CN115403672A - Biotinylated anti-lysozyme antibody and construction method thereof - Google Patents
Biotinylated anti-lysozyme antibody and construction method thereof Download PDFInfo
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- CN115403672A CN115403672A CN202211039680.9A CN202211039680A CN115403672A CN 115403672 A CN115403672 A CN 115403672A CN 202211039680 A CN202211039680 A CN 202211039680A CN 115403672 A CN115403672 A CN 115403672A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
- G01N2333/936—Hydrolases (3) acting on glycosyl compounds (3.2) acting on beta-1, 4 bonds between N-acetylmuramic acid and 2-acetyl-amino 2-deoxy-D-glucose, e.g. lysozyme
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Abstract
The invention discloses a biotinylated anti-lysozyme gold antibody and a construction method of the biotinylated gold antibody. The biotinylated anti-lysozyme gold antibody can be specifically bound with lysozyme and streptavidin, and the gold antibody can still be bound with lysozyme in a complex environment, so that the biotinylated anti-lysozyme gold antibody has the potential of being applied to an immunoassay method. In addition, compared with a natural antibody which is easily inactivated by the influence of environmental temperature, the gold antibody can keep a high inhibition rate on lysozyme activity after high-temperature pretreatment, and has great potential for replacing the natural antibody for application.
Description
Technical Field
The invention relates to the field of nano medicine, in particular to a multifunctional anti-lysozyme gold antibody and a construction method of a biotinylated gold antibody.
Background
Lysozyme is a protein with the molecular weight of 14400Da, and the primary sequence of the lysozyme contains 129 amino acids. The lysozyme can catalyze the hydrolysis of glycosidic bonds of gram-positive bacteria cell walls so as to destroy the cell membranes of the bacteria, so that the lysozyme has a strong bactericidal effect. In human body, lysozyme is a natural antibacterial and anti-infectious substance, and has the functions of diminishing inflammation, resisting virus, etc., and the content of lysozyme in human body can be used as one of indexes for diagnosing various diseases. In the food industry, lysozyme is widely used as a preservative, but lysozyme is also a common allergen, and the content of lysozyme needs to be detected in the production and use processes.
The methods developed at present for detecting lysozyme basically comprise two methods, namely an instrumental detection method and an immunoassay method. The instrument detection method generally uses instruments such as high performance liquid chromatography, liquid chromatography-mass spectrometry and the like, the methods need complex sample pretreatment, the instruments are expensive, and the detection process needs professional personnel. The immunoassay method mainly based on the enzyme-linked immunosorbent assay and the lateral immunochromatography can quickly complete detection and also can detect low-concentration samples with high accuracy, and the immunoassay method can better meet the requirements in real life.
Immunoassays are a broad class of methods established based on the interaction of antibodies with antigens. The core of all immunoassay methods is antibodies, and the currently used natural antibodies generally have the defects of difficult preparation, easy inactivation caused by temperature change and the like, and the defects can cause certain limitation on the application of the immunoassay methods. Therefore, there is a need to develop a natural antibody substitute that is simple in preparation process and has good thermostability.
The anti-lysozyme antibody is a brand new artificial antibody constructed by a conformation engineering method. The gold nanoparticles are grafted with the Complementarity Determining Regions (CDRs) of the natural antibody, the conformation of the CDRs in the natural antibody is reconstructed through conformation engineering, and the gold antibody has the capability of specifically binding lysozyme, has excellent thermal stability and has the potential of becoming a substitute of the natural antibody. The quantity of the polypeptide on the gold antibody is further reduced through polyethylene glycol, the cost of synthesizing the gold antibody is reduced, and the quantity of the coupled antigen on the surface of the gold antibody is reduced. However, monofunctional gold antibodies are difficult to use in immunoassays.
Disclosure of Invention
The invention provides a biotinylation anti-Lysozyme gold antibody and a construction method thereof on the basis of a patent with the publication number of CN105884888A, aiming at solving the problems of multi-functionalization technology and cost of the gold antibody.
In order to achieve the purpose, the invention adopts the following technical scheme:
a biotinylated anti-lysozyme antibody is biotinylated, and the biotinylated anti-lysozyme antibody can simultaneously bind lysozyme and streptavidin for immunoassay. The invention couples the polypeptide from the natural antibody determinant complementary region of lysozyme on the gold nano-particles to prepare the anti-lysozyme gold antibody. The biotinylated anti-lysozyme antibody can be simultaneously specifically combined with lysozyme and streptavidin, and can be directly used for immunoassay.
A construction method of a biotinylated anti-lysozyme gold antibody is characterized by comprising the following steps: in the process of preparing the anti-lysozyme gold antibody, biotin is covalently coupled on gold nanoparticles through polyethylene glycol, and finally, the exposed sites on the gold nanoparticles are sealed by using the polyethylene glycol, so that the biotinylated anti-lysozyme gold antibody capable of specifically binding lysozyme and streptavidin is prepared.
The construction method of the biotinylation anti-lysozyme gold antibody greatly reduces the quantity of polypeptide on the basis of coupling the polypeptide from a lysozyme natural antibody determinant complementary region on gold nanoparticles to prepare the anti-lysozyme gold antibody, and covalently couples biotin on the gold nanoparticles through polyethylene glycol, and finally closes exposed sites on the gold nanoparticles by using the polyethylene glycol to prepare the biotinylation anti-lysozyme gold antibody capable of specifically combining lysozyme and streptavidin. In the preparation process of the original single-function gold antibody, the invention reduces the coupling quantity of polypeptide, simultaneously uses polyethylene glycol to seal the exposed sites on the gold nanoparticles, and covalently couples biotin on the gold nanoparticles through the polyethylene glycol to prepare the biotinylated gold antibody.
Preferably, gold nanoparticles having a particle size of 2 to 100nm are used.
Preferably, the biotin is coupled on the gold nanoparticles through polyethylene glycol, the molecular weight of the polyethylene glycol is 300-10000Da, one end of the polyethylene glycol is biotin, the other end of the polyethylene glycol is a group containing sulfydryl, such as cysteine, and the polyethylene glycol can react with the gold nanoparticles to generate Au-S bonds so as to covalently connect the two.
Preferably, the coupling amount of biotin is calculated according to the surface area of the gold nanoparticles, and the ratio is 1nm 2 The surface area of the gold nano particles is coupled with 0.005-0.5 biotin.
Compared with the prior art, the invention has the following obvious substantive characteristics and remarkable advantages:
1. compared with the natural antibody, the biotinylation anti-lysozyme gold antibody has the advantages that the biotinylation anti-lysozyme gold antibody can be specifically combined with lysozyme, is simple in preparation process and good in thermal stability, and has great potential for being applied to an immunoassay method;
2. compared with the anti-lysozyme antibody, the biotinylated anti-lysozyme antibody can be specifically combined with lysozyme and streptavidin, is more suitable for immunoassay and other applications, and has other advantages of the anti-lysozyme antibody.
Drawings
FIG. 1 is a schematic structural diagram of a biotinylated anti-lysozyme gold antibody of the present invention.
FIG. 2 is a graph showing the change in the mean hydrated particle size of gold antibodies according to a preferred embodiment of the present invention.
FIG. 3 is a graph showing the results of fluorescence quenching experiments according to the preferred embodiment of the present invention.
FIG. 4 is a diagram showing the results of the lysozyme activity test performed in the complex environment according to the preferred embodiment of the present invention.
FIG. 5 is a graph showing the results of the experiment on the thermostability of the gold antibody according to the preferred embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In a preferred embodiment of the present invention, an anti-lysozyme gold antibody having the same specificity as a natural antibody was prepared by grafting a gold nanoparticle with an S-Au bond using Pep1 designed from a polypeptide fragment of CDR3 of cAb-Lys3 disclosed in patent publication No. CN 105884888A. On the basis of the anti-lysozyme gold antibody, biotin is coupled on the gold nanoparticles through polyethylene glycol, so that the gold antibody is biotinylated, meanwhile, the polyethylene glycol is used for sealing exposed sites on the gold nanoparticles, the structural schematic diagram of the biotinylated anti-lysozyme gold antibody is shown in figure I, and the prepared biotinylated anti-lysozyme gold antibody has the capacity of specifically binding lysozyme and streptavidin.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in further detail below.
The above-described embodiments are further illustrated below with reference to specific examples, in which preferred embodiments of the invention are detailed below:
the first embodiment is as follows:
in this example, biotinylated anti-lysozyme gold antibodies were prepared as follows:
the 14nm gold nano-particle is synthesized by the following method: a500 mL round bottom flask was charged with a Teflon stirrer (25X 9.5 mm), 240mL of ultrapure water and 25mL of 39.47mM trisodium citrate were added to the flask, the round bottom flask was put in a constant temperature oil bath at 140 ℃ and after 20min of condensation and reflux, 10mL of a 25mM chloroauric acid solution was rapidly added, and the mixture was stirred uniformly until the solution became wine red and cooled naturally to room temperature. The Teflon stirrer in the round-bottom flask is taken out, and the gold nanoparticles are filtered by a 0.22 mu m filter membrane and then put at 4 ℃ for standby.
Preparation of biotinylated anti-lysozyme gold antibody: 6mL of gold nanoparticle solution was added to a 10mL glass vial containing a 15X 8mm diamond Teflon magnetic stirrer and stirred uniformly at 650rpm to ensure no bubbles were generated during the entire reaction. 1mL of thiol-polyethylene glycol thiol solution (containing 60. Mu.L of 2mM sodium hydroxide) was added and stirring was continued at room temperature for 1h. Add 500. Mu.L of biotin solution and stir at room temperature for 1h. Adding 500 mu L of polypeptide Pep1 solution, continuously stirring at room temperature for 1h, taking out the stirrer, and placing the stirrer into a shaker (100 rpm) for reacting at room temperature for 12h. The biotinylated anti-lysozyme gold antibody was concentrated to 10nM by ultrafiltration centrifugation, gold nanoparticles: mercapto polyethylene glycol mercapto group: biotin: polypeptide Pep1=1:500:5:10 (ratio of final molarity).
Example two:
the present embodiment is substantially the same as the first embodiment, and the special points are that:
in this example, biotinylated anti-lysozyme gold antibody was tested for binding to lysozyme and streptavidin using a dynamic light scattering particle size Detector (DLS) as follows:
the whole test process is carried out at room temperature, and a lysozyme solution and a streptavidin solution with the concentration of 14nM are respectively prepared by using a carbonate buffer solution with the pH value of 0.02M and 9.2 as a buffer solution. Firstly, taking 1mL of gold antibody solution for testing to obtain the average hydrated particle size of the gold antibody, then respectively incubating 1mL of gold antibody (3.5 nM) and 1mL of two target proteins for 15min, and then taking 1mL of mixed solution for testing to obtain the particle size change of the gold antibody; and finally, mixing the gold antibody and the two target proteins at the same time, incubating for 15min, and then testing to obtain the average hydrated particle size of the gold antibody.
FIG. 2 is a graph showing the variation of the average hydrated particle size of the gold antibody, and it can be seen from the graph that the particle size of the gold antibody increases to different extents after the gold antibody and two target proteins are incubated respectively, indicating that the gold antibody can bind to the two target proteins; after incubation with both proteins, the particle size further increased, which may preliminarily suggest that the gold antibody may bind to both proteins of interest simultaneously.
Example three:
the present embodiment is substantially the same as the first embodiment, and the special points are that:
in this example, the binding of gold antibody biotinylated anti-lysozyme to lysozyme and streptavidin was demonstrated by a fluorescence quenching experiment as follows:
the lysozyme and the streptavidin are respectively labeled with Fluorescein Isothiocyanate (FITC) and Tetramethyl Rhodamine Isothiocyanate (TRITC) in advance. By utilizing the characteristic that the gold nanoparticles can quench the surface fluorescent groups, if the gold antibody is combined with two target proteins, the fluorescent groups marked by the target proteins can be quenched. After incubating 0.5FITC-Lysozyme with 0.5mL of Streptavidin solution for 5min at room temperature, 0.5mL of gold antibody solution (10 nM) was added and the incubation was continued for 10min with shaking thoroughly, and 0.5mL of the mixed solution was used for detection. Meanwhile, 0.5mL of Lysozyme and 0.5mL of TRITC-Streptavidin solution were mixed and incubated at room temperature for 5min, then 0.5mL of gold antibody solution (10 nM) was added and sufficiently shaken, and then incubation was continued for 10min, and 0.5mL of the mixed solution was taken for detection. So as to respectively detect the combination of the gold antibody and the two target proteins. When fluorescence quenching of the gold antibody to FITC-Lysozyme is detected, the excitation wavelength is set to 488nm; when TRITC-Streptavidin was detected, the excitation wavelength was set to 547nm. In the whole experiment process, the width of the excitation slit and the width of the emission slit are both 5nm.
FIG. 3 shows the results of fluorescence quenching experiments with Lysozyme in which the gold antibody quenches the fluorophore FITC on Lysozyme regardless of the presence or absence of Streptavidin, i.e., streptavidin does not affect the binding of gold antibody to Lysozyme; similar conclusions can also be drawn for Streptavidin, i.e. Lysozyme does not affect the binding of gold antibodies to Streptavidin. This may further demonstrate that gold antibodies can bind Lysozyme and Streptavidin simultaneously.
Example four:
this embodiment is substantially the same as the previous embodiment, and is characterized in that:
in this example, the binding of a biotinylated anti-lysozyme gold antibody to lysozyme in a complex environment was simulated as follows:
in practical application, many non-specific proteins often exist, for this reason, ribonuclease A (RNase A) with the size and the property extremely similar to those of lysozyme is selected, the concentration of the ribonuclease A is set to be 5 times of that of the lysozyme, and the lysozyme activity test experiment proves the binding capacity of the gold antibody to the lysozyme in a complex environment. Meanwhile, gold nanoparticles and polypeptide Pep1s (with the same amino acid composition as Pep1 but different arrangement order) were used as control groups. FIG. 4 shows the results of the lysozyme activity test experiments, and we can see that, compared with the control group, even if high concentration of nonspecific protein ribonuclease A appears in the environment, the gold antibody can still maintain higher inhibition rate on lysozyme activity, which indicates that the gold antibody has the potential for being applied to a real complex environment.
Example five:
this embodiment is substantially the same as the previous embodiment, and is characterized in that:
in this example, the thermal stability of biotinylated anti-lysozyme gold antibodies was tested as follows:
the natural antibody is easy to be inactivated by the change of environmental temperature, and in order to test the thermal stability of the gold antibody, the gold antibody is pretreated for 1h in water baths at 60, 80 and 100 ℃ respectively, and a lysozyme activity test experiment is carried out after natural cooling. FIG. 5 shows the results of the thermal stability test, and we can see that the biotinylated anti-lysozyme gold antibody maintains a high inhibition rate of 79% without much decrease in the lysozyme activity even after pretreatment at a high temperature of 100 ℃, which indicates that it has excellent thermal stability.
The construction method of the biotinylated anti-lysozyme gold antibody of the above embodiment. Based on the anti-lysozyme gold antibody, the coupling quantity of the polypeptide is reduced, and biotin is coupled on the gold nanoparticles through polyethylene glycol to complete the biotinylation of the gold antibody. The biotinylation anti-lysozyme gold antibody can be specifically combined with lysozyme and streptavidin, and the gold antibody can still be combined with lysozyme in a complex environment, so that the biotinylation anti-lysozyme gold antibody has the potential of being applied to an immunoassay. In addition, compared with a natural antibody which is easily inactivated by the influence of environmental temperature, the gold antibody can keep a high inhibition rate on lysozyme activity after pretreatment at a high temperature of 100 ℃, so that the gold antibody has great potential for replacing the natural antibody for application.
The embodiments of the present invention have been described above with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made according to the purpose of the invention, and any changes, modifications, substitutions, combinations or simplifications made based on the spirit and principle of the technical solution of the present invention shall be equivalent replacement, so long as the invention is in accordance with the purpose of the present invention, and the technical principle and the inventive concept of the present invention shall fall within the protection scope of the present invention.
Claims (5)
1. A biotinylated anti-lysozyme gold antibody characterized by: the anti-lysozyme antibody is biotinylated, and the biotinylated anti-lysozyme antibody can be simultaneously combined with lysozyme and streptavidin for immunoassay.
2. The biotinylated anti-lysozyme gold antibody of claim 1, wherein: the grain diameter of the gold nano-particles is 2-100nm.
3. A construction method of a biotinylated anti-lysozyme gold antibody is characterized by comprising the following steps: in the process of preparing the anti-lysozyme gold antibody, biotin is covalently coupled on gold nanoparticles through polyethylene glycol, and finally, the exposed sites on the gold nanoparticles are sealed by using the polyethylene glycol, so that the biotinylated anti-lysozyme gold antibody capable of specifically binding lysozyme and streptavidin is prepared.
4. The method of claim 3 for the construction of a biotinylated anti-lysozyme gold antibody, characterized in that: the biotin is coupled on the gold nanoparticles through polyethylene glycol, the molecular weight of the polyethylene glycol is 300-10000Da, one end of the polyethylene glycol is biotin, the other end of the polyethylene glycol is a group containing sulfydryl, such as cysteine, and the like, and the polyethylene glycol can react with the gold nanoparticles to generate Au-S bonds so as to be covalently connected with the Au-S bonds.
5. The method of claim 3 for the construction of a biotinylated anti-lysozyme gold antibody, characterized in that: the coupling amount of biotin is calculated according to the surface area of the gold nanoparticles, and the ratio is 1nm 2 The surface area of the gold nano particles is coupled with 0.005-0.5 biotin.
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