A vascular endothelial growth factor inhibitory protein and its preparation method and application

Abstract

The present invention relates to a vascular endothelial growth factor inhibitory protein, its preparation method, and its application, belonging to the field of genetic engineering technology. The invention solves the technical problems of the complex production process and high cost of endothelial growth factor monoclonal antibody drugs in the prior art. The vascular endothelial growth factor inhibitory protein of the present invention comprises the following amino acid sequence: SEQ ID NO: 1. The vascular endothelial growth factor inhibitory protein is safe and stable, has a clear and direct efficacy, has minimal toxic and side effects, is not prone to drug resistance, has relatively mild storage and transportation conditions, is low in preparation cost, and is easy to promote and use in clinical practice.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to a vascular endothelial growth factor inhibitory protein and its preparation method and application, and especially relates to the application of the inhibitory protein in the preparation of a drug for treating retinal neovascular diseases caused by massive angiogenesis. Background Art

[0002] Many retinal diseases are related to neovascularization. The common pathological changes of this type of disease are the formation of neovascularization in the retina due to ischemia and hypoxia, which leads to leakage, proliferation and traction of blood vessels, resulting in recurrent vitreous hemorrhage, tractional retinal detachment and neovascular glaucoma. The disease progresses rapidly and, if not treated in time, will eventually lead to blindness.

[0003] Vascular endothelial growth factor (VEGF) is a highly specific endothelial growth factor that promotes vascular endothelial cell growth, including increased vascular permeability, extracellular matrix degeneration, endothelial cell migration, proliferation, and angiogenesis. VEGFRs primarily include VEGFR1, VEGFR2, and VEGFR3, with VEGFR2 being the primary VEGF signaling receptor involved in angiogenesis and mitosis. The VEGF / VEGFR2-mediated signaling cascade regulates endothelial cell proliferation, migration, and survival, causing changes in vascular permeability and controlling angiogenesis.

[0004] Prior art treatments for retinal diseases associated with neovascularization primarily utilize monoclonal antibodies against endothelial growth factor (VEGF). The principle of treatment is that VEGF and VEGF antibodies specifically bind to each other, competing with VEGF receptors on the cell surface for VEGF. Once VEGF and the antibody form a complex, they no longer activate VEGFRs on the cell surface. This blocks the cell signaling pathways that promote VEGF-mediated angiogenesis, achieving therapeutic efficacy. However, the complex manufacturing process for VEGF monoclonal antibodies leads to high drug costs, ultimately resulting in high prices and significantly increasing the burden on patients.

[0005] In view of this, it is necessary to study a drug based on reducing the angiogenesis-promoting activity of VEGF to solve the technical problems existing in the existing endothelial growth factor monoclonal antibody drugs. Summary of the Invention

[0006] The present invention aims to solve the technical problems of complex process and high cost of endothelial growth factor monoclonal antibody drugs in the prior art, and provides a vascular endothelial growth factor inhibitory protein and its preparation method and application.

[0007] The technical solutions adopted by the present invention to solve the above technical problems are as follows.

[0008] The vascular endothelial growth factor inhibitory protein of the present invention comprises the following amino acid sequence: SEQ ID NO: 1.

[0009] The present invention also provides a method for preparing the above-mentioned vascular endothelial growth factor inhibitory protein:

[0010] The amino acid sequence SEQ ID NO: 1 was cloned into a plasmid, and the resulting recombinant plasmid was transformed into Escherichia coli for induced expression. After the induced expression was completed, the bacteria were collected by centrifugation, and the bacteria were broken and centrifuged again to obtain supernatant protein. The supernatant protein was purified by affinity chromatography, desalting chromatography, and molecular sieve column purification to obtain vascular endothelial growth factor inhibitory protein.

[0011] Preferably, the amino acid sequence SEQ ID NO: 1 is cloned into the PET21b plasmid using NED1 and XhoII.

[0012] Preferably, the temperature for inducing expression is 37°C.

[0013] Preferably, the inducer used for inducing expression is IPTG, and more preferably, the concentration of the inducer is 0.2 mM.

[0014] Preferably, the centrifugal speed for collecting bacterial cells is 4000 rpm.

[0015] Preferably, the device used for breaking the bacteria is an ultrasonic disruptor.

[0016] Preferably, the centrifugal speed for obtaining supernatant protein is 13000 rpm.

[0017] Preferably, the affinity chromatography uses a Ni-NTA affinity chromatography column, washes the impurities with Wash Buffer, and then elutes the protein with Elution Buffer.

[0018] Preferably, desalting chromatography is performed using a G25 desalting column.

[0019] Preferably, the molecular sieve column purification uses a Superdex75 molecular sieve column to purify the protein at a flow rate of 0.5 ml / min.

[0020] The present invention also provides the use of the vascular endothelial growth factor inhibitory protein in the preparation of a drug for treating retinal neovascular diseases.

[0021] Preferably, the concentration of the VEGF inhibitor is >100 pM.

[0022] The present invention also provides a drug for treating retinal neovascularization diseases containing the vascular endothelial growth factor inhibitory protein.

[0023] Preferably, the concentration of the VEGF inhibitor is >100 pM.

[0024] The principle of the present invention is: the vascular endothelial growth factor inhibitory protein of the present invention includes partial extracellular sequences of VEGFR1 and VEGFR2. Therefore, based on the principle that receptors and ligands can specifically bind to each other, the vascular endothelial growth factor inhibitory protein of the present invention can specifically bind to VEGF, thereby reducing the probability of VEGF binding to cell surface VEGFR1 and VEGFR2, thereby reducing VEGF-mediated fundus angiogenesis.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The vascular endothelial growth factor inhibitory protein of the present invention is safe, stable, has clear and direct efficacy, has small toxic and side effects, is not prone to drug resistance, and has relatively mild storage and transportation conditions.

[0027] The preparation method of the vascular endothelial growth factor inhibitor protein of the present invention uses a prokaryotic expression system for preparation, which is simpler and has a much lower cost than that of VEGF monoclonal antibody. Therefore, it is cheap, easy to promote clinical use, and has significant social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the implementation. Obviously, the drawings described below are only some implementations of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0029] Figure 1 The diagram is a schematic diagram showing the origin of the amino acid sequence of the vascular endothelial growth factor inhibitory protein of the present invention.

[0030] Figure 2 The results of Example 1 of the present invention are shown in which the vascular endothelial growth factor inhibitory protein was prepared by induction expression in E. coli at different induction temperatures (16-37° C.) and different IPTG concentrations (0.1-0.3 mM). In the figure, M represents a protein marker.

[0031] Figure 3This is an electrophoresis diagram of the vascular endothelial growth factor inhibitory protein purified by Ni-NTA affinity chromatography in Example 1 of the present invention. In the figure, M represents protein marker, Q represents before induction, 8h represents the whole bacteria 8 hours after induction, TOP represents supernatant protein, CH represents precipitated protein, FL represents the flow-through through the Ni column, W1 represents the washing solution, and E1 represents the VEGFR eluate.

[0032] Figure 4 This is the electrophoresis diagram after fine purification of the protein using a Superdex75 molecular sieve column in Example 1 of the present invention.

[0033] Figure 5 This is a diagram showing the effect of the vascular endothelial growth factor inhibitory protein in Example 1 of the present invention on inhibiting VEGF-mediated HUVEC proliferation. DETAILED DESCRIPTION

[0034] In order to further understand the present invention, preferred embodiments of the present invention are described below. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention.

[0035] like Figure 1 As shown, the vascular endothelial growth factor inhibitory protein of the present invention includes three extracellular functional domains of VEGFR, namely D1, D2 and D3. Among them, one domain (D1) comes from the VEGFR1 protein, and two (D2 and D3) come from the VEGFR2 protein. Specifically, the vascular endothelial growth factor inhibitory protein of the present invention includes the following amino acid sequence: PEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIP DGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRGGGGSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHGGGGTVGERVRIPAKYLGYPPPEIKWYKNGIPLESNHTIKAGHVLTIMEVSERDTGNYTVILTNPIS, recorded as SEQ ID NO: 1.

[0036] A receptor is a protein, often located on the plasma membrane or in the nucleus / cytosol of target organs. The corresponding signaling molecule that binds to it is called a ligand. Different ligands can only bind to their corresponding receptors, initiating the intracellular signaling system and leading to changes in cellular function. VEGF is a ligand, and VEGFR1 and VEGFR2 are receptors. They are transmembrane proteins consisting of intracellular and extracellular portions. The extracellular portion is responsible for binding to free ligand VEGF, thereby activating intracellular signaling pathways and promoting angiogenesis. The vascular endothelial growth factor inhibitory protein of the present invention uses genetic recombinant expression technology to express a novel protein containing partial extracellular sequences of VEGF R1 and VEGFR2 in Escherichia coli. This VEGFR inhibitory protein can bind to the VEGF ligand but does not stimulate intracellular signaling, thereby hindering VEGF-mediated angiogenesis and reducing the incidence of various retinal diseases caused by angiogenesis.

[0037] The preparation method of the vascular endothelial growth factor inhibitory protein of the present invention is as follows:

[0038] The amino acid sequence SEQ ID NO: 1 was cloned into a plasmid, and the resulting recombinant plasmid was transformed into Escherichia coli for induced expression (fusion expression). After the induced expression was completed, the bacteria were collected by centrifugation, and the bacteria were broken and centrifuged again to obtain the supernatant protein. The supernatant protein was purified by affinity chromatography, desalting chromatography, and molecular sieve column purification to obtain vascular endothelial growth factor inhibitory protein.

[0039] In the above technical solution, preferably, NED1 and XhoII are used to clone the amino acid sequence SEQ ID NO: 1 into the PET21b plasmid.

[0040] In the above technical solution, the temperature for inducing expression is preferably 25° C.; and 0.2 mM IPTG is preferably used for inducing expression.

[0041] In the above technical solution, the centrifugal speed for collecting the bacteria is preferably 4000 rpm; the equipment used for crushing the bacteria is preferably an ultrasonic crusher; and the centrifugal speed for obtaining the supernatant protein is preferably 13000 rpm.

[0042] In the above technical solution, affinity chromatography preferably uses a Ni-NTA affinity chromatography column, uses Wash Buffer to wash the impurities, and then uses Elution Buffer to elute the protein; preferably, desalting chromatography is performed on a G25 desalting column; and molecular sieve column purification preferably uses a Superdex75 molecular sieve column to purify the supernatant protein at a flow rate of 0.5 ml / min.

[0043] The E. coli expression system has become the most commonly used system for producing recombinant proteins due to its advantages such as rapid cell reproduction, high yield, and relatively simple IPTG-induced expression. Different vectors are required to express different proteins. Currently known E. coli expression vectors can be divided into two types: non-fusion expression vectors and fusion expression vectors. Fusion expression is the fusion of the target protein or polypeptide with the DNA sequence of another protein or polypeptide fragment and expression within the bacteria. Fusion expression vectors include secretory expression vectors, expression vectors with purification tags, surface-presented expression vectors, and expression vectors with chaperones. The advantages of the E. coli expression system are clear genetic background, rapid reproduction, low cost, high expression level, easy purification of expression products, good stability, strong anti-pollution ability, and a wide range of applications.

[0044] The vascular endothelial growth factor inhibitory protein of the present invention can be used in the preparation of medicines for treating retinal neovascular diseases.

[0045] In the above technical solution, the concentration of vascular endothelial growth factor inhibitory protein is greater than 100 pM.

[0046] The present invention also provides a drug for treating retinal neovascularization diseases containing the vascular endothelial growth factor inhibitory protein.

[0047] In the above technical solution, the concentration of vascular endothelial growth factor inhibitory protein is greater than 100 pM.

[0048] The terms used in the present invention generally have the meanings commonly understood by those skilled in the art, unless otherwise specified. In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the embodiments.

[0049] In the following examples, various processes and methods not described in detail are conventional methods known in the art. Unless otherwise specified, the materials, reagents, devices, instruments, equipment, etc. used in the following examples can be obtained from commercial sources.

[0050] The present invention is further described below with reference to the examples.

[0051] Example 1

[0052] (1) The amino acid sequence SEQ ID NO: 1 was cloned into the PET21b plasmid using NED1 and XhoII, and the recombinant plasmid was then transformed into BL21 Escherichia coli.

[0053] (2) The amount of inducer IPTG used was 0.1mM, 0.15mM, 0.2mM, and 0.3mM, and the induction temperature was 16°C, 20°C, 25°C, and 37°C. The above induction conditions were used to induce the expression of vascular endothelial growth factor inhibitor protein.

[0054] (3) After the induction expression is completed, the bacteria are collected by centrifugation at 4000 rpm, and then the bacteria are broken using an ultrasonic disruptor, and then centrifuged at 13000 rpm to obtain the supernatant protein.

[0055] (4) The supernatant protein was passed through a Ni-NTA affinity chromatography column, and the impurities were washed with Wash Buffer. The protein was then eluted with Elution Buffer. The obtained protein was desalted by passing it through a G25 desalting column.

[0056] (5) The protein was finely purified using a Superdex75 molecular sieve column at a flow rate of 0.5 ml / min to obtain vascular endothelial growth factor inhibitory protein.

[0057] SDS-PAGE electrophoresis was used to detect the components and proteins during the purification process. Among them, the vascular endothelial growth factor inhibitor protein was prepared at IPTG concentrations of 0mM, 0.1mM, 0.15mM, 0.2mM, and 0.3mM at 25°C. The purification results are shown in Figure 2. Figure 2 As shown, from Figure 2 It can be seen that when the IPTG concentration is 0.2mM, the vascular endothelial growth factor inhibitory protein prepared has the best effect. Then, when the IPTG concentration is 0.2mM and the temperature is 16℃, 20℃, 25℃, and 37℃, the vascular endothelial growth factor inhibitory protein is prepared. The results are as follows Figure 2 As shown, from Figure 2 It can be seen that when the temperature is 25°C, the prepared vascular endothelial growth factor inhibitory protein has the best effect.

[0058] The components and proteins in the purification process were detected by SDS-PAGE electrophoresis. The vascular endothelial growth factor inhibitor protein was prepared with an IPTG concentration of 0.2 mM and a temperature of 25°C. The crude protein was as follows: Figure 3 As shown, the protein was finely purified using Superdex75 molecular sieve, and the obtained protein was as shown in Figure 4 As shown, it can be seen that high-purity VEGF inhibitory protein was obtained.

[0059] The obtained vascular endothelial growth factor inhibitor protein was quantitatively analyzed using an ultraviolet spectrophotometer and sterilized using a 0.22 μm filter for subsequent activity detection. Human vascular endothelial cells (HU VEC) were used as research cells, and the cells were cultured at 5×10 4 / ml density was seeded in a 24-well plate. The next day, the maintenance medium was replaced with DMEM (containing 0.05% serum). 1 ng / ml VEGF was added to the culture medium, and then 25 pM, 50 pM, 75 pM, 100 pM, 150 pM, and 200 pM of VEGFR inhibitory protein were added respectively. The next day, CCK-8 was used to detect the effect of VEGFR inhibitory protein in inhibiting VEGF-mediated HUVEC proliferation. The results are shown in the figure. Figure 5 As shown. Figure 5 It can be seen that when the concentration of VEGFR inhibitory protein is greater than 100 pM, the effect of inhibiting VEGF-mediated HUVEC proliferation is most obvious.

[0060] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the embodiments. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all embodiments here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A vascular endothelial growth factor inhibitory protein, characterized in that: The amino acid sequence is: SEQ ID NO:

1.

2. The method for preparing the vascular endothelial growth factor inhibitory protein according to claim 1, characterized in that: A nucleic acid molecule encoding the amino acid sequence SEQ ID NO: 1 is cloned into a plasmid, and the resulting recombinant plasmid is transformed into Escherichia coli for induced expression. After the induced expression is completed, the bacteria are collected by centrifugation, and the bacteria are broken and then centrifuged again to obtain supernatant protein. The supernatant protein is purified by affinity chromatography, desalting chromatography, and molecular sieve column purification to obtain vascular endothelial growth factor inhibitory protein.

3. The method for preparing the vascular endothelial growth factor inhibitory protein according to claim 2, characterized in that: The nucleic acid molecule encoding the amino acid sequence SEQ ID NO: 1 was cloned into the PET21b plasmid using NED1 and XhoII.

4. The method for preparing the vascular endothelial growth factor inhibitory protein according to claim 2, wherein: The temperature for inducing expression is 25°C; The inducing agent used for the induced expression is IPTG, with a concentration of 0.2 mM.

5. The method for preparing the vascular endothelial growth factor inhibitory protein according to claim 2, characterized in that: The centrifugal speed for collecting the bacterial cells is 4000 rpm; The equipment used for bacterial cell crushing is an ultrasonic crusher; The centrifugal speed for obtaining supernatant protein by centrifugation is 13000 rpm.

6. The method for preparing the vascular endothelial growth factor inhibitory protein according to claim 2, characterized in that: Affinity chromatography was performed using a Ni-NTA affinity column, and impurities were washed with Wash Buffer, followed by elution of the protein with Elution Buffer. Desalting chromatography was performed on a G25 desalting column; Molecular sieve column purification The protein was purified using a Superdex75 molecular sieve column at a flow rate of 0.5 ml / min.

7. Use of the VEGF inhibitory protein according to claim 1 or the VEGF inhibitory protein prepared by the preparation method of any one of claims 2 to 6 in the preparation of a medicament for treating retinal neovascular diseases, wherein the concentration of the VEGF inhibitory protein is >100 pM.

8. A drug for treating retinal neovascularization diseases comprising the vascular endothelial growth factor inhibitory protein according to claim 1 or the vascular endothelial growth factor inhibitory protein prepared by the preparation method of the vascular endothelial growth factor inhibitory protein according to any one of claims 2 to 6.

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