentrations. After the cells were fully adhered, each dilution was added to a 96-well plate, 100 μL per well, respectively. The integrin blocker polypeptide was used as an administration group, and Avastin® (bevacizumab) was used as a positive control group, and a culture liquid containing no drug was used as a blank control group, which were incubated in a 5% CO.sub.2 incubator at 37° C. for 48 hours. 20 μL of 5 mg/mL MTT was added to each well of a 96-well plate, and incubation was continued for 4 hours. The medium was aspirated and 100 μL of DMSO was added per well for dissolution. The absorbance was measured at 570 nm with a microplate reader with a reference wavelength of 630 nm, and the proliferation inhibition (PI) was calculated. The formula was as follows:

(152) $PI\left(\%\right)=1-\frac{N_{\mathrm{test}}}{N_{\mathrm{control}}}\times 100\%$

(153) where N.sub.test is the OD value of the test group and N.sub.control is the OD value of the blank control group.

(154) Data Statistics:

(155) The test was repeated 5 times independently. The results obtained from the test were calculated as mean±SD, and statistical t-test was performed. P<0.05 was considered as a significant difference, and P<0.01 was considered as an extremely significant difference. The experimental results are shown in Table 17.

(156) TABLE-US-00019 TABLE 17 Inhibitory effect of protein I and protein II on proliferation of human retinal vascular endothelial cell (HRCEC) Dose Group (n = 5) (μg/mL) A570 nm/A630 nm PI (%) Protein I 1 1.0639 ± 0.0162 12.94% 2 1.0025 ± 0.0329 17.96% 4 1.0750 ± 0.0324* 12.03% 8 0.9567 ± 0.0467* 21.71% 16 0.8459 ± 0.0731* 30.78% 32 0.7126 ± 0.0486** 41.69% 64 0.5618 ± 0.0222** 54.03% 128 0.4567 ± 0.0181** 62.63% 256 0.3274 ± 0.01.6** 73.21% Protein II 1 1.0588 ± 0.0247 13.36% 2 1.0079 ± 0.0392 17.52% 4 1.0380 ± 0.0712*  5.06% 8 0.9911 ± 0.0697 18.90% 16 0.8865 ± 0.0143* 27.45% 32 0.7142 ± 0.0199** 41.55% 64 0.5911 ± 0.0478** 51.63% 128 0.4632 ± 0.0597** 62.09% 256 0.3354 ± 0.0858** 72.55% Avastin ® 5 0.4544 ± 0.0288** 62.82% (bevacizumab) control — 1.2222 ± 0.0464  0.00% *P < 0.05, **P < 0.01 vs control.

(157) The results showed that fusion proteins I and II could significantly inhibit the proliferation inhibition of human retinal vascular endothelial cells (HRCECs) in a dose-dependent manner. At a concentration of 64 μg/mL, the inhibition rate reached 50% or more.

Example 12

(158) Activity of Fusion Proteins I and II to Inhibit Angiogenesis In Vivo Analyzed by Chicken Embryo Chorioallantoic Membrane (CAM)

(159) In this study, CAM assay was used to investigate the activity of fusion protein I and protein II to inhibit angiogenesis in vivo. The study has shown that the biosynthesis rate of collagen reached the maximum on the 8th to 11th day of chicken embryo development, which was the most vigorous stage of angiogenesis, and the body's immune system had not yet been fully established at that time, and thus the chicken embryos developed to the 8th day was selected to be administered. Considering that the polypeptide on drug-loaded paper had a certain diffusion range limitation on the chicken embryo chorioallantoic membrane, only the number of new blood vessels within a radius of 5 mm from the edge of the paper was counted in the test. The following steps were used:

(160) (1) The White Leghorn chicken embryos on day 6 were cultured in a 37° C. incubator at 60%-70% humidity for two days.

(161) (2) A 1.0 cm×1.0 cm window was drilled above the chicken embryo air sac, and the inner membrane was torn off with forceps to expose the chorioallantoic membrane. Lens paper having a diameter of 5 mm was used as a loading carrier, and was placed on the chorioallantoic membrane of the chicken embryo air sac. Filter paper with PBS was used as a blank group, and an administration group was added with different doses of fusion protein. The positive control was Avastin® (bevacizumab).

(162) (3) The chicken embryo air sac was sealed with a sterile transparent tape, and after culturing at 37° C. for 72 hours, the chicken embryo air sac was opened, and a fixative (formaldehyde:acetone=1:1) was added for fixation for 15 minutes. The chorioallantoic membrane to which the lens paper was adhered was taken out, the distribution of the new blood vessels was observed, and the new blood vessels were counted and photographed. Five replicates were set for each dose and the results were statistically analyzed.

(163) The analysis results of the activity of fusion protein to inhibit angiogenesis in vivo by the chicken embryo chorioallantoic membrane (CAM) assay were as follows: negative control was treated with PBS, the dose of positive control Avastin® (bevacizumab) was 10 fusion protein I and protein II was used to treat the chicken embryos at high, medium and low doses of 128 μg, 32 μg and 8 μg, respectively. The results are shown in Table 18.

(164) TABLE-US-00020 TABLE 18 Inhibitory effect of protein I and protein II on angiogenesis of chicken embryo chorioallantoic membrane Group (n = 5) Dose (μg) Blood vessel number PI (%) Protein I 8 89 ± 8  32.58% 32 81 ± 3  38.64% 128  55 ± 10** 58.33% Protein II 8 105 ± 12  20.45% 32 86 ± 13 34.85% 128 69 ± 8* 47.73% Avastin ® 10  63 ± 17** 52.27% (bevacizumab) control — 132 ± 15  0.00% *P < 0.05, **P > 0.01 vs control.

(165) The results showed that fusion protein I and protein II could inhibit angiogenesis in CAM, and had a strong inhibitory effect (nearly 50%) at high dose.

Example 13

(166) Effect of Fusion Proteins I and II on Corneal Neovascularization in Mice

(167) (1) Preparation of Corneal Neovascularization Model Induced by Alkali Burn in BALB/c Mice:

(168) 15 healthy male BALB/c mice with weight of 20-25 g were examined under a slit lamp microscope for the anterior segment of both eyes and the appendage to exclude ocular lesions. The eyes were given 0.3% loxacin eye drops 1 day before the preparation of alkali burn model, twice a day. After the mice were anesthetized by intraperitoneal injection of 1.8% Avertin, single-layer filter paper with a diameter of 2 mm was clamped with tweezers, and immersed in a 1 mol/L sodium hydroxide solution to reach a saturated state, and the excess liquid was removed. The filter paper was placed in the central corneal of BALB/c mice for 40 s and then discarded, and the burned area and conjunctival sac were immediately rinsed with 1 mL of PBS for 1 min. Excess water was wiped away with cotton swabs, and under an operating microscope, the corneal epithelium was vortically scraped off by paralleling a corneal scraping knife to the limbus corneae. The subcutaneous stromal layer and limbus corneae was carefully not to be injured, and after surgery, an erythromycin eye ointment was applied into the conjunctival sac to prevent infection.

(169) (2) Experimental Animal Grouping and Sample Acquisition:

(170) 15 mice were randomly divided into fusion protein I and protein II groups and a control group, with 5 rats in each group. After alkali burn, 64 μg of fusion protein I and 64 μg of protein II and saline were given once via intravitreal injection every 7 days, and the inflammatory reaction and neovascularization of the cornea in each group were observed under a slit lamp microscope on day 1, day 7, and day 14 after alkali burn. On day 14 after alkali burn, the corneal neovascularization in each group was photographed and recorded under the slit lamp microscope for photographing anterior segment of the eye. Then, all the mice were sacrificed by cervical dislocation and the eyeballs were removed. The blood was washed with physiological saline, and the eyeballs were fixed in 4% paraformaldehyde for 1.5 h, dehydrated in PBS containing 30% sucrose overnight, embedded in an OCT tissue freezing medium, stored in a refrigerator at −80° C., subjected to cryosection at 8 μm, and detected by immunohistochemistry for CD31 expression.

(171) (3) Quantitative Measurement for Microvessel Density of Corneal Tissue:

(172) Microvessel density (MVD) is an indicator for evaluating angiogenesis. An anti-CD31 antibody immunohistochemistry was used to label vascular endothelial cells and the number of microvessels per unit area was counted to measure the extent of neovascularization. Standards for counting microvessels were that under a microscope, the endothelial cells or cell clusters which were clearly demarcated from adjacent tissues in the corneal tissue and were stained tan or brown were counted as the new blood vessels. The number of new blood vessels in the entire section was counted under a 10×20 microscope. After the corneal tissue was photographed, the area of the entire corneal tissue was calculated by image processing software Image J, and the density of new blood vessels in the entire section in this example was determined. The results are shown in Table 19.

(173) TABLE-US-00021 TABLE 19 MVD count showing effect of protein I and protein II on corneal neovascularization in mice Group (n = 5) Dose (μg) MVD Protein I 64 27.38 ± 6.13* Protein II 64 23.98 ± 4.50* control — 52.11 ± 7.85* *P < 0.05, **P < 0.01 vs control.

(174) The results showed that CD31 was used as a microvascular marker, which was mainly expressed in the cytoplasm of vascular endothelial cells. The stained positive cells were vascular endothelial cells stained tan or brown without background staining. The number of CD31-positive new blood vessels in fusion protein I and II experimental groups was significantly reduced compared with that of the control group. Fusion protein I and II groups had significant difference compared with the control group. The experimental results showed that fusion protein I and II could inhibit the growth of corneal new blood vessels, and can be used as a drug for the treatment of corneal neovascular eye diseases.

Example 14

(175) Effect of Fusion Proteins I and II on Iris Neovascularization in Rabbits

(176) The argon ion laser at 577 nm was used to occlude the major branch vein of rabbit retina, and a success venous occlusion was confirmed by fundus fluorescein angiography (FFA). After 5-12 days, the iris fluorescein angiography (IFA) showed that the fluorescein leakage was obvious in the iris vessels compared with the normal control group, confirming the formation of the animal model of the iris neovascularization (NVI).

(177) 9 eyes successful in modeling were randomly divided into 3 groups with 3 eyes for each group. They were labeled as a negative control group, and fusion protein I and II treatment groups, respectively, which were respectively given physiological saline, 128 μg of fusion protein I and 128 μg of fusion protein II via intravitreal injection once every 7 days for 2 weeks. The observation was performed with an optical and electron microscope on the third week.

(178) Results: under the optical microscope, it was observed that the anterior surface of the iris was a fibrous vascular membrane remnant mainly consisting of fibrous tissue, and there were few open vascular lumens. Vascular residues can be seen in the iris matrix, which are necrotic cells and cell debris. The iris surface of the control eye under a light microscope is a fibrous vascular membrane with branches and potential lumens.

(179) The ultrastructure of the iris in the treatment group showed a series of degenerative changes. The endothelial cells of the large blood vessels in the middle of the iris matrix had normal nucleus, cytoplasm and cell junctions. There were capillary residues in the iris matrix and on the anterior surface of the iris, which were surrounded by cell debris and macrophage infiltration. No capillary with potential lumens and degenerated parietal cells indicated regression of new blood vessels.

(180) Through animal model experiments of iris neovascularization, it was demonstrated that fusion protein I and fusion protein II could inhibit neovascularization and regress the formed blood vessels.

Example 15

(181) Effect of Fusion Proteins I and II on Choroidal Blood Flow in Rabbit Eyes

(182) New Zealand white rabbits with weight of 2.5-3.0 kg were randomly divided into 3 groups, which were labeled as a control group, and fusion protein I and II groups. White rabbits in each group were anesthetized with 35 mg/kg xylazine via intramuscular injection, and then anesthesia was maintained with half of the initial amount via intramuscular injection per hour. The intraocular pressure of the left eye was increased to 40 mmHg, under which the blood flow can be reduced to ⅓ of the normal value. The left ventricle was cannulated through right carotid artery for injection of microspheres (for the calculation of ocular blood flow), and the femoral artery was cannulated for blood collection. Each group was given physiological saline, 128 μg of fusion protein I and 128 μg of fusion protein II via intravitreal injection. The ocular blood flow of rabbit eyes with high intraocular pressure was measured by a color microsphere technique at 0, 30, and 60 minutes after administration. At each time point, 0.2 mL (about 2 million) of microspheres were injected. Immediately after the microspheres were injected, blood was collected through the femoral artery for 60 seconds, and placed in a heparinized anticoagulant tube, and the amount of blood collected was recorded. After the last blood collection, the animals were sacrificed with 100 mg/kg phenobarbital via intravenous infusion. The eyeballs were removed, and the retina, choroid, iris and ciliary body were separated, and the tissue weight was recorded. The tissue blood flow at each time point was calculated with the following formula: Qm=(Cm×Qr)/Cr, where Qm represented tissue blood flow in μL/min/mg; Cm was the number of microspheres per milligram of tissue; Qr was blood flow in μL/min; and Cr was the number of blood microspheres as a reference. The experimental results are shown in Table 20.

(183) TABLE-US-00022 TABLE 20 Effect of protein I and protein II on choroidal blood flow in white rabbit eyes Blood flow Group (n = 3) Dose (μg) Time (mm) (μL/min/mg) Protein I 128 0 23.1 ± 2.6 128 30 17.5 ± 3.1 128 60 14.3 ± 1.9 Protein II 128 0 23.3 ± 2.7 128 30 15.6 ± 1.2 128 60 14.6 ± 1.4 control — 0 12.7 ± 1.4 — 30  9.6 ± 1.8 — 60  5.8 ± 1.9

(184) The results showed that choroidal blood flow was significantly increased in the fusion proteins I and II treatment groups at all observation time points.

Example 16

(185) Effect of Fusion Proteins I and II on Retinal Blood Vessels in OIR Mice

(186) Establishment of the OIR model: young mice and their mothers were exposed to 75% hyperoxic environment from day 7 to day 12 after birth of C57/B16 mice so that capillaries in the central retina rapidly disappeared. On day 12, the mice were returned to indoor air and the retinal blood vessels exposed to hyperoxia rapidly disappeared, which caused extensive abnormal neovascularization, and the central part of the retina remained largely avascular for a long time. After the blood vessels disappeared completely, the fusion protein (administration group, the doses of fusion proteins I and II were both 64 μg) or physiological saline (negative group) was injected into the vitreous body on day 13. Retinal vessels were evaluated on day 17 (labeled as unclosed vessels, 50 mL of Texas Red-labeled tomato lectin was injected into the left ventricle and cycled for 5 minutes). The experimental results are shown in Table 21.

(187) TABLE-US-00023 TABLE 21 Effect of protein I and protein II on retinal blood vessels in OIR mice Group (n = 5) Dose (μg) Area (mm.sup.2) Reduce (%) Protein I 64 0.111 ± 0.027* 52.97% Protein II 64 0.153 ± 0.022* 35.17% control — 0.236 ± 0.039   0.00% *P < 0.05, **P < 0.01 vs control.

(188) The results showed that the administration of fusion proteins I and II to OIR mice could alleviate pathological neovascularization. Compared with the negative control, the neovascular clusters in the retina of OIR mice treated with fusion proteins I and II were significantly reduced, and the areas occupied by neovascular clusters were decreased by 52.97% and 35.17%, respectively.

Example 17

(189) Effect of Fusion Proteins I and II on Neovascularization in Premature Rat Retinopathy Model

(190) A fluctuating oxygen-induced animal model was adopted, and newborn rats (within 12 hours) spontaneously delivered on the same day were randomly divided into three groups: an oxygen model group, an oxygen treatment group, and a normal control group. The oxygen model was subdivided into three model subgroups, which were placed in a semi-closed oxygen chamber made of plexiglass together with the treatment group. The medical oxygen was introduced into the chamber, and the oxygen concentration was adjusted to 80%±2% with an oxygen meter. After 24 hours, nitrogen gas was introduced into the oxygen chamber, and then the oxygen concentration was rapidly adjusted to 10%±2% and maintained for 24 hours. The operation was repeated, the oxygen concentration in the oxygen chamber was maintained to be alternated between 80% and 10% every 24 hours for 7 days, and then the rats were transferred to the air and fed. The oxygen concentration was monitored 8 times a day, and the ambient temperature in the chamber was controlled to 23° C.±2° C. The litter was replaced, food was added, water was changed, and mother rat was replaced once. The normal control group was placed in an animal house feeding environment. Compared with the control group, if the retinal stretched preparation stained with ADPase in the model group showed obvious vascular changes, the nucleus count of vascular endothelial cells that broke through the inner limiting membrane of the retina into the vitreous body was increased, and the difference was statistically significant, the model was successfully established.

(191) The oxygen treatment group was divided into two subgroups. On day 7 of modeling, the administration was performed via intravitreal injection, in which the fusion proteins I and II were administered at a dose of 100 μg, respectively. The rats in the oxygen model group and the control group were given only physiological saline for one week.

(192) On day 14, after the rats was sacrificed with ether anesthesia, the eyeballs were removed and fixed in a 40 g/L paraformaldehyde solution for 24 hours. The eyeballs were dehydrated with gradient alcohol and hyalinized with xylene. After being immersed in wax, the eyeballs were continuously sectioned to a thickness of 4 μm, avoiding the surrounding of the optic disc as much as possible. The sections were parallel to the sagittal plane of the cornea to the optic disc. 10 sections were randomly selected from each eyeball to be stained with hematoxylin and eosin, and the nucleus of vascular endothelial cells that broke through the inner limiting membrane of the retina was counted (only the nucleus of vascular endothelial cells closely related to the inner limiting membrane were counted), and the average number of cells per section per eyeball was counted.

(193) Results: no or few nucleus of vascular endothelial cells that broke through the inner limiting membrane of the retina into the vitreous body was found in the control group. More nucleuses of vascular endothelial cell that broke through the inner limiting membrane of the retina were found in the model group, some of which appeared alone, some clustered, and some nucleuses of vascular endothelial cells were found to be adjacent to the deep retinal vessels on some sections, confirming that they were originated from the retina instead of the vitreous body or other tissues in eyes. Only a few nucleuses of vascular endothelial cell that broke through the inner limiting membrane of the retina were found in the sections of the treatment group. The experimental results are shown in Table 22.

(194) TABLE-US-00024 TABLE 22 Nucleus count of vascular endothelial cells in each group Group Dose (μg) Nucleus number Protein I 100  6.693 ± 2.109 Protein II 100  7.333 ± 1.263 Model group — 28.392 ± 2.220 control —  1.315 ± 0.321

(195) The results showed that the nucleus counts of retinal vascular endothelial cells in the fusion proteins I and II treatment groups were 6.693±2.109 and 7.333±1.263, compared with the oxygen model group (28.392±2.220), the nucleus counts of retinal vascular endothelial cells were significantly reduced, which proved that they can inhibit the neovascularization in the oxygen-induced neonatal rat retinopathy model to a certain extent.

Example 19

(196) Effect of Fusion Proteins I and II on Neovascularization in Diabetic Retinopathy Rat Model

(197) The experimental diabetic rat model was established with streptozotocin STZ. STZ was dissolved in 0.1 mol/L citrate buffer at pH 4.5 to prepare a 2% solution. All experimental Wistar rats were fasted for 12 h before injection, and each rat was intraperitoneally injected with a 2% STZ solution at a dose of 65 mg/kg. After the injection, the rats were fed in single cages, and urine sugar and blood sugar were detected at the 48th. When urine sugar was +++ or above, and blood glucose was higher than 16.7 mmol/L, the model establishment requirement is reached. The diabetic retinopathy model was successfully established by detecting blood glucose, urine glucose and urine volume detection and retinal VEGF immunohistochemistry.

(198) 15 rats were randomly divided into three groups, which were labeled as a control group, a fusion protein I treatment group and a fusion protein II treatment group. The administration was performed via intravitreal injection once every 5 days for 2 weeks, in which the control group was injected with physiological saline (0.1 mL), and the fusion protein I and protein II were all administered with 100 μg (0.1 mL). Observation was performed on week 4, week 8, and week 12. The experimental results are shown in Table 23.

(199) TABLE-US-00025 TABLE 23 Effect of protein I and protein II on neovascularization in a diabetic retinopathy rat model Group (n = 5) Week 4 Week 8 Week 12 Protein I 182.03 ± 3.42 211.04 ± 3.33 252.36 ± 1.34 Protein II 188.26 ± 2.23 212.33 ± 4.59 257.92 ± 3.88 control 211.88 ± 4.36 227.52 ± 1.54 188.48 ± 3.89

(200) The results showed that under an optical microscope, the number of ganglion cells in 10 retina of posterior pole in each eyeball was counted, and the thickness of 10 retina of posterior pole in each eyeball was measured. Compared with the control group, the thickness of each layer of the retinal tissue of the rats in the experimental group was increased. Compared with the control group, the number of ganglion cells in the retinal of rats in the experimental group was increased. Compared with the control group, the number of visual cells in the treatment group was increased. It was indicated that fusion proteins I and II could produce a certain therapeutic effect on diabetic retinopathy at 100 μg dose.