CN106699890B - Gold nanoparticle-based artificial antibody targeting Epidermal Growth Factor Receptor (EGFR) and preparation method thereof - Google Patents
Gold nanoparticle-based artificial antibody targeting Epidermal Growth Factor Receptor (EGFR) and preparation method thereof Download PDFInfo
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- CN106699890B CN106699890B CN201610927137.0A CN201610927137A CN106699890B CN 106699890 B CN106699890 B CN 106699890B CN 201610927137 A CN201610927137 A CN 201610927137A CN 106699890 B CN106699890 B CN 106699890B
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
Abstract
The invention relates to an artificial antibody targeting epidermal growth factor receptor EGFR (epidermal growth factor receptor) based on gold nanoparticles and a preparation method thereof. The artificial antibody is characterized by consisting of gold nanoparticles and a section of designed polypeptide sequence, and the nano-gold artificial antibody is prepared by reconstructing the conformation of the polypeptide on the surface of the nano-gold. The artificial antibody has the performance of specifically recognizing EGFR, strong binding force and good stability, so that the EGFR over-expressed on the surface of a target tumor cell by utilizing the specificity of the artificial antibody can be utilized, and the artificial antibody with the function of specifically recognizing EGFR has wide application prospects in the fields of disease diagnosis, target treatment, photothermal treatment, drug carrying and the like.
Description
Technical Field
The invention relates to an artificial antibody targeting epidermal growth factor receptor EGFR (epidermal growth factor receptor) and a preparation method thereof. Is characterized in that the artificial antibody of the targeted epidermal growth factor receptor EGFR based on gold nanoparticles and the preparation method thereof.
Background
The directional delivery of nanoparticles to tumor sites is of great importance to the fields of molecular imaging and targeted therapy of nano-drugs in vivo. In recent years, in order to solve the problems of poor binding force and low specificity of nanoparticles and target sites, there have been increasing attempts to utilize the properties of high specificity and strong binding force of antibodies and antigens by binding natural antibodies to nanoparticles, thereby directionally delivering the nanoparticles to the target sites and extending the circulation time of nanoparticles in vivo.
The research shows that the high expression of EGFR exists in a plurality of solid tumors, and the EGFR has a certain relation with the proliferation, angiogenesis, tumor invasion, metastasis and apoptosis of tumor cells. Therefore, EGFR is increasingly studied and utilized as an important target for targeting tumor cells, and cetuximab and panitumumab, which are natural antibodies to EGFR, have been successfully applied to clinical treatment of tumors. However, natural antibodies have self-defects such as poor stability and high price, so that the synthesis of an artificial antibody which has good stability and can specifically bind to EGFR has important value for molecular imaging, targeted therapy and drug carrier of nano-drugs in organisms.
The nano gold has the excellent characteristics of good biocompatibility, large specific surface area, easy surface modification and the like, so that the nano gold has wide application value in the aspects of cell detection, drug synthesis and transportation, photochemotherapy and the like. Therefore, the preparation of nano-drugs capable of targeting EGFR by functionalized nanoparticles has been widely studied, however, these nano-drugs are mainly delivered to tumor sites by modifying protein antibodies with the function of specifically recognizing EGFR onto the surfaces of the nanoparticles and utilizing the tumor targeting property of the modified protein antibodies. Although the nano-drug synthesized by the method has better tumor targeting, the nano-drug synthesized by the method has low binding force with EGFR, poor stability and high preparation cost due to the difficult extraction and poor stability of the macromolecular protein antibody.
Disclosure of Invention
One of the purposes of the invention is to provide an artificial receptor targeting epidermal growth factor receptor EGFR based on gold nanoparticles.
The second object of the present invention is to provide a method for preparing the artificial receptor.
In order to achieve the purpose, the invention adopts the following technical scheme:
an artificial receptor targeting Epidermal Growth Factor Receptor (EGFR) based on gold nanoparticles, which consists of gold nanoparticles and polypeptides, and is characterized in that the polypeptides consist of an identification region, two regulation and control regions and two fixing regions; both ends of the recognition area are respectively connected with a regulation area; and a fixing region is connected to each end of the regulatory region; the polypeptide is fixed on the surface of the gold nano-particle through fixing zones at two ends of the polypeptide so as to form a ring-shaped structure;
the regulation and control region is used for regulating the length of the polypeptide and is formed by randomly arranging 0-8 amino acid sequences which are not cysteine;
the immobilization region spontaneously forms S-Au bonds through sulfydryl and the surface of the nanogold, so that the polyAu is bondedPeptide with two ends fixed on the surface of gold nano-particle, every 1nm2And 0.05-2 polypeptides are fixed on the surface area of the gold nanoparticles, so that the polypeptides form a ring conformation on the surface of the gold nanoparticles.
The amino acid sequence of the recognition region is AWYGTLYEYD, AWYGTVYEYD, SWYGTLYEYD or AWYGSLYEYD.
The fixing area is as follows: cysteine or polyethylene glycol with sulfhydryl groups are modified at two ends of the regulation and control region by dehydration condensation to form a fixed region.
The amino acids constituting the regulatory region are: glycine, serine, alanine, glutamine and asparagine. .
A preparation method of an artificial antibody targeting epidermal growth factor receptor EGFR based on gold nanoparticles is characterized by comprising the following specific steps: according to each 1nm2The surface area of the gold nanoparticles is fixed with the proportion of 0.05-2 polypeptides, the polypeptide solution is dripped into the nano-gold sol, and the nano-gold artificial antibody with the targeted epidermal growth factor receptor EGFR is obtained after mixing and reacting for 1-180min at normal temperature.
According to the method, as long as the polypeptide is ensured to have specific cyclic conformation on the surface of the gold nanoparticles, the polypeptide can be modified on the surfaces of the gold nanoparticles with different shapes and sizes, such as: nano gold ball with diameter of 2-200nm, nano gold rod with length of 20-200nm and diameter of 5-100nm or star-shaped nano gold with diameter of 10-200 nm.
The artificial antibody has the performance of specifically recognizing EGFR, strong binding force and good stability, so that the EGFR which is overexpressed or abnormally expressed on the surface of a tumor cell can be targeted by utilizing the specificity of the artificial antibody. Therefore, the artificial antibody with the function of specifically recognizing EGFR has wide application prospects in the fields of disease diagnosis, targeted therapy, photothermal therapy, drug carrier and the like.
The designed polypeptide is modified on the surface of the nanogold through an S-Au bond, and the designed polypeptide forms a specific annular conformation on the surface of the nanogold, so that the nano artificial antibody with a target protein EGFR (epidermal growth factor receptor) is prepared. Since many tumor cell surface EGFR are overexpressed, the synthesized nanogold antibody can be used for specifically targeting the EGFR with high expression on the tumor cell surface. Therefore, the nano artificial antibody synthesized by the method has wide application prospects in the fields of disease diagnosis, targeted therapy, photothermal therapy, drug carrying and the like.
Drawings
Fig. 1 is a schematic diagram of an artificial antibody targeting epidermal growth factor receptor EGFR based on gold nanoparticles.
FIG. 2 shows the specific binding performance of the artificial antibody AuNP (4nm) + P1 to the protein EGFR.
FIG. 3 is a graph representing the kinetic performance of binding of the artificial antibody AuNP (4nm) + P1 to EGFR in example one.
FIG. 4 shows the thermodynamic stability of the artificial antibody AuNP (4nm) + P1 detected in the first example.
FIG. 5 shows the specific binding performance of the artificial antibody AuNP (4nm) + P2 to the protein EGFR in the second detection example.
FIG. 6 is a graph depicting the kinetic performance of binding of the artificial antibody AuNP (4nm) + P2 to EGFR in example II.
FIG. 7 shows the thermodynamic stability of the artificial antibody AuNP (4nm) + P2 detected in example two.
FIG. 8 shows specific binding performance of AuNP (4nm) + P3 as an artificial antibody to EGFR in the third detection example.
FIG. 9 shows the kinetics of binding of the artificial antibody AuNP (4nm) + P3 to EGFR in the examples.
FIG. 10 shows the thermodynamic stability of the artificial antibody AuNP (4nm) + P3 detected in example III.
FIG. 11 shows specific binding performance of the artificial antibody AuNP (30nm) + P1 to the protein EGFR in the fourth detection example.
FIG. 12 is a graph depicting the kinetic performance of binding of the artificial antibody AuNP (30nm) + P1 to EGFR in example IV.
FIG. 13 shows thermodynamic stability of the example IV detection artificial antibody AuNP (30nm) + P1.
FIG. 14 shows the specific binding performance of the artificial antibody AuNP (30nm) + P2 and the protein EGFR in the fifth detection example.
Figure 15 is an example five characterization of the kinetics of binding of the artificial antibody AuNP (30nm) + P2 to EGFR.
FIG. 16 shows the thermodynamic stability of the example V detection artificial antibody AuNP (30nm) + P2.
FIG. 17 shows the specific binding performance of the artificial antibody AuNP (30nm) + P3 and the protein EGFR in the sixth detection example.
Figure 18 is an example six of the kinetic performance of the artificial antibody AuNP (30nm) + P3 in binding to EGFR.
FIG. 19 shows the thermodynamic stability of the example VI test artificial antibody AuNP (30nm) + P3.
FIG. 20 shows the specific binding performance of the artificial antibodies AuNP (4nm) + P4 and AuNP (4nm) + P2 to the protein EGFR in example seven.
FIG. 21 shows the specific binding performance of the artificial antibodies AuNP (4nm) + P5 and AuNP (4nm) + P2 to the protein EGFR in the eighth detection example.
Detailed Description
The following examples are given to further illustrate the invention. It should be noted that the following examples should not be construed as limiting the scope of the present invention, and that the skilled person in the art would still fall within the scope of the present invention if he or she made some insubstantial modifications and adjustments to the present invention, such as minor mutations to the amino acid sequence of the designed polypeptide portion, based on the above disclosure.
The first embodiment is as follows:
1. design of polypeptide fragments
Directly combines the recognition area and the fixed area, the number of amino acids of the regulatory area is 0, and a polypeptide P1: CAWYGTLYEYDC is designed, as shown in figure 1. The designed polypeptide P1 can be fixed on the surface of the nanogold through sulfydryl in cysteine at two ends, so that a specific conformation is formed.
2. Preparation of gold nano artificial antibody AuNP (4nm) + P1 with EGFR targeting performance
According to 1nm2The surface area of the nanogold ball is fixed with the proportion of 0.05-2 polypeptides, the polypeptides are fixed on the surface of a nanogold ball with the diameter of 4nm through S-Au bonds, and more than 10-150 polypeptides are arranged on each nanogold surfaceA peptide. Thus, an artificial antibody AuNP (4nm) + P1 capable of specifically binding to EGFR was obtained.
3. Characterization of specific binding Performance of the Artificial antibody AuNP (4nm) + P1 to the protein EGFR
To test the ability of the artificial antibody AuNP (4nm) + P1 to specifically bind to EGFR, we measured the binding of AuNP (4nm) + P1 to EGFR, EGF and BSA, as shown in figure 2. The artificial antibody AuNP (4nm) + P1 has obvious binding with EGFR but no obvious binding with EGF and BSA, which indicates that the artificial antibody AuNP (4nm) + P1 has the property of specifically binding with EGFR.
4. Characterization of the kinetics of binding of the Artificial antibody AuNP (4nm) + P1 to EGFR
To characterize the kinetic performance of the synthesized nanogold antibody AuNP (4nm) + P1 in binding with EGFR, we determined the kinetic profile and binding constant K of AuNP (4nm) + P1 in binding with EGFRDValues, as shown in fig. 3. Binding constant K of artificial antibody AuNP (4nm) + P1 to EGFRD=1.89E-11, the binding constant of the artificial antibody AuNP (4nm) + P1 to EGFR was found by comparison to be three orders of magnitude higher than that of the natural antibody to EGFR. These demonstrate that the artificial antibody AuNP (4nm) + P1 has better kinetic properties for binding to EGFR than the natural antibody.
5. Detection of thermodynamic stability of AuNP (4nm) + P1
To demonstrate that the artificial antibody has better thermodynamic stability than the natural antibody, we put the artificial antibody AuNP (4nm) + P1 into boiling water bath for 1h, and then measure the binding performance of the two antibodies to EGFR before and after the water bath, as shown in FIG. 4. By comparing the binding performance of the artificial antibody AuNP (4nm) + P1 with EGFR before and after water bath, the binding performance of the artificial antibody after the water bath to the EGFR is not obviously reduced compared with that before the water bath, however, most natural antibodies have irreversible changes in the structure after being heated by boiling water, so that the natural antibodies lose activity. The artificial antibody thus has superior thermodynamic stability compared to the natural antibody.
Example two:
1. design of polypeptide fragments
The polypeptide P2: CSAWYGTLYEYDGC was designed by adding one serine and one glycine to each of the two regulatory regions at both ends of the recognition region, as shown in FIG. 1. The designed polypeptide can be fixed on the surface of the nanogold through sulfydryl in cysteine of two fixing regions, so that a specific conformation is formed.
2. Preparation of gold nano artificial antibody with targeting EGFR (epidermal growth factor receptor) performance
According to 1nm2The surface area of the gold nano-particles is fixed with the proportion of 0.05-2 polypeptides, the polypeptides are fixed on the surface of a 4nm nano-gold ball through S-Au bonds, and each nano-gold surface is provided with 10-150 polypeptides. Thus, an artificial antibody AuNP (4nm) + P2 capable of specifically binding to EGFR was obtained.
3. Characterization of specific binding Performance of the Artificial antibody AuNP (4nm) + P2 to the protein EGFR
To test the ability of the artificial antibody AuNP (4nm) + P2 to specifically bind to EGFR, we measured the binding of AuNP (4nm) + P2 to EGFR, EGF and BSA, as shown in fig. 5. The artificial antibody AuNP (4nm) + P2 has obvious binding with EGFR but no obvious binding with EGF and BSA, which indicates that the artificial antibody AuNP (4nm) + P2 has the property of specifically binding with EGFR.
4. Characterization of the kinetics of binding of the Artificial antibody AuNP (4nm) + P2 to EGFR
To characterize the kinetic performance of the synthesized nanogold antibody AuNP (4nm) + P2 in binding with EGFR, we determined the kinetic profile and binding constant K of AuNP (4nm) + P2 in binding with EGFRDValues, as shown in fig. 6. Binding constant K of artificial antibody AuNP (4nm) + P2 to EGFRDAnd = 4.04E-11. The binding constant of the artificial antibody AuNP (4nm) + P2 to EGFR was found by comparison to be three orders of magnitude higher than that of the natural antibody to EGFR. These demonstrate that the artificial antibody AuNP (4nm) + P2 has better kinetic properties for binding to EGFR than the natural antibody.
6. Detection of thermodynamic stability of AuNP (4nm) + P2
To demonstrate that the artificial antibody has better thermodynamic stability than the natural antibody, we placed the artificial antibody AuNP (4nm) + P2 in a boiling water bath for 1h, and then measured the binding performance of the two antibodies to EGFR before and after the water bath, as shown in FIG. 7. By comparing the binding performance of the artificial antibody AuNP (4nm) + P2 with EGFR before and after water bath, the binding performance of the artificial antibody after the water bath to the EGFR is not obviously reduced compared with that before the water bath, however, most natural antibodies have irreversible changes in the structure after being heated by boiling water, so that the natural antibodies lose activity. The artificial antibody thus has superior thermodynamic stability compared to the natural antibody.
Example three:
1. design of polypeptide fragments
The polypeptide P3: CGAGASAWYGTLYEYDGSASAC was designed by adding 5 amino acids to each of the two regulatory regions at the two ends of the recognition region, as shown in FIG. 1. The designed polypeptide can be fixed on the surface of the nanogold through sulfydryl in cysteine of two fixing regions, so that a specific conformation is formed.
2. Preparation of gold nano artificial antibody with targeting EGFR (epidermal growth factor receptor) performance
According to 1nm2The surface area of the gold nano-particles is fixed with the proportion of 0.05-2 polypeptides, the polypeptides are fixed on the surface of a 4nm nano-gold ball through S-Au bonds, and each nano-gold surface is provided with 10-150 polypeptides. Thus, an artificial antibody AuNP (4nm) + P3 capable of specifically binding to EGFR was obtained.
3. Characterization of specific binding Performance of the Artificial antibody AuNP (4nm) + P3 to the protein EGFR
To test the ability of the artificial antibody AuNP (4nm) + P3 to specifically bind to EGFR, we measured the binding of AuNP (4nm) + P3 to EGFR, EGF and BSA, as shown in fig. 8. The artificial antibody AuNP (4nm) + P3 has obvious binding with EGFR but no obvious binding with EGF and BSA, which indicates that the artificial antibody AuNP (4nm) + P3 has the property of specifically binding with EGFR.
4. Characterization of the kinetics of binding of the Artificial antibody AuNP (4nm) + P3 to EGFR
To characterize the kinetic performance of the synthesized nanogold antibody AuNP (4nm) + P3 in binding with EGFR, we determined the kinetic profile and binding constant K of AuNP (4nm) + P3 in binding with EGFRDValues, as shown in fig. 9. Binding constant K of artificial antibody AuNP (4nm) + P3 to EGFRDAnd 5.96E-11. The binding constant of the artificial antibody AuNP (4nm) + P3 to EGFR was found by comparison to be three orders of magnitude higher than that of the natural antibody to EGFR. These demonstrate that the artificial antibody AuNP (4nm) + P3 has better kinetic properties for binding to EGFR than the natural antibody.
7. Detection of thermodynamic stability of AuNP (4nm) + P3
To demonstrate that the artificial antibody has better thermodynamic stability than the natural antibody, we placed the artificial antibody AuNP (4nm) + P3 in a boiling water bath for 1h, and then measured the binding performance of the two antibodies to EGFR before and after the water bath, as shown in FIG. 10. By comparing the binding performance of the artificial antibody AuNP (4nm) + P3 with EGFR before and after water bath, the binding performance of the artificial antibody after the water bath to the EGFR is not obviously reduced compared with that before the water bath, however, most natural antibodies have irreversible changes in the structure after being heated by boiling water, so that the natural antibodies lose activity. The artificial antibody thus has superior thermodynamic stability compared to the natural antibody.
Example four:
1. design of polypeptide fragments
Directly combines the recognition area and the fixed area, the number of amino acids of the regulatory area is 0, and a polypeptide P1: CAWYGTLYEYDC is designed, as shown in figure 1. The designed polypeptide P1 can be fixed on the surface of the nanogold through sulfydryl in cysteine at two ends, so that a specific conformation is formed.
2. Preparation of gold nano artificial antibody AuNP (30nm) + P1 with EGFR targeting performance
According to 1nm2The surface area of the gold nano-particles is fixed with the proportion of 0.05-2 polypeptides, the polypeptides are fixed on the surface of the gold nano-particles with the diameter of 30nm through S-Au bonds, and each nano-gold surface is provided with 10-150 polypeptides. Thus, an artificial antibody AuNP (4nm) + P1 capable of specifically binding to EGFR was obtained.
3. Characterization of specific binding Performance of the Artificial antibody AuNP (30nm) + P1 to the protein EGFR
To test the ability of the artificial antibody AuNP (30nm) + P1 to specifically bind to EGFR, we measured binding of AuNP (30nm) + P1 to EGFR, EGF and BSA, as shown in fig. 11. The artificial antibody AuNP (30nm) + P1 has obvious binding with EGFR but no obvious binding with EGF and BSA, which indicates that the artificial antibody AuNP (30nm) + P1 has the property of specifically binding with EGFR.
4. Characterization of the kinetics of binding of the Artificial antibody AuNP (30nm) + P1 to EGFR
To characterize the kinetic performance of the synthesized nanogold antibody AuNP (30nm) + P1 in binding to EGFR, we determined the kinetic profile and binding constant K of AuNP (30nm) + P1 in binding to EGFRDValues, as shown in fig. 12. Binding constant K of artificial antibody AuNP (30nm) + P1 to EGFRD= 9.80E-11. The binding constant of the artificial antibody AuNP (30nm) + P1 to EGFR was found by comparison to be three orders of magnitude higher than that of the natural antibody to EGFR. These demonstrate that the artificial antibody AuNP (30nm) + P1 has better kinetic properties for binding to EGFR than the natural antibody.
5. Detection of thermodynamic stability of AuNP (30nm) + P1
To demonstrate that the artificial antibody has better thermodynamic stability than the natural antibody, we placed the artificial antibody AuNP (30nm) + P1 in a boiling water bath for 1h, and then measured the binding performance of the two antibodies to EGFR before and after the water bath, as shown in FIG. 13. By comparing the binding performance of the artificial antibody AuNP (30nm) + P1 with EGFR before and after water bath, the binding performance of the artificial antibody with EGFR after the water bath is not obviously reduced compared with that before the water bath, however, most natural antibodies have irreversible changes in antibody structure after being heated by boiling water, so that the natural antibodies lose activity. The artificial antibody thus has superior thermodynamic stability compared to the natural antibody.
Example five:
1. design of polypeptide fragments
The polypeptide P2: CSAWYGTLYEYDGC was designed by adding one serine and one glycine to each of the two regulatory regions at both ends of the recognition region, as shown in FIG. 1. The designed polypeptide can be fixed on the surface of the nanogold through sulfydryl in cysteine of two fixing regions, so that a specific conformation is formed.
2. Preparation of gold nano artificial antibody with targeting EGFR (epidermal growth factor receptor) performance
According to 1nm2The surface area of the gold nano-particles is fixed with the proportion of 0.05-2 polypeptides, the polypeptides are fixed on the surface of the gold nano-particles with the length of 4nm through S-Au bonds, and each nano-gold surface is provided with 10-150 polypeptides. Thus, an artificial antibody AuNP (30nm) + P2 capable of specifically binding to EGFR was obtained.
3. Characterization of specific binding Performance of the Artificial antibody AuNP (30nm) + P2 to the protein EGFR
To test the ability of the artificial antibody AuNP (30nm) + P2 to specifically bind to EGFR, we measured binding of AuNP (30nm) + P2 to EGFR, EGF and BSA, as shown in fig. 14. The artificial antibody AuNP (30nm) + P2 has obvious binding with EGFR but no obvious binding with EGF and BSA, which indicates that the artificial antibody AuNP (30nm) + P2 has the property of specifically binding with EGFR.
4. Characterization of the kinetics of binding of the Artificial antibody AuNP (30nm) + P2 to EGFR
To characterize the kinetic performance of the synthesized nanogold antibody AuNP (30nm) + P2 in binding to EGFR, we determined the kinetic profile and binding constant K of AuNP (30nm) + P2 in binding to EGFRDValues, as shown in fig. 15. Binding constant K of artificial antibody AuNP (30nm) + P2 to EGFRD= 4.92E-11. The binding constant of the artificial antibody AuNP (30nm) + P2 to EGFR was found by comparison to be three orders of magnitude higher than that of the natural antibody to EGFR. These demonstrate that the artificial antibody AuNP (30nm) + P2 has better kinetic properties for binding to EGFR than the natural antibody.
8. Detection of thermodynamic stability of AuNP (30nm) + P2
To demonstrate that the artificial antibody has better thermodynamic stability than the natural antibody, we placed the artificial antibody AuNP (30nm) + P2 in a boiling water bath for 1h, and then measured the binding performance of the two antibodies to EGFR before and after the water bath, as shown in FIG. 16. By comparing the binding performance of the artificial antibody AuNP (30nm) + P2 with EGFR before and after water bath, the binding performance of the artificial antibody with EGFR after the water bath is not obviously reduced compared with that before the water bath, however, most natural antibodies have irreversible changes in antibody structure after being heated by boiling water, so that the natural antibodies lose activity. The artificial antibody thus has superior thermodynamic stability compared to the natural antibody.
Example six:
1. design of polypeptide fragments
The polypeptide P3: CGAGASAWYGTLYEYDGSASAC was designed by adding 5 amino acids to each of the two regulatory regions at the two ends of the recognition region, as shown in FIG. 1. The designed polypeptide can be fixed on the surface of the nanogold through sulfydryl in cysteine of two fixing regions, so that a specific conformation is formed.
2. Preparation of gold nano artificial antibody with targeting EGFR (epidermal growth factor receptor) performance
According to 1nm2The surface area of the gold nano-particles is fixed with the proportion of 0.05-2 polypeptides, the polypeptides are fixed on the surface of the gold nano-particles with the diameter of 30nm through S-Au bonds, and each nano-gold surface is provided with 10-150 polypeptides. Thus, an artificial antibody AuNP (30nm) + P3 capable of specifically binding to EGFR was obtained.
3. Characterization of specific binding Performance of the Artificial antibody AuNP (30nm) + P3 to the protein EGFR
To test the ability of the artificial antibody AuNP (30nm) + P3 to specifically bind to EGFR, we measured binding of AuNP (30nm) + P3 to EGFR, EGF and BSA, as shown in fig. 17. The artificial antibody AuNP (30nm) + P3 has obvious binding with EGFR but no obvious binding with EGF and BSA, which indicates that the artificial antibody AuNP (4nm) + P3 has the property of specifically binding with EGFR.
4. Characterization of the kinetics of binding of the Artificial antibody AuNP (30nm) + P3 to EGFR
To characterize the kinetic performance of the synthesized nanogold antibody AuNP (30nm) + P3 in binding to EGFR, we determined the kinetic profile and binding constant K of AuNP (30nm) + P3 in binding to EGFRDValues, as shown in fig. 18. Binding constant K of artificial antibody AuNP (30nm) + P3 to EGFRDAnd 7.38E-11. The binding constant of the artificial antibody AuNP (30nm) + P3 to EGFR was found by comparison to be three orders of magnitude higher than that of the natural antibody to EGFR. These demonstrate that the artificial antibody AuNP (30nm) + P3 has better kinetic properties for binding to EGFR than the natural antibody.
5. Detection of thermodynamic stability of AuNP (30nm) + P3
To demonstrate that the artificial antibody has better thermodynamic stability than the natural antibody, we placed the artificial antibody AuNP (30nm) + P3 in a boiling water bath for 1h, and then measured the binding performance of the two antibodies to EGFR before and after the water bath, as shown in FIG. 19. By comparing the binding performance of the artificial antibody AuNP (30nm) + P3 with EGFR before and after water bath, the binding performance of the artificial antibody with EGFR after the water bath is not obviously reduced compared with that before the water bath, however, most natural antibodies have irreversible changes in antibody structure after being heated by boiling water, so that the natural antibodies lose activity. The artificial antibody thus has superior thermodynamic stability compared to the natural antibody.
Example seven:
1. design of polypeptide fragments
Ensuring that AWYGTLYEYD key amino acid residue YEYD in the recognition region is unchanged, and carrying out single-point mutation on other amino acids in the recognition region to obtain AWYGTVYEYD; the polypeptide P4: CSAWYGTVYEYDGC was designed by adding one serine and one glycine to each of the two regulatory regions at both ends of the recognition region, as shown in FIG. 1. The designed polypeptide can be fixed on the surface of the nanogold through sulfydryl in cysteine of two fixing regions, so that a specific conformation is formed.
2. Preparation of gold nano artificial antibody with targeting EGFR (epidermal growth factor receptor) performance
According to 1nm2The surface area of the gold nano-particles is fixed with the proportion of 0.05-2 polypeptides, the polypeptides are fixed on the surface of a 4nm nano-gold ball through S-Au bonds, and each nano-gold surface is provided with 10-150 polypeptides. Thus, an artificial antibody AuNP (4nm) + P4 capable of specifically binding to EGFR was obtained.
3. Characterization of specific binding Performance of the Artificial antibodies AuNP (4nm) + P4 and AuNP (4nm) + P2 to the protein EGFR
To examine that the specific binding of the artificial antibodies AuNP (4nm) + P4 to EGFR was not much affected after single point mutation on the polypeptide recognition region, we determined the binding of AuNP (4nm) + P4 and AuNP (4nm) + P2 to EGFR, EGF and BSA, respectively, as shown in fig. 20. The artificial antibodies AuNP (4nm) + P4 and AuNP (4nm) + P2 have obvious binding with EGFR but have no obvious binding with EGF and BSA, which shows that the artificial antibody AuNP (4nm) + P4 has the property of specifically binding with EGFR.
Example eight:
1. design of polypeptide fragments
Two regulatory regions at two ends of a recognition region are respectively added with a serine and a glycine, polyethylene glycol (0.3K) with sulfhydryl groups is modified at two ends of the polypeptide by dehydration condensation to form fixed regions, and the polypeptide P5: SH-PEGSAWYGTVYEYDGPEG-SH is designed, as shown in figure 1. The designed polypeptide can be fixed on the surface of the nanogold through sulfydryl in cysteine of two fixing regions, so that a specific conformation is formed.
2. Preparation of gold nano artificial antibody with targeting EGFR (epidermal growth factor receptor) performance
According to 1nm2The surface area of the gold nano-particles is fixed with the proportion of 0.05-2 polypeptides, the polypeptides are fixed on the surface of a 4nm nano-gold ball through S-Au bonds, and each nano-gold surface is provided with 10-150 polypeptides. Thus, an artificial antibody AuNP (4nm) + P5 capable of specifically binding to EGFR was obtained.
3. Characterization of specific binding Performance of the Artificial antibodies AuNP (4nm) + P5 and AuNP (4nm) + P2 to the protein EGFR
To examine the effect of polypeptide anchor region changes on the specific binding of the artificial antibodies AuNP (4nm) + P5 to EGFR, we measured the binding of AuNP (4nm) + P5 and AuNP (4nm) + P2 to EGFR, EGF and BSA, respectively, as shown in fig. 21. The artificial antibodies AuNP (4nm) + P6 and AuNP (4nm) + P2 have obvious binding with EGFR but have no obvious binding with EGF and BSA, which shows that the artificial antibody AuNP (4nm) + P5 has the property of specifically binding with EGFR.
<110> university at Shanghai
<120> gold nanoparticle-based artificial antibody targeting epidermal growth factor receptor EGFR and preparation method thereof
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1
Claims (4)
1. An artificial antibody targeting Epidermal Growth Factor Receptor (EGFR) based on gold nanoparticles, which consists of gold nanoparticles and polypeptides, and is characterized in that:
the polypeptide consists of a recognition area, two regulation areas and two fixing areas;
both ends of the recognition area are respectively connected with a regulation area;
and a fixing region is connected to each end of the regulatory region;
the polypeptide is fixed on the surface of the gold nano-particle through fixing zones at two ends of the polypeptide so as to form a ring-shaped structure;
the regulation and control region is used for regulating the length of the polypeptide and is formed by randomly arranging 0-5 amino acid sequences which are not cysteine;
the immobilization region spontaneously forms S-Au bonds through sulfydryl and the surface of the nano-gold, so that two ends of the polypeptide are immobilized on the surface of the gold nano-particle, and each 1nm of the immobilization region2Fixing 0.05-2 polypeptides on the surface area of the gold nanoparticles, so that the polypeptides form a ring conformation on the surface of the gold nanoparticles;
the amino acid sequence of the recognition region is AWYGTLYEYD or AWYGTVYEYD;
the amino acids that make up the regulatory region are: glycine, serine or alanine.
2. The gold nanoparticle-based Epidermal Growth Factor Receptor (EGFR) -targeting artificial antibody according to claim 1, characterized in that: the fixed area is as follows: cysteine or polyethylene glycol with sulfhydryl groups are modified at two ends of the regulation and control region by dehydration condensation to form a fixed region.
3. The gold nanoparticle-based Epidermal Growth Factor Receptor (EGFR) -targeting artificial antibody according to claim 1, characterized in that: the gold nanoparticles are nano gold spheres with the diameter of 2-200nm, nano gold rods with the length of 20-200nm and the diameter of 5-100nm or star-shaped nano gold with the diameter of 10-200 nm.
4. The gold nanoparticle-based Epidermal Growth Factor Receptor (EGFR) -targeting artificial antibody according to claim 1, characterized in that: the surface of each gold nanoparticle is provided with 10-150 polypeptides.
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