A long-chain pegylated click chemistry covalent conjugate with low steric hindrance, and a preparation method and application thereof

Abstract

The present application relates to a kind of low steric hindrance long-chain PEGylation click chemistry covalent coupling and its preparation method and application, the covalent coupling includes the target protein, first PEG linker, click chemistry connecting bond, second PEG linker and horseradish peroxidase connected in sequence.The present application increases the space interval by the way of increasing PEG linker between target protein and horseradish peroxidase, effectively avoids steric hindrance, so as to realize the improvement of detection sensitivity and the combination activity of antibody Fc section.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and in particular to a long-chain PEGylated click chemical covalent conjugate with low steric hindrance, its preparation method, and its application. Background Technology

[0002] Horseradish peroxidase (HRP) covalent conjugates with antibodies or Fc-binding proteins (such as Protein G and Protein A) are widely used in detection techniques such as enzyme-linked immunosorbent assay (ELISA), Western blotting (WB), and immunohistochemistry. Traditional HRP labeling methods typically employ the sodium periodate oxidation method: sodium periodate oxidizes the glycosyl groups on the HRP surface to aldehyde groups, which then react with the primary amino groups on the target protein (such as antibodies or Protein G) to form a Schiff base, which is then stabilized with a reducing agent. This method is simple to operate and yields good results for labeling most antibodies.

[0003] However, research has found that the traditional sodium periodate oxidation method is a zero-length coupling method. When labeling Protein G or Protein A using the above-mentioned traditional sodium periodate method, the reaction process is as follows: sodium periodate oxidizes the glycosyl group on the surface of HRP to generate an aldehyde group (-CHO), the aldehyde group reacts with the primary amino group (-NH2) on the surface of Protein G to form a Schiff base (-CH=N-), which is then reduced to obtain a stable secondary amine (-CH2-NH-). In this product, there is no additional linking arm between HRP and Protein G; they are directly connected by a methylene group (-CH2-), with a distance of only about 1.5 Å. Such a short distance causes the HRP molecule or its linking site to directly partially or completely obscure the Fc binding domain of Protein G, resulting in severe steric hindrance, thus severely reducing or even completely eliminating the Fc binding activity of the resulting coupling. Specifically, in Western blotting experiments, the Protein G-HRP conjugate prepared by traditional methods could not detect the target band at a concentration of 2 μg / mL, while free Protein G could efficiently bind to antibody Fc at the nanomolar level.

[0004] Current reports indicate the use of glutaraldehyde, a bifunctional cross-linking agent, whose two functional groups can bind to the amino groups on HRP and antibodies, respectively, forming HRP-GA-IgG conjugates. However, this method still lacks a flexible spacer between HRP and Protein G, and the steric hindrance problem remains unresolved. Other literature reports introducing PEG chains into HRP to improve its stability, or using click chemistry to couple antibodies and enzymes, but none of these approaches offer a systematic solution to the specific steric hindrance problem of Protein G / Protein A.

[0005] Therefore, there is an urgent need in the field for a highly sensitive HRP labeling method that can effectively eliminate steric hindrance and retain the Fc binding activity of Protein G or Protein A. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a long-chain PEGylated click chemical covalent conjugate with low steric hindrance, its preparation method, and its application. This invention increases the spatial interval by adding a PEG linker between the target protein and horseradish peroxidase, effectively avoiding steric hindrance.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a long-chain PEGylated click chemical covalent conjugate with low steric hindrance, the covalent conjugate comprising a target protein, a first PEG linker, a click chemical linker, a second PEG linker, and horseradish peroxidase connected in sequence.

[0009] In this invention, it was discovered that the traditional sodium periodate method is a zero-length coupling mechanism, lacking sufficient spatial spacing between the HRP and the Fc binding domains of Protein G (or Protein A). This results in the HRP molecule or its linker sites directly blocking the binding of Protein G (or Protein A) to the Fc domain, causing severe steric hindrance. This problem is particularly prominent with Protein G, but no effective solution has been found in the prior art.

[0010] Based on the above, the present invention adds a first PEG connector and a second PEG connector, which greatly increases the spatial distance between the Fc binding domain and the HRP, effectively avoiding spatial steric hindrance.

[0011] The estimated length of the PEG spacer arm (total number of units: 16) selected in this invention is approximately 48-64 Å in aqueous solution. In contrast, the spatial scale of a single Fc binding domain of Protein G (composed of approximately 55-56 amino acid residues, whose crystal structure has been resolved, PDB number 1FCC) is approximately tens of Å (radius of gyration approximately 33 Å). Therefore, the spatial span of this PEG spacer arm significantly exceeds the spatial scale of the target protein's active site itself, which is sufficient to provide adequate physical separation between HRP and the target protein, thereby effectively eliminating the loss of Fc binding activity caused by steric hindrance of the coupling agent.

[0012] Further investigation revealed that when the total number of PEG units is small, the spacer arms are short, failing to effectively eliminate all steric hindrance. Conversely, when the total number of PEG units exceeds 24, excessively long flexible chains may increase non-specific interactions or purification difficulties in the conjugates, and significantly increase costs. Considering these factors and experimental verification, it can be understood that improvements are achieved when the total number of PEG units is within the range of 12-24, with 16 being the optimal value verified experimentally. A total PEG unit count of 16 is the optimal value, achieving the best balance between Fc binding activity, detection sensitivity, and practical application convenience. Those skilled in the art can select an appropriate PEG chain length within the range of 12-24 based on the steric hindrance of the specific target protein.

[0013] Preferably, the structure of the first PEG connector is -(OCH2CH2)a-.

[0014] Preferably, the structure of the second PEG connector is -(OCH2CH2)n-.

[0015] The numbers n and a represent the number of -(OCH2CH2)- atoms in the first PEG connector and the second PEG connector, respectively. The number of n atoms is 0-24, and the number of a atoms is 0-24. These 0-24 numbers can be, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

[0016] Preferably, the sum of n in the structure of the first PEG connector and a in the structure of the second PEG connector is 12-24. The 12-24 can be, for example, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24, etc.

[0017] More preferably, the sum of n in the structure of the first PEG connector and a in the structure of the second PEG connector is 16-24. The 16-24 can be, for example, 16, 17, 18, 19, 20, 21, 22, 23 or 24, etc.

[0018] Preferably, the number of n is 1-16, and the number of a is 1-16. The 1-16 can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0019] Based on the PEG chain length and structure-activity relationship, the spacer arm length of 12 PEG units is approximately 36-48 Å, that of 18 PEG units is approximately 54-72 Å, and that of 24 PEG units can reach 72-96 Å (approximately 70-100 Å). When the PEG spacer arm length reaches 70-100 Å, it is sufficient to provide adequate space between the active site of the target protein and the HRP, thereby effectively eliminating steric hindrance.

[0020] Preferably, the target protein includes any one or a combination of at least two of Protein G, Protein A, Fc-binding protein Z domain, streptavidin, or nanobody.

[0021] Preferably, the click chemical linker includes a triazole ring linker.

[0022] In this invention, a stable triazole ring linker is formed by a copper-free strain-promoted azido-yne cycloaddition reaction (SPAAC) between the azido group (-N3) at the end of the first PEG linker and the cyclooctyne group (preferably dibenzocyclooctyne, DBCO) at the end of the second PEG linker. Alternatively, the functional groups can be interchanged (i.e., DBCO is linked to the target protein side and azido group is linked to the HRP side), and the same linker is formed via SPAAC.

[0023] Preferably, the connection between the target protein and the first PEG linker includes maleimide-thioether bonds and / or amide bonds.

[0024] In this invention, when the target protein contains free thiol groups (such as those introduced through engineering or naturally occurring), the linkage is a maleimide-thioether bond (formed by the reaction of maleimide and thiol groups); when the target protein does not contain free thiol groups, the linkage is an amide bond (formed by the reaction of NHS ester and amino groups).

[0025] Preferably, the second PEG connector is connected to the horseradish peroxidase via an amide bond.

[0026] In this invention, the connection method is to covalently link the PEG chain to a chemical group on the HRP, preferably an amide bond (formed by the reaction of NHS ester and amino groups on the HRP).

[0027] In a second aspect, the present invention provides a method for preparing a long-chain PEGylated click chemical covalent conjugate with low steric hindrance according to the first aspect, the method comprising: (1) mixing maleimide-first PEG linker-dibenzocyclooctylene or succinimide-first PEG linker-dibenzocyclooctylene with the target protein in a buffer solution, and purifying after reaction to obtain a first reactant; (2) dissolving horseradish peroxidase in a solvent to obtain a horseradish peroxidase solution, mixing N3-second linker-NHS with the horseradish peroxidase solution and incubating, and purifying to obtain a second reactant; (3) placing the first reactant obtained in step (1) and the second reactant obtained in step (2) into a reaction solution, and clicking chemically conjugating to obtain a crude product; (4) purifying the crude product obtained in step (3) to obtain a covalent conjugate.

[0028] Preferably, in step (1), the molar ratio of maleimide-first PEG linker-dibenzocyclooctylene or succinimidide-first PEG linker-dibenzocyclooctylene to the target protein is (5-10):1. The (5-10) can be, for example, 5, 6, 7, 8, 9 or 10.

[0029] Preferably, in step (1), the reaction temperature is 20℃-35℃ and the time is 1-4 h. The 20℃-35℃ can be, for example, 20℃, 22℃, 24℃, 25℃, 26℃, 28℃, 30℃, 32℃, 34℃, or 35℃. The 1-4 h can be, for example, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, or 4 h.

[0030] Preferably, in step (1), the buffer solution includes PBS buffer.

[0031] Preferably, in step (2), the concentration of the horseradish peroxidase solution is 1-10 mg / mL. The 1-10 mg / mL can be, for example, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, or 10 mg / mL, etc.

[0032] Preferably, in step (2), the molar ratio of the N3-second linker peptide-NHS to horseradish peroxidase is (5-20):1. The (5-20) can be, for example, 5, 6, 8, 10, 12, 14, 16, 18 or 20.

[0033] Preferably, in step (2), the temperature for the mixed incubation is 20℃-35℃, and the time is 1-4 h. The 20℃-35℃ can be, for example, 20℃, 22℃, 24℃, 25℃, 26℃, 28℃, 30℃, 32℃, 34℃, or 35℃. The 1-4 h can be, for example, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, or 4 h.

[0034] Preferably, in step (2), the solvent comprises a 0.05-0.3 M sodium bicarbonate solution. The 0.05-0.3 M can be, for example, 0.05 M, 0.1 M, 0.15 M, 0.2 M, 0.25 M, or 0.3 M.

[0035] Preferably, in step (2), the mixing incubation is a light-protected reaction.

[0036] Preferably, in step (3), the temperature for the click chemical coupling is 20℃-35℃, and the time is 10-20 h. The 20℃-35℃ can be, for example, 20℃, 22℃, 24℃, 25℃, 26℃, 28℃, 30℃, 32℃, 34℃, or 35℃. The 10-20 h can be, for example, 10 h, 12 h, 14 h, 16 h, 18 h, or 20 h.

[0037] Preferably, in step (3), the reaction solution includes PBS buffer.

[0038] Preferably, in step (4), the purification method includes any one or a combination of at least two of size exclusion chromatography, affinity chromatography, or salting out.

[0039] Preferably, the preparation method further includes pretreatment of the target protein.

[0040] Preferably, the pretreatment includes dissolving the target protein, reducing it with DTT, and then ultrafiltration to exchange the solution into the ultrafiltrate.

[0041] Preferably, the concentration of the target protein is 1-3 mg / mL. The 1-3 mg / mL concentration can be, for example, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, or 3 mg / mL.

[0042] Preferably, the concentration of DTT is 1-6 mM. The 1-6 mM can be, for example, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, or 6 mM.

[0043] Preferably, the reduction reaction is carried out at a temperature of 20℃-30℃ for a time of 0.5-3 h. The 20℃-30℃ can be, for example, 20℃, 22℃, 24℃, 26℃, 28℃, or 30℃. The 0.5-3 h can be, for example, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, or 3 h.

[0044] Preferably, the ultrafiltrate comprises PBS buffer containing EDTA.

[0045] Preferably, the concentration of EDTA in the ultrafiltrate is 2-15 mM. The 1-15 mM can be, for example, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 8 mM, 10 mM, 12 mM, 14 mM, or 15 mM.

[0046] Thirdly, the present invention provides a method for preparing a long-chain PEGylated click chemical covalent conjugate with low steric hindrance according to the first aspect or the long-chain PEGylated click chemical covalent conjugate with low steric hindrance as described in the second aspect, and its application in the preparation of antibody detection reagents.

[0047] Preferably, the antibody includes any one or a combination of at least two of human IgG antibodies, mouse IgG antibodies, or rabbit IgG antibodies.

[0048] Compared with the prior art, the present invention has at least the following beneficial effects:

[0049] 1. The present invention adds a first PEG linker and a second PEG linker to the target protein and horseradish peroxidase, which greatly increases the spatial distance between the Fc binding domain and HRP, effectively avoiding steric hindrance.

[0050] 2. Compared with existing technologies, the covalent conjugate prepared by this invention significantly improves the binding activity of the antibody Fc fragment and increases the sensitivity by 27 times, successfully solving the technical problem of specific protein labeling failure.

[0051] 3. The structure of this invention has been experimentally verified to be effective against both Protein G and Protein A. Based on the same principle, it can be reasonably extended to other Fc-binding proteins. Attached Figure Description

[0052] Figure 1 It is Protein G-PEG prepared in Example 1 16 - Fc binding activity assay of HRP conjugate and commercial Protein G-HRP conjugate.

[0053] Figure 2 Protein G-PEG prepared in Example 116 Western blotting results of the Protein G-HRP conjugate and the commercial Protein G-HRP conjugate, where Figure A shows the results of the Protein G-HRP conjugate prepared by the conventional sodium periodate method, and Figure B shows the results of the Protein G-PEG conjugate prepared in Example 1. 16 Figure C shows the results of the HRP conjugate, while Figure D shows the results of the commercial Protein G-HRP conjugate.

[0054] Figure 3 Protein G-PEG prepared in Example 1 16 Immunoprecipitation assay of PEG-HRP conjugate and commercial Protein G-HRP conjugate, where Figure A shows Protein G-PEG. 16 Figure B shows the results of the HRP conjugate, while Figure B shows the results of the commercial Protein G-HRP conjugate.

[0055] Figure 4 Protein A-PEG prepared in Example 3 16 - Fc binding activity assay of HRP conjugate and commercial Protein A-HRP conjugate.

[0056] Figure 5 Protein A-PEG prepared in Example 3 16 Western blotting results of the -HRP conjugate and the commercial Protein A-HRP conjugate, where Figure A shows Protein A-PEG. 16 Figure B shows the results of the HRP conjugate, while Figure B shows the results of the commercial Protein A-HRP conjugate. Detailed Implementation

[0057] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0058] This invention designs a long-chain PEGylated click chemiluminescence covalent conjugate with low steric hindrance. The covalent conjugate comprises a target protein, a first PEG linker, a click chemiluminescence linker, a second PEG linker, and horseradish peroxidase, sequentially linked. The structure of the first PEG linker is -(OCH2CH2)a-, and the structure of the second PEG linker is -(OCH2CH2)n-; the sum of 'n' in the structure of the first PEG linker and 'a' in the structure of the second PEG linker is 12-24. This invention aims to increase the spatial interval between the target protein and horseradish peroxidase by adding a PEG linker, effectively avoiding steric hindrance, thereby improving detection sensitivity and antibody Fc fragment binding activity. Verification showed that a significant improvement was achieved when the sum of 'n' in the structure of the first PEG linker and 'a' in the structure of the second PEG linker was 12-24. In a specific experiment of this invention, the first PEG linker contained 4 'a's, and the second PEG linker contained 12 'n's.

[0059] The sources of reagents used in the following examples are:

[0060] Recombinant Protein G with cysteine ​​residues: Produced by Sinopharm (Catalog No.: 13103-PNAE), obtained by introducing cysteine ​​residues at the end through genetic engineering;

[0061] Recombinant Protein A without cysteine ​​residues, manufactured by Sinocare (Catalog No.: 10600-P07E).

[0062] Mal-PEG4-DBCO: Maleimide-PEG4-dibenzocyclooctylene, purchased from MCE;

[0063] N3-PEG 12 -NHS: Azide-PEG 12 -NHS ester, purchased from BroadPharm;

[0064] NHS-PEG4-DBCO: Purchased from BroadPharm;

[0065] HRP: Purchased from Shanghai Sangon Biotech;

[0066] DTT: Purchased from Shanghai Sangon Biotech.

[0067] Example 1

[0068] This embodiment describes the preparation of a long-chain PEGylated click chemical covalent conjugate with low steric hindrance.

[0069] (1) Reduction of protein G

[0070] Using PBS buffer at pH 7.4, the concentration of Protein G was adjusted to 2 mg / mL. DTT was added to the solution to a final concentration of 4 mM, and the reaction was carried out at 26 °C for 1 h. The solution was then ultrafiltered to PBS buffer at pH 7.4 containing 10 mM EDTA, and the concentration of Protein G was adjusted to 2 mg / mL to obtain reduced Protein G containing free thiol groups.

[0071] (2) Modification of Protein G

[0072] Mal-PEG4-DBCO (the number of 'a's in the structure of the first PEG connector can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16, and good results are obtained within the above ranges; in this embodiment, 'a' is 4) was mixed with Protein G obtained in step (1) at a molar ratio of 10:1 in PBS buffer (pH 7.2) and reacted at 25°C for 1 h. After the reaction, Protein G-PEG4-DBCO was obtained by purification using a desalting column.

[0073] (3) HRP modification

[0074] HRP was dissolved in a 0.1 M sodium bicarbonate solution at pH 8.3 to a concentration of 5 mg / mL. N3-PEG was then added. 12 -NHS (the number of 'n' in the structure of the second PEG connector includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16, etc., and good results are obtained within the above ranges. In this embodiment, 'n' is 12) and HRP are reacted at a molar ratio of 10:1 in 0.1 M sodium bicarbonate solution (pH 8.3) at 25°C in the dark for 1 h. After the reaction is complete, the HRP-PEG is obtained by purification by a desalting column. 12 -Azides.

[0075] (4) SPAAC coupling

[0076] The above Protein G-PEG4-DBCO and HRP-PEG 12 The azides were mixed in equimolar ratio and reacted in PBS buffer (pH 7.4) at 25°C for 16 h. After the reaction, the mixture was purified by size exclusion chromatography to obtain Protein G-PEG. 16 -HRP conjugate, the structure of which is: Protein GS-maleimide-(CH2)2-(OCH2CH2)4-triazole-(OCH2CH2) 12 -(CH2)2-CO-NH-HRP.

[0077] Example 2

[0078] This embodiment investigates the effect of the prepared covalently coupled compound.

[0079] This embodiment uses Protein G-PEG prepared in Example 1. 16 -HRP conjugates were compared with Protein G-HRP prepared by the conventional sodium periodate oxidation method and commercial Protein G-HRP (purchased from Merck, catalog number 18-161). The structure of Protein G-HRP prepared by the conventional sodium periodate oxidation method is Protein G—NH—CH2—HRP, where -NH- comes from the ε-amino group of the lysine residue on Protein G, and -CH2- comes from the aldehyde group (-CHO) generated after the oxidation of the HRP glycosyl group reacts with the amino group to form a Schiff base (-CH=N-), which is then reduced to a secondary amine (-CH2-NH-). HRP (oxidized glycosyl group) indicates that the aldehyde group comes from the oxidized sugar chain on the surface of HRP.

[0080] (1) Fc binding activity

[0081] SARS-CoV / SARS-CoV-2 Nucleocapsid Antibody, RabbitMab antibody, were coated with Protein G-PEG from Example 1 at different concentration gradients. 16 -HRP conjugates and commercial Protein G-HRP conjugates were detected, and EC50 was calculated. Specific results are as follows: Figure 1 As shown, the Protein G-PEG prepared in Example 1 can be seen... 16 The EC50 value of the binding of the -HRP conjugate to antibody Fc was 7.08 ng / mL; the EC50 value of the commercial Protein G-HRP conjugate was 190.38 μg / mL; the EC50 value of the conjugate prepared by the conventional sodium periodate method was undetectable under the same conditions. The relative binding activity of the conjugate of this invention is 27 times that of the commercial Protein G-HRP conjugate.

[0082] (2) Western Blot sensitivity

[0083] In the Western blot (WB) assay for detecting rabbit IgG, equal volumes of HeLa and HepG2 cell lysates were loaded, and primary antibody Beta-Actin Loading Control Antibody was added. Protein G-PEG from Example 1 was used. 16Protein G-HRP conjugates, commercially available Protein G-HRP conjugates, and Protein G-HRP conjugates prepared using the traditional sodium periodate method were analyzed. Enzyme-linked immunosorbent assay (ELISA) was used to determine the results under identical conditions. Figure 2 As shown, Protein G-PEG prepared in Example 1 16 -HRP conjugates produced clear bands at a concentration of 0.4 μg / mL, such as Figure 2 As shown in Figure B, commercially available Protein G-HRP conjugate requires 5 μg / mL to achieve the same colorimetric intensity, such as... Figure 2 As shown in Figure C, the traditional sodium periodate method for preparing Protein G-HRP conjugates still showed no visible bands at 2 μg / mL. Figure 2 As shown in Figure A, lane 1 contains HeLa cell lysate, and lane 2 contains HepG2 cell lysate.

[0084] (3) Immunoprecipitation

[0085] A431 cells were lysed, and anti-SMAC antibody was added to the MCF-7 cell lysate. After incubation, magnetic beads coated with secondary antibody were added to capture the antigen-antibody complex. The mixture was then boiled, loaded onto the electrophoresis apparatus, and detected using a protein G-HRP conjugate. Specific results are as follows: Figure 3 As shown, Figure 3 As shown in Figures A and B, Protein G-PEG prepared in Example 1 16 The HRP conjugate achieved the same colorimetric intensity as the commercial Protein G-HRP conjugate at a concentration of 0.4 μg / mL at a concentration of 0.05 μg / mL. Lane 1 contained A431 cell lysate, and lane 2 contained MCF-7 cell lysate.

[0086] The above results show that the Protein G-PEG prepared in this invention... 16 -HRP conjugates exhibit high Fc binding activity and detection sensitivity. SPAAC reaction conditions are mild, require no metal catalysts, and have minimal impact on protein activity.

[0087] Example 3

[0088] This embodiment investigates the effect of the amide bond between the target protein and the first PEG linker on the efficacy.

[0089] This embodiment is based on Example 1, but directly uses Protein A, i.e., Protein A is not terminally linked to cysteine. Mal-PEG4-DBCO is replaced with NHS-PEG4-DBCO, Protein A is not reduced, and the linkage is performed through the amino group of Protein A. Everything else is the same as in Example 1. The structure of the obtained conjugate is: Protein A-NH-CO-(CH2)2-(OCH2CH2)4-triazole-(OCH2CH2) 12 -(CH2)2-CO-NH-HRP. Protein A-PEG is obtained. 16 -HRP conjugate, using the method of Example 1, was used to detect Protein A-PEG. 16 The Fc binding activity and Western blotting sensitivity of -HRP conjugates and commercial Protein A-HRP are shown in the following figures. Figure 4 and Figure 5 As shown, Protein A-PEG can be seen. 16 The EC50 of the -HRP conjugate is 5.71 ng / mL, while the EC50 of commercial Protein A-HRP is 6.82 μg / mL. For example... Figure 5 As shown in Figures A and B, Protein A-PEG in WB sensitivity... 16 The HRP conjugate concentration was 0.5 μg / mL, while the commercially available Protein A-HRP concentration was 4 μg / mL. This demonstrates that the present invention is also effective for Protein A, with lane 1 containing HeLa cell lysate and lane 2 containing HepG2 cell lysate.

[0090] Example 4

[0091] This embodiment explores the effects of functional group interchange.

[0092] This embodiment is based on Example 1, but the linkers of DBCO and the azide group are interchanged: Protein G is modified with N3-PEG4-NHS, and DBCO-PEG is used. 12 -NHS-modified HRP, coupled via SPAAC reaction, maintains a total PEG ratio of 16. The resulting conjugate also exhibits good Fc binding activity. This demonstrates that the core of this invention lies in "long-chain PEG spacer arms + SPAAC site-specific coupling," and that functional group allocation does not affect the final effect.

[0093] Example 5

[0094] This embodiment explores the effect of different purification methods on the results.

[0095] (1) Effect of different purification methods on the effect

[0096] This embodiment is based on the method of Example 1, but the size exclusion chromatography purification method in step (4) is adjusted to affinity chromatography or salting out. The specific steps are as follows:

[0097] Affinity chromatography purification: After the coupling reaction, the reaction mixture was loaded onto an IgG affinity chromatography column (an agarose gel column conjugated with human IgG). Five column volumes were washed with PBS buffer; subsequently, elution was performed with a low-pH buffer (0.1 M glycine-HCl, pH 2.7), and the eluent was collected and immediately adjusted to neutral with neutralization buffer (1 M Tris-HCl, pH 9.0). Further purification could be achieved using size exclusion chromatography. As verified by the Fc binding activity in Example 1, the EC50 of the affinity-purified Protein G-HRP conjugate reached 6.83 ng / mL, comparable to the activity of the conjugate in Example 1.

[0098] Salting-out purification: After the coupling reaction, the mixture was placed in an ice bath, and solid ammonium sulfate was added to 50% saturation with slow stirring. The mixture was allowed to stand at 4°C for 2 hours, then centrifuged at 10000 g for 15 minutes to collect the precipitate. The precipitate was washed twice with 50% ammonium sulfate, centrifuged again to discard the supernatant, and finally reconstituted with an appropriate amount of PBS buffer (pH 7.4). For further purification, size exclusion chromatography or affinity chromatography can be used. The purified Protein G-HRP conjugate achieved an EC50 of 7.51 ng / mL, comparable to the conjugate in Example 1, as verified by the Fc binding activity in Example 1.

[0099] In summary, the addition of a first PEG linker and a second PEG linker to the target protein and horseradish peroxidase significantly increases the spatial distance between the Fc binding domain and HRP, effectively avoiding steric hindrance, thereby improving detection sensitivity and antibody Fc fragment binding activity.

[0100] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A low-steric-hindrance long-chain PEGylated click chemical covalent conjugate, characterized in that, The covalent conjugate consists of a target protein, a first PEG linker, a click chemical linker, a second PEG linker, and horseradish peroxidase connected in sequence. The structure of the first PEG connector is -(OCH2CH2)a-; The structure of the second PEG connector is -(OCH2CH2)n-; The target protein includes any one or a combination of at least two of the following: Protein G, Protein A, or Fc binding protein Z domain. The click chemical link is a triazole ring link; The target protein is connected to the first PEG linker via a maleimide-thioether bond and / or an amide bond. The second PEG connector is connected to the horseradish peroxidase via an amide bond. The sum of n in the structure of the first PEG connector and a in the structure of the second PEG connector is 12-24; The number of -(OCH2CH2)- in the first PEG connector and the second PEG connector are respectively, with n ranging from 0 to 24 and a ranging from 0 to 24.

2. The low steric hindrance long-chain PEGylated click chemocovalent conjugate according to claim 1, characterized in that, The sum of n in the structure of the first PEG connector and a in the structure of the second PEG connector is 16-24.

3. The method for preparing the low steric hindrance long-chain PEGylated click chemical covalent conjugate according to claim 1 or 2, characterized in that, The preparation method includes: (1) Maleimide-first PEG linker-dibenzocyclooctylene or succinimide-first PEG linker-dibenzocyclooctylene are mixed with the target protein in a buffer solution, and the first reactant is obtained after purification. (2) Horseradish peroxidase was dissolved in a solvent to obtain a horseradish peroxidase solution. The N3-second linker peptide-NHS was mixed with the horseradish peroxidase solution and incubated. The solution was then purified to obtain the second reactant. (3) Place the first reactant obtained in step (1) and the second reactant obtained in step (2) into the reaction solution, and click chemical coupling to obtain the crude product; (4) The crude product obtained in step (3) is purified to obtain a covalent conjugate.

4. The method for preparing the low steric hindrance long-chain PEGylated click chemical covalent conjugate according to claim 3, characterized in that, In step (1), the molar ratio of maleimide-first PEG linker-dibenzocyclooctylene or succinimide-first PEG linker-dibenzocyclooctylene to the target protein is 10:1; In step (1), the reaction temperature is 25°C and the time is 1 h; In step (2), the concentration of the horseradish peroxidase solution is 5 mg / mL; In step (2), the molar ratio of N3-second linker-NHS to horseradish peroxidase is 10:1; In step (2), the temperature for the mixed incubation is 25°C and the time is 1 h.

5. The method for preparing the low steric hindrance long-chain PEGylated click chemical covalent conjugate according to claim 3, characterized in that, In step (3), the temperature for the click chemical coupling is 25°C and the time is 16 h; In step (4), the purification method includes any one or a combination of at least two of size exclusion chromatography, affinity chromatography or salting out.

6. The method for preparing the low steric hindrance long-chain PEGylated click chemical covalent conjugate according to claim 3, characterized in that, The preparation method further includes pretreatment of the target protein; The pretreatment includes dissolving the target protein, reducing it with DTT, and then ultrafiltration to change the solution into the ultrafiltrate. The concentration of the target protein is 2 mg / mL; The concentration of DTT was 4 mM; The reduction reaction was carried out at a temperature of 26°C for 1 hour. The ultrafiltration solution includes a PBS buffer containing EDTA; The concentration of EDTA in the ultrafiltrate is 10 mM.

7. The application of the preparation method of the low steric hindrance long-chain PEGylated click chemical covalent conjugate according to claim 1 or 2, or the low steric hindrance long-chain PEGylated click chemical covalent conjugate according to any one of claims 3-6, in the preparation of antibody detection reagents.

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