CN106699890A - Artificial antibody for targeting EGFR (epidermal growth factor receptor) based on gold nanoparticles and preparation method thereof - Google Patents
Artificial antibody for targeting EGFR (epidermal growth factor receptor) based on gold nanoparticles and preparation method thereof Download PDFInfo
- Publication number
- CN106699890A CN106699890A CN201610927137.0A CN201610927137A CN106699890A CN 106699890 A CN106699890 A CN 106699890A CN 201610927137 A CN201610927137 A CN 201610927137A CN 106699890 A CN106699890 A CN 106699890A
- Authority
- CN
- China
- Prior art keywords
- egfr
- aunp
- artificial antibody
- polypeptide
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to an artificial antibody for targeting an EGFR (epidermal growth factor receptor) based on gold nanoparticles and a preparation method thereof. The artificial antibody is characterized by consisting of the gold nanoparticles and a section of designed polypeptide sequence; the conformation of the polypeptide on the nanometer gold surface is reconstructed, so as to prepare the artificial antibody based on the gold nanoparticles. The artificial antibody has the advantages that the property of specifically identifying the EGFR is realized, the bonding ability is strong, and the stability is good; the artificial antibody is used for specially targeting the over-expressed EGFR on the tumor cell surface, and the artificial antibody with the special EGFR identifying function has broad application prospect in the fields of disease diagnosis, targeting therapy, photo-thermal therapy, and medicine carriers.
Description
Technical field
The present invention relates to a kind of targeting epidermal growth factor receptor EGFR（epidermal growth factor
receptor）Artificial antibody and preparation method thereof.It is characterized in that a kind of targeting epidermal growth factor based on golden nanometer particle is received
Artificial antibody of body EGFR and preparation method thereof.
Background technology
Orientation conveying nano particle reaches tumor locus for Nano medication molecular imaging and targeted therapy in vivo
It is most important Deng field.In recent years, in order to solve the problems, such as nano particle and target position adhesion difference and specific low, people
More and more it is attached on nano particle by by natural antibody, using the high specific and strong adhesion of antibody and antigen
Property, so as to nano particle orientation to be transported to target position and can extend nano particle circulation time in vivo.
Research finds the expression high that there is EGFR in many entity tumors, propagation of the EGFR with tumour cell, blood vessel life
There is certain relation into, tumor invasion, transfer and Apoptosis.Therefore, EGFR is used as the important of targets neoplastic cells
Target spot is studied and utilized by increasing, and EGFR natural antibody Cetuximab and Victibix by successfully
It is applied in the clinical treatment of tumour.But natural antibody has the defect of itself, such as stability is poor, expensive, therefore
Synthesize a kind of good stability and can specifically bind the artificial antibody of EGFR for Nano medication in vivo molecule into
Picture, targeted therapy and medicine carrier band have important value.
Because nm of gold has good biocompatibility, specific surface area big and is easy to the good characteristics such as surface modification, this makes it
It is with a wide range of applications at the aspect such as cell detection, pharmaceutical synthesis and transport, photochemical therapy.Therefore received by functionalization
The Nano medication that targeting EGFR is capable of in rice corpuscles preparation is widely studied, but these Nano medications are mainly by inciting somebody to action
Nanoparticle surface is arrived in protein antibody modification with specific recognition EGFR functions, using the swollen of the protein antibody modified
Knurl targeting by Nano medication so that be transported to tumor locus.Although the Nano medication of this method synthesis has preferable tumour
Targeting, but because macro-molecular protein antibody is extracted, difficult and stability is poor so that the nanometer medicine of this method synthesis
Thing is low with the adhesion of EGFR, stability is poor and preparation cost is high.
The content of the invention
An object of the present invention is to provide a kind of targeting epidermal growth factor receptor EGFR based on golden nanometer particle
Artificial receptors.
The second object of the present invention is the preparation method for providing the artificial receptors.
To reach above-mentioned purpose, the present invention is adopted the following technical scheme that：
A kind of artificial receptors of the targeting epidermal growth factor receptor EGFR based on golden nanometer particle, by golden nanometer particle with
And polypeptide composition, it is characterised in that described polypeptide is made up of cog region, two control regions and two fixed areas；Described identification
The two ends in area respectively connect a control region；And respectively connect a fixed area at the two ends of control region；The polypeptide is by its two ends
Fixed area is fixed on golden nanometer particle surface so as to form cyclic structure；
Described control region is used for adjusting the length of polypeptide, by 0~8 amino acid sequence random alignment of non-cysteine
Composition；
Described fixed area spontaneously forms S-Au keys by sulfydryl and nanometer gold surface, so as to the polypeptide two ends be fixed
On golden nanometer particle surface, per 1nm20.05~2 polypeptide is fixed on golden nanometer particle surface area, so that polypeptide is in gold
Nanoparticle surface forms cyclic conformation.
The amino acid sequence of above-mentioned cog region be AWYGTLYEYD, AWYGTVYEYD, SWYGTLYEYD or
AWYGSLYEYD。
Above-mentioned fixed area is：Cysteine modifies in control region the polyethylene glycol with sulfydryl by dehydrating condensation
Two ends formed fixed area.
The amino acid of above-mentioned composition control region is：Glycine, serine, alanine, glutamine and asparagine..
A kind of preparation method of the artificial antibody of the targeting epidermal growth factor receptor EGFR based on golden nanometer particle, it is special
It is concretely comprising the following steps for the method to levy：According to every 1nm20.05~2 ratio of polypeptide is fixed on golden nanometer particle surface area,
Polypeptide solution is added dropwise in nano gold sol, at normal temperatures hybrid reaction 1-180min, that is, obtains being grown with targeting epidermal
The nm of gold artificial antibody of factor acceptor EGFR.
As long as ensureing that polypeptide has specific cyclic conformation in nanometer gold surface according to above-mentioned method, polypeptide can be repaiied
Adorn on different shape and the golden nanometer particle surface of size, such as：The nano gold spherical of a diameter of 2-200nm, length are 20-200nm,
The nanometer gold bar of a diameter of 5-100nm or the star-like nm of gold of 10-200nm.
There is the performance of specific recognition EGFR due to the artificial antibody, and adhesion is strong, good stability, therefore can be with
Using the specific targets neoplastic cells surface overexpression of this artificial antibody or the EGFR of unconventionality expression.So this with spy
The artificial antibody of opposite sex identification EGFR functions has wide in fields such as medical diagnosis on disease, targeted therapy, photo-thermal therapy and medicine carrier bands
Wealthy application prospect.
The polypeptide that the present invention will be designed is modified to nanometer gold surface by S-Au keys, and the polypeptide of design is in nanometer gold surface shape
Into specific cyclic conformation, so as to prepare with targeting protein EGFR（epidermal growth factor receptor）
Nano artificial antibody.Because many tumor cell surface EGFR are overexpression, it is possible to use this nm of gold of synthesis
The EGFR of antibody specificity ground targets neoplastic cells surface expression high.Therefore, the nano artificial antibody that this method synthesis is obtained exists
The fields such as medical diagnosis on disease, targeted therapy, photo-thermal therapy and medicine carrier band have broad application prospects.
Brief description of the drawings
Fig. 1 is the schematic diagram of the artificial antibody of the targeting epidermal growth factor receptor EGFR based on golden nanometer particle.
Fig. 2 is the detection artificial antibody of embodiment one AuNP（4nm）+ P1 and protein EGFR specifically binds performance.
Fig. 3 is that embodiment one characterizes artificial antibody AuNP（4nm）The dynamic performance that+P1 is combined with EGFR.
Fig. 4 is the detection artificial antibody of embodiment one AuNP（4nm）The thermodynamic stability of+P1.
Fig. 5 is the detection artificial antibody of embodiment two AuNP（4nm）+ P2 and protein EGFR specifically binds performance.
Fig. 6 is that embodiment two characterizes artificial antibody AuNP（4nm）The dynamic performance that+P2 is combined with EGFR.
Fig. 7 is the detection artificial antibody of embodiment two AuNP（4nm）The thermodynamic stability of+P2.
Fig. 8 is the detection artificial antibody of embodiment three AuNP（4nm）+ P3 and EGFR specifically binds performance.
Fig. 9 is that embodiment three characterizes artificial antibody AuNP（4nm）The dynamic performance that+P3 is combined with EGFR.
Figure 10 is the detection artificial antibody of embodiment three AuNP（4nm）The thermodynamic stability of+P3.
Figure 11 is example IV detection artificial antibody AuNP（30nm）+ P1 and protein EGFR specifically binds performance.
Figure 12 is that example IV characterizes artificial antibody AuNP（30nm）The dynamic performance that+P1 is combined with EGFR.
Figure 13 is example IV detection artificial antibody AuNP（30nm）The thermodynamic stability of+P1.
Figure 14 is the detection artificial antibody of embodiment five AuNP（30nm）+ P2 and protein EGFR specifically binds performance.
Figure 15 is that embodiment five characterizes artificial antibody AuNP（30nm）The dynamic performance that+P2 is combined with EGFR.
Figure 16 is the detection artificial antibody of embodiment five AuNP（30nm）The thermodynamic stability of+P2.
Figure 17 is the detection artificial antibody of embodiment six AuNP（30nm）+ P3 and protein EGFR specifically binds performance.
Figure 18 is that embodiment six characterizes artificial antibody AuNP（30nm）The dynamic performance that+P3 is combined with EGFR.
Figure 19 is the detection artificial antibody of embodiment six AuNP（30nm）The thermodynamic stability of+P3.
Figure 20 is the detection artificial antibody of embodiment seven AuNP（4nm）+ P4 and AuNP（4nm）+ P2 is special with protein EGFR
Property binding ability.
Figure 21 is the detection artificial antibody of embodiment eight AuNP（4nm）+ P5 and AuNP（4nm）+ P2 is special with protein EGFR
Property binding ability.
Specific embodiment
Embodiment is given below so that the invention will be further described.Be necessary it is pointed out here that be that following examples can not
Be interpreted as limiting the scope of the invention, if the person skilled in the art in the field according to the invention described above content to this hair
It is bright to make some nonessential modifications and adaptations, small mutation for example is carried out to the polypeptide portion amino acid sequence for designing, still
Belong to the scope of the present invention.
Embodiment one：
1st, the design of polypeptide fragment
Directly cog region and fixed area are combined, regulatory region amino acid number is 0, devises polypeptide P1:
CAWYGTLYEYDC, as shown in Figure 1.The polypeptide P1 of design can be fixed on nm of gold by the sulfydryl in the cysteine at two ends
Surface, so as to form specific conformation.
2nd, the preparation of gold nano artificial antibody AuNP (the 4nm)+P1 with targeting EGFR performance
According to 1nm2The 0.05-2 ratio of polypeptide is fixed on nano gold spherical surface area, polypeptide is scheduled on by S-Au keyings
The nano gold spherical surface of 4nm, each nanometer of gold surface has 10-150 polypeptide.So as to obtain what can be specifically bound with EGFR
Artificial antibody AuNP (4nm)+P1.
3rd, artificial antibody AuNP is characterized（4nm）The specific binding performance of+P1 and protein EGFR
In order to detect artificial antibody AuNP（4nm）The ability of+P1 and EGFR specific bindings, we determine AuNP
（4nm）The combination of+P1 and EGFR, EGF and BSA, as shown in Figure 2.Artificial antibody AuNP（4nm）+ P1 and EGFR has obvious knot
Close, but do not combined significantly with EGF and BSA, illustrate artificial antibody AuNP（4nm）+ P1 has and EGFR specific bindings
Performance.
4th, artificial antibody AuNP is characterized（4nm）The dynamic performance that+P1 is combined with EGFR
In order to characterize the nm of gold antibody A uNP of synthesis（4nm）The dynamic performance that+P1 is combined with EGFR, we determine
AuNP（4nm）Dynamics spectrogram and binding constant K that+P1 is combined with EGFRDValue, as shown in Figure 3.Artificial antibody AuNP
（4nm）The binding constant K of+P1 and EGFRD=1.89E-11, artificial antibody A uNP is had found by contrasting（4nm）+ P1 and EGFR's
Binding constant is higher by three orders of magnitude than the binding constant of natural antibody and EGFR.These demonstrate that artificial antibody AuNP（4nm）+
P1 has the dynamic performance of combination EGFR more more preferable than natural antibody.
5th, AuNP is detected（4nm）The thermodynamic stability of+P1
In order to prove that artificial antibody has more preferable thermodynamic stability than natural antibody, we are by artificial antibody AuNP
（4nm）+ P1 is put into water-bath 1h in boiling water, then determines the performance of water-bath both front and back antibody combination EGFR, as shown in Figure 4.It is logical
Cross contrast artificial antibody AuNP（4nm）With the binding ability of EGFR before and after+P1 water-baths, it is found that the artificial antibody after water-bath combines
After the performance of EGFR is not reduced significantly compared to before water-bath, but most of natural antibodies are heated by boiling water, resist
Body structure can occur irreversible change so as to lose activity.Therefore the artificial antibody has superior heat compared to natural antibody
Mechanical stability.
Embodiment two：
1st, the design of polypeptide fragment
Two regulatory regions at cog region two ends respectively add a serine and a glycine, devise polypeptide P2:
CSAWYGTLYEYDGC, as shown in Figure 1.The polypeptide of design can be fixed on by the sulfydryl in two fixed area cysteines and be received
Rice gold surface, so as to form specific conformation.
2nd, the preparation of the gold nano artificial antibody with targeting EGFR performance
According to 1nm2The 0.05-2 ratio of polypeptide is fixed on golden nanometer particle surface area, polypeptide is scheduled on by S-Au keyings
The nano gold spherical surface of 4nm, each nanometer of gold surface has 10-150 polypeptide.So as to obtain what can be specifically bound with EGFR
Artificial antibody AuNP (4nm)+P2.
3rd, artificial antibody AuNP is characterized（4nm）The specific binding performance of+P2 and protein EGFR
In order to detect artificial antibody AuNP（4nm）The ability of+P2 and EGFR specific bindings, we determine AuNP
（4nm）The combination of+P2 and EGFR, EGF and BSA, as shown in Figure 5.Artificial antibody AuNP（4nm）+ P2 and EGFR has obvious knot
Close, but do not combined significantly with EGF and BSA, illustrate artificial antibody AuNP（4nm）+ P2 has and EGFR specific bindings
Performance.
4th, artificial antibody AuNP is characterized（4nm）The dynamic performance that+P2 is combined with EGFR
In order to characterize the nm of gold antibody A uNP of synthesis（4nm）The dynamic performance that+P2 is combined with EGFR, we determine
AuNP（4nm）Dynamics spectrogram and binding constant K that+P2 is combined with EGFRDValue, as shown in Figure 6.Artificial antibody AuNP
（4nm）The binding constant K of+P2 and EGFRD=4.04E-11.Artificial antibody A uNP is found by contrasting（4nm）+ P2 and EGFR's
Binding constant is higher by three orders of magnitude than the binding constant of natural antibody and EGFR.These demonstrate that artificial antibody AuNP（4nm）+
P2 has the dynamic performance of combination EGFR more more preferable than natural antibody.
6th, AuNP is detected（4nm）The thermodynamic stability of+P2
In order to prove that artificial antibody has more preferable thermodynamic stability than natural antibody, we are by artificial antibody AuNP
（4nm）+ P2 is put into water-bath 1h in boiling water, then determines the performance of water-bath both front and back antibody combination EGFR, as shown in Figure 7.It is logical
Cross contrast artificial antibody AuNP（4nm）With the binding ability of EGFR before and after+P2 water-baths, it is found that the artificial antibody after water-bath combines
After the performance of EGFR is not reduced significantly compared to before water-bath, but most of natural antibodies are heated by boiling water, resist
Body structure can occur irreversible change so as to lose activity.Therefore the artificial antibody has superior heat compared to natural antibody
Mechanical stability.
Embodiment three：
1st, the design of polypeptide fragment
Two regulatory regions at cog region two ends respectively add 5 amino acid, devise polypeptide P3:
CGAGASAWYGTLYEYDGSASAC, as shown in Figure 1.The polypeptide of design can be by the sulfydryl in two fixed area cysteines
A nanometer gold surface is fixed on, so as to form specific conformation.
2nd, the preparation of the gold nano artificial antibody with targeting EGFR performance
According to 1nm2The 0.05-2 ratio of polypeptide is fixed on golden nanometer particle surface area, polypeptide is determined by S-Au keyings
On the nano gold spherical surface of 4nm, each nanometer of gold surface has 10-150 polypeptide.So as to obtain being specifically bound with EGFR
Artificial antibody AuNP (4nm)+P3.
3rd, artificial antibody AuNP is characterized（4nm）The specific binding performance of+P3 and protein EGFR
In order to detect artificial antibody AuNP（4nm）The ability of+P3 and EGFR specific bindings, we determine AuNP
（4nm）The combination of+P3 and EGFR, EGF and BSA, as shown in Figure 8.Artificial antibody AuNP（4nm）+ P3 and EGFR has obvious knot
Close, but do not combined significantly with EGF and BSA, illustrate artificial antibody AuNP（4nm）+ P3 has and EGFR specific bindings
Performance.
4th, artificial antibody AuNP is characterized（4nm）The dynamic performance that+P3 is combined with EGFR
In order to characterize the nm of gold antibody A uNP of synthesis（4nm）The dynamic performance that+P3 is combined with EGFR, we determine
AuNP（4nm）Dynamics spectrogram and binding constant K that+P3 is combined with EGFRDValue, as shown in Figure 9.Artificial antibody AuNP
（4nm）The binding constant K of+P3 and EGFRD=5.96E-11.Artificial antibody A uNP is found by contrasting（4nm）+ P3 and EGFR's
Binding constant is higher by three orders of magnitude than the binding constant of natural antibody and EGFR.These demonstrate that artificial antibody AuNP（4nm）+
P3 has the dynamic performance of combination EGFR more more preferable than natural antibody.
7th, AuNP is detected（4nm）The thermodynamic stability of+P3
In order to prove that artificial antibody has more preferable thermodynamic stability than natural antibody, we are by artificial antibody AuNP
（4nm）+ P3 is put into water-bath 1h in boiling water, then determines the performance of water-bath both front and back antibody combination EGFR, as shown in Figure 10.It is logical
Cross contrast artificial antibody AuNP（4nm）With the binding ability of EGFR before and after+P3 water-baths, it is found that the artificial antibody after water-bath combines
After the performance of EGFR is not reduced significantly compared to before water-bath, but most of natural antibodies are heated by boiling water, resist
Body structure can occur irreversible change so as to lose activity.Therefore the artificial antibody has superior heat compared to natural antibody
Mechanical stability.
Example IV：
1st, the design of polypeptide fragment
Directly cog region and fixed area are combined, regulatory region amino acid number is 0, devises polypeptide P1:
CAWYGTLYEYDC, as shown in Figure 1.The polypeptide P1 of design can be fixed on nm of gold by the sulfydryl in the cysteine at two ends
Surface, so as to form specific conformation.
2nd, the preparation of gold nano artificial antibody AuNP (the 30nm)+P1 with targeting EGFR performance
According to 1nm2The 0.05-2 ratio of polypeptide is fixed on golden nanometer particle surface area, polypeptide is scheduled on by S-Au keyings
The golden nanometer particle surface of 30nm, each nanometer of gold surface has 10-150 polypeptide.So as to obtain being tied with EGFR specificity
Artificial antibody AuNP (4nm)+P1 of conjunction.
3rd, artificial antibody AuNP is characterized（30nm）The specific binding performance of+P1 and protein EGFR
In order to detect artificial antibody AuNP（30nm）The ability of+P1 and EGFR specific bindings, we determine AuNP
（30nm）The combination of+P1 and EGFR, EGF and BSA, as shown in figure 11.Artificial antibody AuNP（30nm）+ P1 and EGFR has significantly
With reference to, but do not combined significantly with EGF and BSA, illustrate artificial antibody AuNP（30nm）+ P1 has and EGFR specificity knots
The performance of conjunction.
4th, artificial antibody AuNP is characterized（30nm）The dynamic performance that+P1 is combined with EGFR
In order to characterize the nm of gold antibody A uNP of synthesis（30nm）The dynamic performance that+P1 is combined with EGFR, we determine
AuNP（30nm）Dynamics spectrogram and binding constant K that+P1 is combined with EGFRDValue, as shown in Figure 12.Artificial antibody AuNP
（30nm）The binding constant K of+P1 and EGFRD=9.80E-11.Artificial antibody A uNP is found by contrasting（30nm）+ P1 and EGFR
Binding constant of the binding constant than natural antibody and EGFR be higher by three orders of magnitude.These demonstrate that artificial antibody AuNP
（30nm）+ P1 has the dynamic performance of combination EGFR more more preferable than natural antibody.
5th, AuNP is detected（30nm）The thermodynamic stability of+P1
In order to prove that artificial antibody has more preferable thermodynamic stability than natural antibody, we are by artificial antibody AuNP
（30nm）+ P1 is put into water-bath 1h in boiling water, then determines the performance of water-bath both front and back antibody combination EGFR, as shown in figure 13.
By contrasting artificial antibody AuNP（30nm）With the binding ability of EGFR before and after+P1 water-baths, the artificial antibody after water-bath is found
Do not reduced significantly compared to before water-bath with reference to the performance of EGFR, but most of natural antibodies heat it by boiling water
Afterwards, antibody structure can occur irreversible change so as to lose activity.Therefore the artificial antibody has excellent compared to natural antibody
Thermodynamic stability more.
Embodiment five：
1st, the design of polypeptide fragment
Two regulatory regions at cog region two ends respectively add a serine and a glycine, devise polypeptide P2:
CSAWYGTLYEYDGC, as shown in Figure 1.The polypeptide of design can be fixed on by the sulfydryl in two fixed area cysteines and be received
Rice gold surface, so as to form specific conformation.
2nd, the preparation of the gold nano artificial antibody with targeting EGFR performance
According to 1nm2The 0.05-2 ratio of polypeptide is fixed on golden nanometer particle surface area, polypeptide is determined by S-Au keyings
On the golden nanometer particle surface of 4nm, each nanometer of gold surface has 10-150 polypeptide.So as to obtain being tied with EGFR specificity
Artificial antibody AuNP (30nm)+P2 of conjunction.
3rd, artificial antibody AuNP is characterized（30nm）The specific binding performance of+P2 and protein EGFR
In order to detect artificial antibody AuNP（30nm）The ability of+P2 and EGFR specific bindings, we determine AuNP
（30nm）The combination of+P2 and EGFR, EGF and BSA, as shown in figure 14.Artificial antibody AuNP（30nm）+ P2 and EGFR has significantly
With reference to, but do not combined significantly with EGF and BSA, illustrate artificial antibody AuNP（30nm）+ P2 has and EGFR specificity knots
The performance of conjunction.
4th, artificial antibody AuNP is characterized（30nm）The dynamic performance that+P2 is combined with EGFR
In order to characterize the nm of gold antibody A uNP of synthesis（30nm）The dynamic performance that+P2 is combined with EGFR, we determine
AuNP（30nm）Dynamics spectrogram and binding constant K that+P2 is combined with EGFRDValue, as shown in Figure 15.Artificial antibody AuNP
（30nm）The binding constant K of+P2 and EGFRD=4.92E-11.Artificial antibody A uNP is found by contrasting（30nm）+ P2 and EGFR
Binding constant of the binding constant than natural antibody and EGFR be higher by three orders of magnitude.These demonstrate that artificial antibody AuNP
（30nm）+ P2 has the dynamic performance of combination EGFR more more preferable than natural antibody.
8th, AuNP is detected（30nm）The thermodynamic stability of+P2
In order to prove that artificial antibody has more preferable thermodynamic stability than natural antibody, we are by artificial antibody AuNP
（30nm）+ P2 is put into water-bath 1h in boiling water, then determines the performance of water-bath both front and back antibody combination EGFR, as shown in figure 16.
By contrasting artificial antibody AuNP（30nm）With the binding ability of EGFR before and after+P2 water-baths, the artificial antibody after water-bath is found
Do not reduced significantly compared to before water-bath with reference to the performance of EGFR, but most of natural antibodies heat it by boiling water
Afterwards, antibody structure can occur irreversible change so as to lose activity.Therefore the artificial antibody has excellent compared to natural antibody
Thermodynamic stability more.
Embodiment six：
1st, the design of polypeptide fragment
Two regulatory regions at cog region two ends respectively add 5 amino acid, devise polypeptide P3:
CGAGASAWYGTLYEYDGSASAC, as shown in Figure 1.The polypeptide of design can be by the sulfydryl in two fixed area cysteines
A nanometer gold surface is fixed on, so as to form specific conformation.
2nd, the preparation of the gold nano artificial antibody with targeting EGFR performance
According to 1nm2The 0.05-2 ratio of polypeptide is fixed on golden nanometer particle surface area, polypeptide is determined by S-Au keyings
On the golden nanometer particle surface of 30nm, each nanometer of gold surface has 10-150 polypeptide.So as to obtain can with EGFR specificity
With reference to artificial antibody AuNP (30nm)+P3.
3rd, artificial antibody AuNP is characterized（30nm）The specific binding performance of+P3 and protein EGFR
In order to detect artificial antibody AuNP（30nm）The ability of+P3 and EGFR specific bindings, we determine AuNP
（30nm）The combination of+P3 and EGFR, EGF and BSA, as shown in figure 17.Artificial antibody AuNP（30nm）+ P3 and EGFR has significantly
With reference to, but do not combined significantly with EGF and BSA, illustrate artificial antibody AuNP（4nm）+ P3 has and EGFR specificity knots
The performance of conjunction.
4th, artificial antibody AuNP is characterized（30nm）The dynamic performance that+P3 is combined with EGFR
In order to characterize the nm of gold antibody A uNP of synthesis（30nm）The dynamic performance that+P3 is combined with EGFR, we determine
AuNP（30nm）Dynamics spectrogram and binding constant K that+P3 is combined with EGFRDValue, as shown in Figure 18.Artificial antibody AuNP
（30nm）The binding constant K of+P3 and EGFRD=7.38E-11.Artificial antibody A uNP is found by contrasting（30nm）+ P3 and EGFR
Binding constant of the binding constant than natural antibody and EGFR be higher by three orders of magnitude.These demonstrate that artificial antibody AuNP
（30nm）+ P3 has the dynamic performance of combination EGFR more more preferable than natural antibody.
5th, AuNP is detected（30nm）The thermodynamic stability of+P3
In order to prove that artificial antibody has more preferable thermodynamic stability than natural antibody, we are by artificial antibody AuNP
（30nm）+ P3 is put into water-bath 1h in boiling water, then determines the performance of water-bath both front and back antibody combination EGFR, as shown in figure 19.
By contrasting artificial antibody AuNP（30nm）With the binding ability of EGFR before and after+P3 water-baths, the artificial antibody after water-bath is found
Do not reduced significantly compared to before water-bath with reference to the performance of EGFR, but most of natural antibodies heat it by boiling water
Afterwards, antibody structure can occur irreversible change so as to lose activity.Therefore the artificial antibody has excellent compared to natural antibody
Thermodynamic stability more.
Embodiment seven：
1st, the design of polypeptide fragment
AWYGTLYEYD critical amino acid residues YEYD is constant in ensureing cog region, and other amino acid in cog region are entered
Row simple point mutation obtains AWYGTVYEYD；Two regulatory regions at cog region two ends respectively add a serine and a sweet ammonia
Acid, devises polypeptide P4:CSAWYGTVYEYDGC, as shown in Figure 1.The polypeptide of design can be by two fixed area cysteines
In sulfydryl be fixed on a nanometer gold surface, so as to form specific conformation.
2nd, the preparation of the gold nano artificial antibody with targeting EGFR performance
According to 1nm2The 0.05-2 ratio of polypeptide is fixed on golden nanometer particle surface area, polypeptide is scheduled on by S-Au keyings
The nano gold spherical surface of 4nm, each nanometer of gold surface has 10-150 polypeptide.So as to obtain what can be specifically bound with EGFR
Artificial antibody AuNP (4nm)+P4.
3rd, artificial antibody AuNP is characterized（4nm）+ P4 and AuNP（4nm）The specific binding performance of+P2 and protein EGFR
Polypeptide cog region is carried out after simple point mutation to artificial antibody AuNP to detect（4nm）+ P4 and EGFR specificity
Little with reference to influence, we determine AuNP（4nm）+ P4 and AuNP（4nm）+ P2 respectively and EGFR, EGF and BSA combination, such as
Shown in Figure 20.Artificial antibody AuNP（4nm）+ P4 and AuNP（4nm）+ P2 and EGFR have an obvious combination, but with EGF and
BSA is not combined significantly, illustrates artificial antibody AuNP（4nm）+ P4 has the performance specifically bound with EGFR.
Embodiment eight：
1st, the design of polypeptide fragment
Two regulatory regions at cog region two ends respectively add a serine and a glycine, by the poly- second with sulfydryl
Glycol（0.3K）Modified by dehydrating condensation and form fixed area at the two ends of polypeptide, devise polypeptide P5:SH-
PEGSAWYGTVYEYDGPEG-SH, as shown in Figure 1.The polypeptide of design can be by the sulfydryl in two fixed area cysteines
A nanometer gold surface is fixed on, so as to form specific conformation.
2nd, the preparation of the gold nano artificial antibody with targeting EGFR performance
According to 1nm2The 0.05-2 ratio of polypeptide is fixed on golden nanometer particle surface area, polypeptide is scheduled on by S-Au keyings
The nano gold spherical surface of 4nm, each nanometer of gold surface has 10-150 polypeptide.So as to obtain what can be specifically bound with EGFR
Artificial antibody AuNP (4nm)+P5.
3rd, artificial antibody AuNP is characterized（4nm）+ P5 and AuNP（4nm）The specific binding performance of+P2 and protein EGFR
In order to detect that polypeptide fixed area changes to artificial antibody AuNP（4nm）The influence of+P5 and EGFR specific bindings, I
Determine AuNP（4nm）+ P5 and AuNP（4nm）+ P2 respectively and EGFR, EGF and BSA combination, as shown in figure 21.It is artificial anti-
Body AuNP（4nm）+ P6 and AuNP（4nm）+ P2 and EGFR have obvious combination, but are not tied significantly with EGF and BSA
Close, illustrate artificial antibody AuNP（4nm）+ P5 has the performance specifically bound with EGFR.
<110>Shanghai University
<120>Artificial antibody of targeting epidermal growth factor receptor EGFR based on golden nanometer particle and preparation method thereof
<160> 4
<210> 1
<211> 10
<212>Single stranded DNA
<213>Artificial sequence
<400> 1
AWYGT LYEYD 10
<210> 2
<211> 10
<212>Single stranded DNA
<213>Artificial sequence
<400> 2
AWYGT VYEYD 10
<210> 3
<211> 10
<212>Single stranded DNA
<213>Artificial sequence
<400> 3
SWYGT LYEYD 10
<210> 4
<211> 10
<212>Single stranded DNA
<213>Artificial sequence
<400> 4
AWYGS LYEYD 10
1
Claims (4)
1. a kind of artificial receptors of the targeting epidermal growth factor receptor EGFR based on golden nanometer particle, by golden nanometer particle and
Polypeptide is constituted, it is characterised in that described polypeptide is made up of cog region, two control regions and two fixed areas；Described cog region
Two ends respectively connection one control region；And respectively connect a fixed area at the two ends of control region；The polypeptide is consolidated by its two ends
Determine area and be fixed on golden nanometer particle surface so as to form cyclic structure；
Described control region is used for adjusting the length of polypeptide, by 0~8 amino acid sequence random alignment group of non-cysteine
Into；Described fixed area spontaneously forms S-Au keys by sulfydryl and nanometer gold surface, so as to the polypeptide two ends are fixed on into gold
Nanoparticle surface, fixes 0.05~2 polypeptide, so that polypeptide is in Jenner on every 1 nm2 golden nanometer particle surface areas
Rice corpuscles surface forms cyclic conformation.
2. artificial receptors of the targeting epidermal growth factor receptor EGFR based on golden nanometer particle according to claim 1,
It is characterized in that the amino acid sequence of described cog region be AWYGTLYEYD, AWYGTVYEYD, SWYGTLYEYD or
AWYGSLYEYD。
3. artificial receptors of the targeting epidermal growth factor receptor EGFR based on golden nanometer particle according to claim 1,
It is characterized in that described fixed area is：Cysteine modifies in regulation and control the polyethylene glycol with sulfydryl by dehydrating condensation
The two ends in area form fixed area.
4. artificial receptors of the targeting epidermal growth factor receptor EGFR based on golden nanometer particle according to claim 1,
It is characterized in that the amino acid of described composition control region is：Glycine, serine, alanine, glutamine and asparagine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610927137.0A CN106699890B (en) | 2016-10-31 | 2016-10-31 | Gold nanoparticle-based artificial antibody targeting Epidermal Growth Factor Receptor (EGFR) and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610927137.0A CN106699890B (en) | 2016-10-31 | 2016-10-31 | Gold nanoparticle-based artificial antibody targeting Epidermal Growth Factor Receptor (EGFR) and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106699890A true CN106699890A (en) | 2017-05-24 |
| CN106699890B CN106699890B (en) | 2021-05-07 |
Family
ID=58940397
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610927137.0A Active CN106699890B (en) | 2016-10-31 | 2016-10-31 | Gold nanoparticle-based artificial antibody targeting Epidermal Growth Factor Receptor (EGFR) and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106699890B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108794626A (en) * | 2018-06-18 | 2018-11-13 | 上海大学 | A kind of polypeptide and the gold nano antibody that HIV envelope proteins gp120 can be targeted |
| CN110090309A (en) * | 2019-05-28 | 2019-08-06 | 上海大学 | The preparation method of functionalizing erythrocyte membrane |
| CN112062812A (en) * | 2020-06-16 | 2020-12-11 | 上海大学 | Polypeptide, anti-lysozyme artificial antibody and application thereof |
| CN112062841A (en) * | 2020-06-16 | 2020-12-11 | 上海大学 | Polypeptide, preparation method thereof, anti-p 53 protein artificial antibody and application thereof |
| CN112375135A (en) * | 2020-07-06 | 2021-02-19 | 上海大学 | Artificial epidermal growth factor, designed polypeptide and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011139792A2 (en) * | 2010-04-27 | 2011-11-10 | Ventana Medical Systems, Inc. | Antibody-nanoparticle conjugates and methods for making and using such conjugates |
| CN104353082A (en) * | 2014-11-13 | 2015-02-18 | 福州大学 | Functional nano material drug delivery system for identifying, capturing and restraining circulating tumor cells |
| CN104379595A (en) * | 2012-06-25 | 2015-02-25 | Hoya株式会社 | Egfr-binding peptide |
| CN105085632A (en) * | 2015-09-14 | 2015-11-25 | 国家纳米科学中心 | Specific EGFR (epidermal growth factor receptor)-protein-targeted polypeptide and application thereof |
| CN105884888A (en) * | 2016-04-14 | 2016-08-24 | 上海大学 | Artificial antibody construction method based on gold nanoparticles |
| CN105940113A (en) * | 2013-11-13 | 2016-09-14 | 酵活有限公司 | Monovalent antigen binding constructs targeting egfr and/or her2 and uses thereof |
-
2016
- 2016-10-31 CN CN201610927137.0A patent/CN106699890B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011139792A2 (en) * | 2010-04-27 | 2011-11-10 | Ventana Medical Systems, Inc. | Antibody-nanoparticle conjugates and methods for making and using such conjugates |
| CN104379595A (en) * | 2012-06-25 | 2015-02-25 | Hoya株式会社 | Egfr-binding peptide |
| CN105940113A (en) * | 2013-11-13 | 2016-09-14 | 酵活有限公司 | Monovalent antigen binding constructs targeting egfr and/or her2 and uses thereof |
| CN104353082A (en) * | 2014-11-13 | 2015-02-18 | 福州大学 | Functional nano material drug delivery system for identifying, capturing and restraining circulating tumor cells |
| CN105085632A (en) * | 2015-09-14 | 2015-11-25 | 国家纳米科学中心 | Specific EGFR (epidermal growth factor receptor)-protein-targeted polypeptide and application thereof |
| CN105884888A (en) * | 2016-04-14 | 2016-08-24 | 上海大学 | Artificial antibody construction method based on gold nanoparticles |
Non-Patent Citations (2)
| Title |
|---|
| CHIARA PAVIOLO等: "Inhibiting EGFR Clustering and Cell Proliferation with Gold Nanoparticles", 《SMALL NANOMICRO》 * |
| MIR HADI JAZAYERI等: "Various methods of gold nanoparticles (GNPs) conjugation to antibodies", 《SENSING AND BIO-SENSING RESEARCH》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108794626A (en) * | 2018-06-18 | 2018-11-13 | 上海大学 | A kind of polypeptide and the gold nano antibody that HIV envelope proteins gp120 can be targeted |
| CN108794626B (en) * | 2018-06-18 | 2021-10-12 | 上海大学 | Polypeptide and gold nanobody capable of targeting HIV envelope protein gp120 |
| CN110090309A (en) * | 2019-05-28 | 2019-08-06 | 上海大学 | The preparation method of functionalizing erythrocyte membrane |
| CN112062812A (en) * | 2020-06-16 | 2020-12-11 | 上海大学 | Polypeptide, anti-lysozyme artificial antibody and application thereof |
| CN112062841A (en) * | 2020-06-16 | 2020-12-11 | 上海大学 | Polypeptide, preparation method thereof, anti-p 53 protein artificial antibody and application thereof |
| CN112062812B (en) * | 2020-06-16 | 2022-11-18 | 上海大学 | Polypeptide, anti-lysozyme artificial antibody and application thereof |
| CN112062841B (en) * | 2020-06-16 | 2022-11-18 | 上海大学 | Polypeptide, preparation method thereof, p53 protein-resistant artificial antibody and application thereof |
| CN112375135A (en) * | 2020-07-06 | 2021-02-19 | 上海大学 | Artificial epidermal growth factor, designed polypeptide and application thereof |
| CN112375135B (en) * | 2020-07-06 | 2022-12-23 | 上海大学 | Artificial epidermal growth factor, designed polypeptide and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106699890B (en) | 2021-05-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106699890A (en) | Artificial antibody for targeting EGFR (epidermal growth factor receptor) based on gold nanoparticles and preparation method thereof | |
| CN103097403B (en) | Chlorotoxin variants, conjugates, and methods for their use | |
| Di Marco et al. | Overview of the main methods used to combine proteins with nanosystems: absorption, bioconjugation, and encapsulation | |
| Kang et al. | Developing an antibody-binding protein cage as a molecular recognition drug modular nanoplatform | |
| Vannucci et al. | Selective targeting of melanoma by PEG-masked protein-based multifunctional nanoparticles | |
| Shemetov et al. | Molecular interaction of proteins and peptides with nanoparticles | |
| Montenegro et al. | Controlled antibody/(bio-) conjugation of inorganic nanoparticles for targeted delivery | |
| JP2020143104A5 (en) | ||
| CN105884888B (en) | Artificial antibody construction method based on gold nanoparticles | |
| CN103409427B (en) | Aptamer EpCAM C of epithelial cell adhesion molecule and preparation method thereof | |
| CN109843336A (en) | Method and composition for targeting T-cells cancer | |
| Konur et al. | Scientometric overview in nanobiodrugs | |
| CN106257271B (en) | A kind of composite material and preparation method based on Surface enhanced Raman scattering technology | |
| CN105968204A (en) | Single-domain heavy chain antibody for anti-prostate specific membrane antigen | |
| CN106008716A (en) | Respiratory Syncytial Virus Antigenic Compositions and Methods | |
| CN105968201A (en) | Single-domain heavy-chain antibody aiming at prostate specific membrane antigen | |
| CN109553684A (en) | A kind of nano-carrier albumen and its preparation method and application | |
| Ghisaidoobe et al. | Functionalized protein nanocages as a platform of targeted therapy and immunodetection | |
| CN105378479A (en) | Method for predicting clinical effect of immunotherapy | |
| CN108578369A (en) | The preparation and application of the targeting human serum albumins nano-medicament carrier of the double modifications in surface | |
| Cavallaro et al. | Peptides targeting HER2-positive breast cancer cells and applications in tumor imaging and delivery of chemotherapeutics | |
| CN110420334A (en) | A kind of method of drug conjugate and raising drug molecule half-life period | |
| WO2022027829A1 (en) | Protein-type nanoparticle for delivery of multi-specific antibody, application thereof and preparation method therefor | |
| CN105478087A (en) | Method for preparing carboxyl magnetic beads based on glucan coating and application of method | |
| CN105968203A (en) | Single-domain heavy chain antibody for anti-prostate specific membrane antigen extracellular region |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |