Method for coupling two proteins

Abstract

The invention discloses a method for coupling two proteins. The method comprises the following steps: carrying out first modification treatment on a first protein, wherein the first modification is N3 or dibenzyl cyclooctyne modification; performing second modification treatment on the second protein, wherein the second modification is dibenzyl cyclooctyne or N3 modification; carrying out coupling treatment on the modified first protein and second protein under the action of a coupling agent; wherein the first modification treatment is different from the second modification treatment. The two proteins are coupled through the method, natural structures of the proteins are not damaged, and the stability and activity of the proteins are improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically, to a method for coupling luciferase to an antibody, and in particular to a method for coupling two proteins, a protein complex, an antigen detection method, a kit and applications thereof. Background Art

[0002] Luciferase is a commonly used biomarker and reporter gene that can be used as a detection tool for biological molecules. Gaussia luciferase (Gaussia princeps luciferase, Gluc) is a luciferase isolated from the marine copepod Gaussia princeps, with a molecular weight of about 19.9kDa and composed of 185 amino acids. Gaussia luciferase can catalyze the enzymatic oxidation reaction of luciferin substrates (such as coelenterazine and its derivatives) and emit fluorescence with a wavelength between 460-480nm (depending on the substrate, the photon wavelength is different). Gaussia luciferase is different from the firefly luciferase system. It does not require the use of high-energy molecules such as ATP and coenzyme A. The reaction only requires coelenterazine and O2 to participate, which greatly simplifies its application. Based on the specificity of Gaussia luciferase, it can be coupled with antibodies to achieve quantitative and localized analysis of biological molecules, thereby expanding its scope of application.

[0003] Bioconjugation is a process in which a molecule is attached to another molecule through a covalent bond to produce a complex consisting of two molecules connected together. The bioconjugation reaction is usually carried out under the mediation of a chemical coupling agent or enzyme. Since the production cost of enzyme coupling is high and the production process is relatively complicated, chemical coupling methods are usually used for coupling.

[0004] Primary amines (-NH2) are present at the N-terminus of each polypeptide chain (called the α-amino group) and in the side chains of lysine (Lys, K) residues (called the ε-amino group). Because primary amines are positively charged under physiological conditions, they are usually on the outer surface of proteins, making them easier to couple without changing the protein structure. Many chemical reactive groups target primary amines, and the most commonly used chemical reactive group is N-hydroxysuccinimide ester (NHS ester). NHS ester is a reactive group formed by carbodiimide (EDC) activated carboxylic acid molecules. Cross-linkers and labeling compounds with activated NHS esters react with primary amines under weak alkaline conditions to produce stable amide bonds, so that luciferase and antibodies are linked together with stable covalent bonds without affecting their biological activity and spatial conformation. Moreover, the reaction releases N-hydroxysuccinimide (NHS), which can be easily removed by dialysis or desalting.

[0005] Click chemistry was proposed by Barry Sharpless around 2000. It is a simple and reliable chemical reaction form that can react quickly and avoid the formation of by-products. It conforms to the 12 principles of green chemistry and has the characteristics of mild reaction conditions, high reaction yield, simple product separation and no harmful by-products. Click chemistry has good applications in many fields such as drug development, biomedical material optimization, biomolecule labeling and detection. Bertozzi et al. developed the strain-promoted azide-alkyne cycloaddition (SPAAC) reaction in 2004, which does not require the use of metal catalysts, reducing agents or stable ligands. Instead, the reaction uses the enthalpy released by the ring strain to form cyclooctyne to form a stable triazole. The reaction of dibenzylcyclooctyne (DBCO) group with azide group (Azide, N3) is a classic SPAAC reaction. DBCO and N3 groups can react selectively to form triazole bonds, and no additional copper ions need to be introduced, thereby preventing copper-induced damage to proteins. Summary of the invention

[0006] This application is filed by the inventor based on the discovery of the following facts and problems:

[0007] SMCC (succinimidyl 4-(N-maleimidyl) cyclohexane-1-carboxylate) coupling is a technique that uses SMCC coupling agents to cross-link proteins containing a primary amine and a sulfhydryl group. One end of the SMCC coupling agent is N-hydroxysuccinimide ester (NHS esters, NHS ester), and the other end is maleimide. Since the maleimide group in the SMCC molecule needs to react with the free sulfhydryl group in the protein, it is inevitable to destroy the natural structure of the protein, resulting in a decrease in the natural activity of the protein or even inactivation. Since the experimental process is linear, the reaction of HNS ester with luciferase must be carried out first, and then with Ab-Dig (digoxigenin antibody), which greatly reduces the flexibility of experimental design during the development process.

[0008] In view of this, the present invention aims to solve one of the problems in the related art at least to some extent.

[0009] In the first aspect of the present invention, the present invention proposes a method for coupling two proteins. According to an embodiment of the present invention, the method comprises: performing a first modification treatment on a first protein, wherein the first modification is N3 or dibenzylcyclooctyne modification; performing a second modification treatment on a second protein, wherein the second modification is dibenzylcyclooctyne or N3 modification; coupling the modified first protein and the second protein under the action of a coupling agent; wherein the first modification treatment and the second modification treatment are different.

[0010] According to an embodiment of the present invention, the method is to perform N3 and DBCO (dibenzylcyclooctyne) modification on two proteins to be coupled, and to cross-link the two proteins by strain-promoting azide-alkyne cycloaddition reaction (SPAAC) reaction. Since it does not involve a reaction with a free thiol group, the stable disulfide bonds in the natural structure of the protein will not be destroyed, and the stability and activity of the protein may be improved. In addition, since the two proteins are modified separately, the optimization of the reaction conditions in the process development process has greater flexibility and selectivity. It can be widely used in the fields of biomedicine, fluorescence tracing, and other protein engineering related fields.

[0011] According to an embodiment of the present invention, the method for coupling two proteins may further include at least one of the following additional technical features:

[0012] According to an embodiment of the present invention, the first protein and the second protein are independently selected from luciferase and antibody. According to an embodiment of the present invention, by coupling the luciferase modified with N3 or dibenzylcyclooctyne and the antibody modified with dibenzylcyclooctyne or N3, the generated product can be widely used in the field of molecular detection.

[0013] According to an embodiment of the present invention, the luciferase includes at least one selected from Gaussia luciferase and Nano luciferase.

[0014] According to an embodiment of the present invention, the luciferase is Gaussia luciferase.

[0015] According to an embodiment of the present invention, the antibody is selected from at least one of a digoxin antibody, an A base antibody, a G base antibody, a C base antibody or a T base antibody.

[0016] According to an embodiment of the present invention, the digoxigenin antibody is labeled.

[0017] According to an embodiment of the present invention, the coupling agent includes at least one of Azide-PEG8-NHS, Azide-PEG12-NHS, DBCO-PEG4-NHS, DBCO-PEG8-NHS, DBCO-PEG12-NHS, NHS-PEG4-Azide and DBCO-NHS.

[0018] It should be noted that the coupling agent can be generally selected under the conditions where a SPAAC reaction can occur.

[0019] According to an embodiment of the present invention, the coupling treatment is achieved by strain-promoted azide-alkyne cycloaddition reaction. The inventors found that strain-promoted azide-alkyne cycloaddition reaction (SPAAC) does not involve reaction with free sulfhydryl groups, does not destroy the stable disulfide bonds in the native structure of the protein, the stability and activity of the protein can be improved, and the experimental design can be more flexible in the process optimization process.

[0020] According to an embodiment of the present invention, the molar ratio of N3 to luciferase is 1:1 to 15:1. Optionally, the molar ratio of N3 to luciferase is 10:1 or 9:1 or 8:1 or 7:1 or 6:1 or 5:1 or 4:1 or 3:1 or 2:1 or 1:1.

[0021] According to an embodiment of the present invention, the molar ratio of N3 to luciferase is 1: 1, 5: 1 or 10: 1. The inventor screened out the optimal molar ratio of N3 to luciferase based on the fact that the properties of amino acids change after lysine on the protein surface is labeled, which may affect the activity of the protein. The more lysine is labeled, the greater the effect will be.

[0022] According to an embodiment of the present invention, the molar ratio of the dibenzylcyclooctyne to the antibody is 5: 1 to 20: 1. Alternatively, the molar ratio of the dibenzylcyclooctyne to the antibody is 5: 1 or 6: 1 or 7: 1 or 8: 1 or 9: 1 or 10: 1.

[0023] According to an embodiment of the present invention, the molar ratio of the dibenzylcyclooctyne to the antibody is 5:1.

[0024] In the second aspect of the present invention, the present invention provides a protein complex. According to an embodiment of the present invention, the protein complex is prepared by the method described in the first aspect of the present invention. The protein complex prepared by the method described in the first aspect can be widely used in the field of molecular detection.

[0025] In the third aspect of the present invention, the present invention provides a protein complex. According to an embodiment of the present invention, the protein complex comprises luciferase and an antibody, wherein the luciferin and the antibody are connected via a coupling agent, wherein the luciferin and the coupling agent are connected via an amide bond, wherein the antibody and the coupling agent are connected via an amide bond, and wherein the molar ratio of the luciferase to the antibody is 15:1.

[0026] According to an embodiment of the present invention, there are stable disulfide bonds in the protein complex, resulting in higher stability and activity.

[0027] In the fourth aspect of the present invention, the present invention provides an antigen detection method. According to an embodiment of the present invention, the method comprises: contacting the antigen to be detected with the protein complex described in the third aspect of the present invention; determining the antigen based on the signal after the contact treatment; wherein the antigen to be detected carries a luciferase substrate or has binding activity with an antibody.

[0028] According to an embodiment of the present invention, the method determines whether the antigen to be detected exists by detecting the intensity of the fluorescent signal or other related signals, and can quickly detect the interaction between small molecule compounds and macromolecular proteins with high detection sensitivity.

[0029] Exemplary, the above-mentioned antigen detection method based on the specific binding characteristics of antigen and antibody. When the antigen to be detected containing a luciferase substrate is contacted with the protein complex provided by the present invention, if there is specific binding, a complex will be formed, and the substrate will generate a fluorescent signal after binding with the luciferase, and the antigen to be detected can be determined by visualizing or detecting the fluorescent signal.

[0030] According to an embodiment of the present invention, the signal is a fluorescence signal.

[0031] In a fifth aspect of the present invention, the present invention provides a kit. According to an embodiment of the present invention, the kit comprises the protein complex described in the second aspect or the third aspect of the present invention. The kit can detect antigens simply and quickly.

[0032] In a sixth aspect of the present invention, the present invention proposes a use of the kit described in the fifth aspect in detecting antigens. According to an embodiment of the present invention, the kit can be used in the medical field or scientific research direction for antigen detection.

[0033] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as implementation schemes) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the following description of the embodiments in conjunction with the accompanying drawings, in which:

[0035] Figure 1 is an SDS-PAGE image of the raw materials, intermediate products and final products in the diamino coupling preliminary experiment described in Example 1 of the present invention;

[0036] Figure 2is the enzyme activity detection result of Gluc, Gluc-N3-DBCO-AbDig and Gluc-DBCO-N3-AbDig described in Example 1 of the present invention;

[0037] Figure 3 is the electrophoresis diagram of N3-Gluc at different NHS-PEG4-Azide:Gluc molar ratios as described in Example 2 of the present invention;

[0038] Figure 4 It is the enzyme activity test result of N3-Gluc with different NHS-PEG4-Azide:Gluc molar ratios described in Example 2 of the present invention;

[0039] Figure 5 is a DBCO-Ab-Dig solubility detection graph of different DBCO-NHS:Ab-Dig molar ratios described in Example 3 of the present invention;

[0040] Figure 6 is the SDS-PAGE image of Gluc-N3-DBCO-AbDig at different coupling agent to protein molar ratios as described in Example 4 of the present invention;

[0041] Figure 7 is a diagram showing the enzyme activity detection results of Gluc-N3-DBCO-AbDig at different coupling agent to protein molar ratios as described in Example 4 of the present invention;

[0042] Figure 8 is a schematic diagram of the reaction formula of the N3 modified luciferase described in the embodiment of the present invention;

[0043] Fig. 9 is a schematic diagram of the reaction formula of the DBCO-modified antibody described in the embodiments of the present invention;

[0044] Fig.10 It is a schematic diagram of the reaction formula of the SPAAC reaction between the N3-modified luciferase and the DBCO-modified antibody described in the embodiment of the present invention. DETAILED DESCRIPTION

[0045] The embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

[0046] In the description of the present application, it should be understood that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the present application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0047] As used herein, "luciferase" refers to a class of enzymes that catalyze a light-emitting chemical reaction. The substrate of the enzyme is called luciferin. Light emission occurs when luciferin is catalyzed by the luciferase to produce a chemical reaction without the presence of ATP.

[0048] In this article, the maleimide-activated crosslinker and labeling reagent react specifically with sulfhydryl groups (-SH) under near-neutral conditions (pH 6.5-7.5) to form stable thioether bonds. It should be noted that the disulfide bonds in the protein structure must be reduced to free sulfhydryl groups before they can react with the maleimide reagent.

[0049] Based on the existing technology, SMCC (succinimidyl 4-(N-maleimidyl) cyclohexane-1-carboxylate) is conjugated to the primary amine group on Nano luciferase (Nluc) through NHS ester, and then the digoxin antibody (Ab-Dig) is reduced with tri(2-carboxyethyl)phosphine hydrochloride (TECP) to expose the free thiol group, and then Nluc is cross-linked with Ab-Dig through the maleimide on the SMCC molecule to obtain the Nluc-Ab-Dig coupling product. Nluc is a β-barrel structure with only one non-conserved cysteine ​​and no disulfide bond inside the molecule; Gluc contains 10 cysteines and forms 5 pairs of disulfide bonds inside the molecule. Due to the difference in the molecular structure of Gluc and Nluc, the enzymatic activity of the Gluc coupling product cannot be guaranteed. And because only one molecular linker is used in the coupling process, the experimental process is linearized, so the flexibility of experimental design is greatly reduced during the development process.

[0050] Therefore, the present application develops a double amino protein coupling method based on SPAAC reaction to solve the problems of reduced enzyme activity of coupling products and linearization of experimental procedures.

[0051] Specifically, the method includes: performing a first modification treatment on the first protein, wherein the first modification is N3 or dibenzylcyclooctyne modification; performing a second modification treatment on the second protein, wherein the second modification is dibenzylcyclooctyne or N3 modification; performing a coupling treatment on the modified first protein and the second protein under the action of a coupling agent; wherein the first modification treatment and the second modification treatment are different. The conjugate is further used as a raw material in the signal display part in gene sequencing or antigen detection. Compared with the existing thiol coupling method, the dual amino protein coupling method based on the SPAAC reaction has high product stability and high activity, and can be more flexibly designed in the process optimization process.

[0052] For ease of understanding, the method described in this application is explained in detail below through examples, as shown below:

[0053] According to an embodiment of the present invention, the Gluc contains 10 cysteines, forming 5 pairs of disulfide bonds, and has a hydrophobic active cavity for recruiting substrates for catalytic luminescence. Therefore, any chemical modification of Gluc is likely to reduce the catalytic activity of Gluc.

[0054] The present invention modifies two proteins to be coupled (Gluc and Ab-Dig) by N3 and DBCO, removes excess reagents and reaction by-products by ultrafiltration, mixes the two modified proteins for Click reaction, and then removes excess uncoupled proteins by ultrafiltration to obtain a pure Gluc-Ab-Dig conjugate, which still has high activity and stability.

[0055] Among them, the synthetic reaction formula of N3 modified luciferase is as follows Figure 8 As shown, Gluc was covalently cross-linked with N3-NHS ester containing n (arbitrary) PEG interarms at room temperature (25° C.) for 30 min to generate N3-modified Gluc and reaction byproduct NHS.

[0056] The synthetic reaction formula of DBCO modified antibody is as follows Fig. 9 As shown, Ab-Dig was covalently cross-linked with DBCO-NHS ester at room temperature (25° C.) for 30 min to generate DBCO-modified Ab-Dig and reaction byproduct NHS.

[0057] The synthetic reaction formula (SPAAC reaction) of the N3-modified luciferase and the DBCO-modified antibody is as follows: Fig.10 As shown, DBCO-modified Ab-Dig and N3-modified Gluc were reacted by SPAAC overnight at 4°C to generate a conjugate of Ab-Dig and Gluc.

[0058] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used without indicating the manufacturer are all conventional products that can be obtained commercially.

[0059] Example 1: Double amino coupled enzyme activity detection

[0060] In order to verify the difference in enzyme activity caused by different coupling methods, the embodiment of the present invention constructed a coupling method for the two products of Gluc-N3-DBCO-Ab-Dig and Gluc-DBCO-N3-Ab-Dig and performed enzyme activity detection of the products. The specific steps are as follows:

[0061] Preparation of NHS-PEG4-Azide stock solution: Take out the unopened NHS-PEG4-Azide (100 mg, Sigma) from -20℃ and place it at room temperature to equilibrate the temperature for 10 minutes. Add 2.57 ml of anhydrous DMSO (dimethyl sulfoxide, Bio-chem), vortex to mix, prepare a 100 mM stock solution and place it at -20℃ for use.

[0062] Preparation of DBCO-NHS stock solution: Take out the unopened DBCO-NHS (50 mg) from -20°C and place it at room temperature to equilibrate the temperature for 10 minutes. Add 12.4254 ml of anhydrous DMSO (Sangon), vortex to mix, prepare a 10 mM stock solution and place it at -20°C for use.

[0063] NHS ester cross-linking reaction: Add the diluted coupling agent to Gluc (3.65 mg / ml, MGI) and Ab-Dig (3.4 mg / ml, MGI) according to Table 1, react at room temperature for 30 min, and mix by inversion every 10 min.

[0064] Table 1: NHS ester cross-linking reaction ratio

[0065]

[0066] SPAAC reaction: NHS ester cross-linked Gluc and Ab-Dig were placed in 10k and 30k ultrafiltration tubes (Merck ), add 3 ml of 4°C pre-cooled PBS, centrifuge at 3900 rpm for 20 min at 4°C, repeat 3 times. Mix N3-modified Gluc and DBCO-modified Ab-Dig, mix DBCO-modified Gluc and N3-modified Ab-Dig, react at room temperature for 30 min and at 4°C overnight.

[0067] Ultrafiltration liquid exchange: Transfer the reaction product to a 30k or 50k ultrafiltration tube (Merck ), add 3 ml of 4°C pre-cooled PBS, centrifuge at 3900 rpm at 4°C for 15 min, and repeat 3 times.

[0068] Gluc-Ab-Dig and all intermediates were run on SDS-PAGE. The results are as follows: Figure 1 shown.

[0069] Enzyme activity detection: Take untreated Gluc and Gluc-N3-DBCO-Ab-Dig and Gluc-DBCO-N3-Ab-Dig after liquid exchange, use Gen5 Synergy H1 microplate reader Take3 plate (Bio Tek) for A280 protein quantification, dilute it to 1ng / ul with luciferase buffer (formula see Table 2), and add 10ul of the diluted product to each well of a black 96-well plate. Dilute the Gluc substrate CTZ (5mM, BGI) to 50uM with Luciferase buffer for later use. Use the microplate reader perfusion program to detect the luminescence intensity, add 100ul of substrate to each well, and immediately detect the luminescence, and adjust the luminescence gain to 75 ( Figure 2 ).

[0070] Table 2: Luciferase buffer recipe

[0071] Luciferase buffer 500ml 1M Tris-HCl pH 8.0 25ml 5M NaCl 50ml Tween 20 0.5ml <![CDATA[H2O]]> 424.5ml

[0072] The results showed that the SPAAC reaction at 4°C overnight was more thorough than the reaction at room temperature for 30 min; 30k ultrafiltration could not effectively remove excess Gluc, while 50k ultrafiltration could completely remove excess Gluc; the N3-modified Gluc coupling product was more active than the DBCO-modified Gluc coupling product, therefore, N3-modified Gluc was used in subsequent examples.

[0073] Example 2: N3 modification of Gluc and detection of enzyme activity

[0074] In the embodiment of the present invention, Gluc is subjected to N3 modification treatment to obtain Gluc-N3 with different N3 modification ratios, and substrate is added to detect enzyme activity on a microplate reader. The specific steps are as follows:

[0075] Preparation of NHS-PEG4-Azide stock solution: Take out the unopened NHS-PEG4-Azide (100 mg, Sigma) from -20°C and place it at room temperature to equilibrate the temperature for 10 minutes. Add 2.57 ml of anhydrous DMSO (Sangon), vortex to mix, prepare a 100 mM stock solution and place it at -20°C for use.

[0076] N3 modification of Gluc: Gluc (MGI) at a concentration of 3.65 mg / ml was mixed evenly with different volumes of NHS-PEG4-Azide 100 mM stock solution (Table 3), and reacted at room temperature for 30 min, with inversion mixing every 10 min.

[0077] Ultrafiltration liquid replacement: The reaction product was transferred to a 10K ultrafiltration tube (Merck ), add 2 ml 4℃ pre-cooled PBS, centrifuge at 3900 rpm for 8 min at 4℃, repeat 3 times, and run SDS-PAGE to verify the sample in the tube ( Figure 3 ).

[0078] Table 3: Ratios of reagents for N3 modification of Gluc

[0079]

[0080] Enzyme activity detection: The product after the liquid change was quantified using the Gen5 Synergy H1 microplate reader Take3 plate (Bio Tek), diluted to 1 ng / ul with luciferase buffer (see Table 2), and 10ul of the diluted product was added to each well of a black 96-well plate. The Gluc substrate CTZ (5mM, BGI) was diluted to 50uM with Luciferase buffer for later use. The luminescence intensity was detected using the microplate reader perfusion program, 100ul of substrate was added to each well, and the luminescence was detected immediately. The luminescence gain was adjusted to 75 ( Figure 4 ).

[0081] Example 3: DBCO modification of Ab-Dig

[0082] In this embodiment, Ab-Dig is subjected to DBCO modification treatment to obtain Ab-Dig-DBCO with different DBCO modification ratios. The specific steps are as follows:

[0083] Preparation of DBCO-NHS stock solution: Take out the unopened DBCO-NHS (50 mg) from -20°C and place it at room temperature to equilibrate the temperature for 10 minutes. Add 12.4254 ml of anhydrous DMSO (Sangon), vortex to mix, prepare a 10 mM stock solution and place it at -20°C for use.

[0084] DBCO modification of Ab-Dig: Take Ab-Dig (MGI) with a concentration of 3.4 mg / ml and mix it evenly with different volumes of DBCO-HNS 10 mM stock solution (see Table 4 for the ratio), react at room temperature for 30 min, invert and mix every 10 min, and observe the precipitation state of the product after the reaction ( Figure 5 ).

[0085] Ultrafiltration liquid replacement: The reaction product was transferred to a 30K ultrafiltration tube (Merck ), add 2 ml of 4°C pre-cooled PBS, centrifuge at 3900 rpm at 4°C for 15 min, and repeat 3 times.

[0086] Table 4: Ab-Dig DBCO modification reagent ratio

[0087]

[0088] Example 4: SPAAC reaction of N3-modified Gluc and DBCO-modified Ab-Dig

[0089] In this example, a series of Gluc-N3-DBCO-AbDig coupling products were obtained by cross-linking Gluc-N3 with different N3 modification ratios and Ab-Dig-DBCO with different DBCO modification ratios, and enzyme activity was detected.

[0090] SPAAC reaction: Based on the results of Example 1 and Example 2, N3-modified Gluc with N3:Gluc (molar ratio) of 1, 5, 10, and 15 and DBCO-modified Ab-Dig with DBCO:Ab-Dig (molar ratio) of 5, 10, 15, and 20 were selected and mixed in a molar ratio of 20:1 (experimental grouping, see Table 5) to ensure that N3-modified Gluc was in excess, and the reaction was carried out at 4°C overnight.

[0091] Table 5: SPAAC reaction experiment grouping

[0092] Group Name N3:Gluc (molar ratio) DBCO:Ab-Dig (molar ratio) 1-5 1 5 1-10 1 10 1-15 1 15 1-20 1 20 5-5 5 5 5-10 5 10 5-15 5 15 5-20 5 20 10-5 10 5 10-10 10 10 10-15 10 15 10-20 10 20 15-5 15 5 15-10 15 10 15-15 15 15 15-20 15 20

[0093] Ultrafiltration and liquid replacement: To remove the excess unreacted N3-modified Gluc in the reaction system, transfer the SPAAC reaction product to a 50k ultrafiltration tube, add 4 ml of 4°C pre-cooled PBS, centrifuge at 3900 rpm for 15 min at 4°C, and repeat 3 times. The remaining sample in the tube after ultrafiltration was run for SDS-PAGE verification ( Figure 6 ).

[0094] The enzyme activity detection method is the same as in Example 1.

[0095] The results are as follows Figure 7 As shown, among them, the enzymatic activity of the coupling products of group 1-5 (molar ratio: N3:Gluc=1:1, DBCO:Ab-Dig=5:1), group 5-5 (molar ratio: N3:Gluc=5:1, DBCO:Ab-Dig=5:1), and group 10-5 (molar ratio: N3:Gluc=10:1, DBCO:Ab-Dig=5:1) was higher.

[0096] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0097] The above embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present application. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the attached claims.

Claims

1. A method for coupling two proteins, characterized in that: include: Performing a first modification treatment on the first protein, wherein the first modification is N3 or dibenzylcyclooctyne modification; Performing a second modification on the second protein, wherein the second modification is dibenzylcyclooctyne or N3 modification; The modified first protein and the second protein are subjected to coupling treatment under the action of a coupling agent; Among them, the first modification process and the second modification process are different.

2. The method according to claim 1, characterized in that The first protein and the second protein are independently selected from luciferase and antibodies, respectively.

3. The method according to claim 2, characterized in that The luciferase comprises at least one selected from Gaussia luciferase and Nano luciferase; Preferably, the luciferase is Gaussia luciferase.

4. The method according to claim 2, characterized in that: The antibody is selected from at least one of digoxin antibody, A base antibody, G base antibody, C base antibody or T base antibody; Optionally, the digoxigenin antibody is labeled.

5. The method according to claim 1, characterized in that The coupling agent includes at least one of Azide-PEG8-NHS, Azide-PEG12-NHS, DBCO-PEG4-NHS, DBCO-PEG8-NHS, DBCO-PEG12-NHS, NHS-PEG4-Azide and DBCO-NHS.

6. The method according to claim 1, characterized in that The coupling process is achieved via a strain-promoted azide-alkyne cycloaddition reaction.

7. The method according to claim 2, characterized in that The molar ratio of N3 to luciferase is 1:1 to 15:1; Preferably, the molar ratio of N3 to luciferase is 1:1, 5:1 or 10:

1.

8. The method according to claim 2, characterized in that: The molar ratio of the dibenzylcyclooctyne to the antibody is 5:1 to 20:1; Preferably, the molar ratio of dibenzylcyclooctyne to antibody is 5:

1.

9. A protein complex, characterized in that The protein complex is prepared by the method according to any one of claims 1 to 8.

10. A protein complex, characterized in that The invention comprises luciferase and an antibody, wherein the luciferase and the antibody are connected via a coupling agent, the luciferase and the coupling agent are connected via an amide bond, the antibody and the coupling agent are connected via an amide bond, and the molar ratio of the luciferase and the antibody is 15:

1.

11. An antigen detection method, characterized in that: include: contacting the antigen to be detected with the protein complex according to claim 9 or 10; Based on the signal after the contact treatment, the antigen is determined; Wherein, the antigen to be detected carries a luciferase substrate or has binding activity with an antibody; Optionally, the signal is a fluorescent signal.

12. A kit, characterized in that: Comprising the complex according to claim 9 or 10.

13. Use of the kit according to claim 12 in detecting antigens.

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