A bovine serum albumin-cyclic beta-1,2-glucan conjugate, and a preparation method and application thereof

Abstract

The application discloses a bovine serum albumin-cyclic beta-1,2-glucan conjugate and a preparation method and application thereof. The bovine serum albumin-cyclic beta-1,2-glucan conjugate is prepared by a chemical coupling reaction of a bromoamide type cyclic beta-1,2-glucan (CbetaG-Br) or an activated CbetaG-DSG and bovine serum albumin (BSA). The preparation method comprises the following steps: fermentation preparation and purification of CbetaG, synthesis of a CbetaG derivative and synthesis of a BSA-CbetaG conjugate. Two synthesis routes are provided, and the routes are coupled by using a thiol group (route a) or an amino group (route b) of the BSA and a reactive functional group of the CbetaG. The method has mild reaction conditions and simple operation, the prepared BSA-CbetaG conjugate has a clear structure and a high grafting rate, and can be used as a hapten-carrier complex for development of a brucellosis detection test strip, and has important biomedical application value.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and diagnostic reagents, and in particular to a bovine serum albumin-cyclic β-1,2-glucan conjugate (BSA-CβG), its preparation method and application, especially the application of this conjugate in the detection and diagnosis of brucellosis antibodies. Background Technology

[0002] Brucellosis is a zoonotic infectious disease caused by Brucella bacteria. The genus Brucella comprises 6 species and 19 biotypes. Surface antigens differ among species, and conventional antigen diagnostic reagents have limitations, making it difficult to effectively distinguish between vaccinated animals and naturally infected animals.

[0003] CN109627320A discloses a Brucella antigen oligosaccharide protein conjugate, its preparation method, and its uses. The oligosaccharide protein conjugate has the structure of general formula I:

[0004] The oligosaccharide residues are selected from any oligosaccharide residue in general formula (II) or general formula (III):

[0005]

[0006] a is any integer from 1 to 6; b is any integer from 1 to 10; X is selected from: -CH2~-NH-, -S-, One of them;

[0007] Linker is a structural part obtained by directly or indirectly linking oligosaccharides to a carrier, selected from:

[0008]

[0009]

[0010] One of them; j1, j2, j3, j3 are any integers from 1 to 10; n represents the number of oligosaccharides linked to the carrier, and n is any integer from 1 to 30;

[0011] The carriers are selected from: bovine serum albumin (BSA), human serum albumin, hemocyanin, tetanus toxin, non-toxic mutant diphtheria toxin, Mycobacterium tuberculosis secretory protein, or monophosphorylated lipid A (LipidA). This oligosaccharide-protein conjugate uses linear oligosaccharide residues to bind to carrier residues via linkers. However, this binding strength is relatively weak, and the preparation method for the oligosaccharide-protein conjugate is complex, hindering industrial production.

[0012] Studies have shown that the key to persistent infection in all Brucella species lies in their ability to synthesize cyclic β-1,2-glucans (CβG). CβG is a cyclic oligosaccharide composed of glucose monosaccharide units linked by β-1,2-glycosidic bonds, with a degree of polymerization (DP) typically between 17 and 23. CβG is one of the key virulence factors in brucellosis, playing a crucial role in the process of bacterial invasion of cells and escape from the host's immune system. Because CβG itself is a small molecule hapten, it lacks immunogenicity and cannot directly stimulate the body to produce antibodies. It must bind with a carrier protein to form a complete antigen to acquire immunogenicity, thus enabling its use in the preparation of specific antibodies.

[0013] Bovine serum albumin (BSA) is a structurally well-defined and chemically stable carrier protein with a molecular weight of approximately 66 kDa. It exhibits good biocompatibility and is commonly used as a carrier for small molecule haptens. Covalently linking haptens to BSA via chemical conjugation is a common method for preparing specific antibodies. However, methods for the chemical conjugation of CβG with BSA have not yet been systematically reported.

[0014] Therefore, there is an urgent need to develop a method for preparing BSA-CβG conjugates with mild reaction conditions and well-defined product structures, in order to overcome the technical bottleneck in the development of brucellosis diagnostic reagents. Summary of the Invention

[0015] The technical problem this invention aims to solve is to overcome the shortcomings of existing technologies and provide a bovine serum albumin-cyclic β-1,2-glucan conjugate (BSA-CβG), its preparation method, and its applications. By establishing a highly efficient "cyclic oligosaccharide-protein" grafting reaction technology, this invention provides core antigen materials for the immunodiagnosis of brucellosis. One of the technical solutions adopted by this invention to solve its technical problem is:

[0016] A bovine serum albumin-cyclic β-1,2-glucan conjugate is formed by coupling a cyclic β-1,2-glucan derivative with the thiol or amino groups of bovine serum albumin.

[0017] Specifically, the cyclic β-1,2-glucan derivative is a bromoamide-type cyclic β-1,2-glucan (CβG-Br) or activated CβG-DSG, and is prepared by chemical coupling reaction between the cyclic β-1,2-glucan derivative and bovine serum albumin (BSA).

[0018] Another technical solution adopted by the present invention to solve its technical problem is:

[0019] A method for preparing bovine serum albumin-cyclic β-1,2-glucan conjugate includes the following steps:

[0020] (1) Fermentation preparation and purification of cyclic β-1,2-glucan (CβG); (2) Synthesis of CβG derivatives: chemical derivatization of CβG to synthesize CβG derivatives with reactive functional groups; (3) Coupling reaction: the CβG derivatives obtained in step (2) are coupled with bovine serum albumin (BSA) and purified to obtain bovine serum albumin-cyclic β-1,2-glucan conjugate.

[0021] Preferably, the specific operation of step (1) includes: fermentation with Rhizobium radiobacter ATCC13333 as the production strain; the fermentation broth is boiled, centrifuged, and subjected to three-step alcohol precipitation to obtain crude oligosaccharide; then, it is purified by Sevag reagent deproteinization, solid phase extraction (SPE) desalting and depigmentation, and DEAE-agarose gel column chromatography to obtain high-purity CβG.

[0022] This invention provides two CβG chemical modification and coupling technology routes:

[0023] Route a: Activate the 6-hydroxyl group of CβG with CDI, introduce an amino group with HDA, and bromoacetyl bromide to obtain bromoamide CβG (CβG-Br); at the same time, break the disulfide bond of BSA with TCEP reducing agent, and use the exposed mercapto group to carry out a click reaction with the bromine group of CβG-Br to prepare BSA-CβG conjugate.

[0024] In one exemplary embodiment, the CβG derivative in step (2) is a bromoamide type CβG (CβG-Br). The synthesis method of CβG-Br is as follows: N,N'-carbonyldiimidazole (CDI) is used to activate the 6-hydroxyl group of CβG, reacts with 1,6-hexanediamine (HDA) to introduce an amino group, and then bromination is carried out with bromoacetyl bromide to obtain CβG-Br.

[0025] Accordingly, the coupling reaction in step (3) is as follows: the disulfide bond of BSA is opened by using tris(2-carboxyethyl)phosphine (TCEP) reducing agent to expose the thiol group; under weakly alkaline conditions, the bromine group on CβG-Br prepared in step (2) undergoes a nucleophilic substitution reaction with the thiol group on BSA to obtain the BSA-CβG coupling compound.

[0026] Route b: Activate the 6-hydroxyl group of CβG with CDI, introduce an amino group with HDA, and then condense the NHS active ester of DSG with the amino amide of CβG to obtain CβG-DSG; use the NHS active ester at the other end to couple with the amino group of BSA to prepare BSA-CβG-DSG conjugate.

[0027] In one exemplary embodiment, the CβG derivative in step (2) is an activated CβG-DSG. The method for synthesizing the activated CβG-DSG is as follows: the 6-hydroxyl group of CβG is activated by CDI, and an amino group is introduced by reacting with HDA. Then, the amino group of CβG is amide condensed with the NHS active ester of disuccinimide glutarate (DSG) to obtain the activated CβG-DSG.

[0028] Accordingly, the coupling reaction in step (3) is as follows: under weakly alkaline conditions, the NHS active ester on the activated CβG-DSG directly undergoes an amide condensation reaction with the amino group on BSA to obtain the BSA-CβG-DSG conjugate.

[0029] In this invention, the degree of polymerization (DP) of the CβG is 17-22, with DP=19 as the main component.

[0030] Another technical solution adopted by the present invention to solve its technical problem is:

[0031] Application of bovine serum albumin-cyclic β-1,2-glucan conjugate in the preparation of Brucella diagnostic antibodies or immunoassay reagents.

[0032] Since cyclic β-1,2-glucan CβG is one of the key virulence factors in brucellosis, and is a conserved specific polysaccharide epitope of Brucella, it is a core target for Brucella recognition and detection. Furthermore, bovine serum albumin (BSA) is a typical T-cell-dependent antigen, rich in free amino and carboxyl groups on its molecular surface, which can stably bind to glucan through cross-linking reactions, preventing antigen dissociation in vivo. After immunization, antigen-presenting cells can phagocytose and process the conjugate. On the one hand, BSA is degraded into polypeptide fragments, which are presented to CD4⁺ helper T cells via MHC-II molecules, activating T cell proliferation and differentiation and secreting cytokines such as IL-4 and IL-2, providing a crucial co-stimulatory signal for B cell activation. On the other hand, the intact cyclic β-1,2-glucan epitope in the conjugate can specifically bind to the B cell surface receptor (BCR), providing a specific recognition signal for B cell activation.

[0033] Under the synergistic activation of dual signals, B cells complete proliferation and differentiation, antibody class switching, and somatic affinity maturation, ultimately differentiating into plasma cells that stably secrete high-affinity specific IgG antibodies. Simultaneously, memory B cells are generated, forming a long-lasting immune response. Since cyclic β-1,2-glucan is a conserved structure unique to Brucella, antibodies induced in this manner can accurately recognize Brucella somatic antigens, showing no significant cross-reactivity with other closely related pathogens and exhibiting good species specificity, thus meeting the application requirements for Brucella detection, identification, and immunological diagnosis.

[0034] The beneficial effects of this invention, including a bovine serum albumin-cyclic β-1,2-glucan conjugate, its preparation method, and its applications, are as follows:

[0035] This invention relates to bovine serum albumin-cyclic β-1,2-glucan conjugates, which utilize the thiol group (route a) or amino group (route b) of BSA to the active group of CβG. The substrates are widely available, can be prepared on a large scale, and have low production costs.

[0036] This invention discloses a method for preparing bovine serum albumin-cyclic β-1,2-glucan conjugates. A large-scale, high-purity fermentation preparation and purification system for CβG was established. The purified yield reached 0.3 g / L. The obtained CβG has a clear structure and a degree of polymerization (DP) of 17-22, with DP=19 being the most common.

[0037] Secondly, both coupling methods involve mild reaction conditions and are easy to operate. Route a achieves specific grafting of BSA onto a single CβG molecule via a click reaction of mercapto-bromine; Route b utilizes the coupling of NHS active esters with amino groups to achieve efficient grafting of BSA onto 4-6 CβG molecules, which can significantly improve the immunogenicity of the conjugate.

[0038] The BSA-CβG conjugate prepared in this invention has a well-defined structure and can be used as an immunogen to prepare Brucella-specific polyclonal or monoclonal antibodies, providing key technical support for the development of Brucella disease test strips. The prepared bovine serum albumin-cyclic β-1,2-glucan conjugate thus provides a new and feasible solution for Brucella disease test strips. Attached Figure Description

[0039] Figure 1 This is a process flow diagram for the extraction and purification of CβG from the fermentation broth of this invention; Figure 2 This is a schematic diagram of the structure of CβG purified in this invention;

[0040] Figure 3 TLC images of CβG eluted on a graphite SPE column according to the present invention: (a) pigment and salt elution; (b) CβG elution; (c) other oligosaccharide elution.

[0041] Figure 4 TLC chromatograms collected for the CβG purification process of this invention: (a) 10-fold volume alcohol precipitation; (b) graphite column elution; (c) DEAE gel column elution; (d) DEAE gel column elution of other components.

[0042] Figure 5 (a) MALDI-TOF-MS spectrum and (b) ¹H NMR spectrum of CβG purified according to this invention; Figure 6 The FTIR spectrum of CβG in this invention; Figure 7The diagram shows the design of two reaction routes for the CβG derivative of this invention.

[0043] Figure 8 The MALDI-TOF-MS spectrum of the CβG derivative of this invention;

[0044] Among them: (A): CβG -CDI; (B): CβG -HDA; (C): CβG -Br; (D): CβG -DSG;

[0045] Figure 9 This is a structural characterization analysis diagram of the CβG derivative of this invention;

[0046] (A) ¹H NMR; (B) ¹³C NMR spectrum: (a): CβG -CDI; (b): CβG -HDA; (c): CβG -Br;

[0047] Figure 10 The FTIR spectrum of the CβG derivative of this invention;

[0048] (a): CβG; (b): CβG -CDI; (c): CβG -HDA; (d): CβG -Br

[0049] Figure 11 This is a schematic diagram of the synthesis of BSA-CβG conjugate from CβG-Br and BSA according to the present invention (reaction route a). Figure 12 This is a schematic diagram (reaction route b) showing the synthesis of BSA-CβG-DSG conjugate from CβG-DSG and BSA according to the present invention. Figure 13 (A) MALDI-TOF-MS spectrum and (B) FTIR spectrum of the BSA-CβG conjugate (route a) of the present invention; Figure 14 The image shows the MALDI-TOF-MS spectrum of the BSA-CβG-DSG conjugate (route b) of this invention.

[0050] Table 1 shows the measured molecular weight (theoretical molecular weight) of the MALDI-TOF-MS spectrum of CβG-CDI of the present invention. a DP: Degree of aggregation; b TNS: Total number of imidazole ester substituents;

[0051] Table 2 shows the measured molecular weight (theoretical molecular weight) of the MALDI-TOF-MS spectrum of CβG-HDA of the present invention. a DP: Degree of aggregation; b TNS: Total number of substitutions in the amination reaction;

[0052] Table 3 shows the measured molecular weight (theoretical molecular weight) of the MALDI-TOF-MS spectrum of CβG-Br in this invention.a DP: Degree of aggregation; b TNS: Total number of substitutions in bromination reactions;

[0053] Table 4 shows the measured molecular weight (theoretical molecular weight) of the MALDI-TOF-MS spectrum of CβG-DSG of the present invention. a DP: Degree of aggregation; b TNS: Total number of substitutions in amide condensation reaction; Detailed Implementation

[0054] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0055] I. Materials of this invention: Bovine serum albumin was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China); N,N-carbonyldiimidazole was obtained from Myriel Biochemical Technology Co., Ltd. (Shanghai, China); tris(2-carbonylethyl)phosphohydrochloric acid was purchased from Great Pharmaceutical Technology Co., Ltd. (Suzhou, China); and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

[0056] Rhizobium radiobacter ATCC13333 was purchased from the Guangzhou Institute of Microbiology.

[0057] II. Structural characterization methods for CβG derivatives and BSA-CβG conjugates:

[0058] 1) MALDI-TOF-MS analysis

[0059] CβG derivative: 1 μL of water-soluble CβG derivative was spotted onto the fixed position on the target plate. After drying, 1 μL of matrix (2,5-dihydroxybenzoic acid, DHB containing Na+) was added. After drying, the sample was loaded onto the instrument and the reflective cation mode was used. The molecular weight range was set to 2000 ~ 4500 Da.

[0060] BSA-CβG conjugate: 1 μL of water-soluble BSA-CβG conjugate and 1 μL of matrix (2,5-dihydroxyacetanilide, DHAP) were spotted at the same position on the target plate, mixed thoroughly, dried, and then processed by mass spectrometry. Mass spectrometry conditions: reflectance cation mode; molecular weight range: 25000 ~ 90000 Da.

[0061] 2) Fourier Transform Infrared (FTIR) Spectroscopy

[0062] A small amount of dry sample powder was placed directly onto the ATR crystal and secured with a clamping rod to ensure tight contact. Subsequently, FTIR (Thermo Nicolet, USA) was used for infrared spectral analysis in the spectral range of 400–4000 cm⁻¹, equipped with a diamond ATR accessory. Acquisition parameters included 32 scans at a resolution of 4 cm⁻¹. Blank spectra were acquired against an air background for each measurement, and the raw data underwent baseline correction and atmospheric compensation.

[0063] 3) Nuclear magnetic resonance (NMR) proton and carbon (¹H NMR, ¹³C NMR) analysis

[0064] 30 mg of CβG derivative was dissolved in 0.5 mL of deuterated dimethyl sulfoxide (DMSO-d6), and 1H NMR and 13C NMR spectra were obtained at 25 °C (Avance III 60, Bruker Instruments).

[0065] Inc., Billerica, MA, USA).

[0066] Example 1

[0067] A bovine serum albumin-cyclic β-1,2-glucan conjugate is formed by coupling a cyclic β-1,2-glucan derivative with the thiol or amino groups of bovine serum albumin.

[0068] The cyclic β-1,2-glucan derivative is a bromoamide-type cyclic β-1,2-glucan (CβG-Br), which is prepared by chemical coupling reaction between the bromoamide-type cyclic β-1,2-glucan (CβG-Br) and the thiol group of bovine serum albumin (BSA).

[0069] This embodiment describes a method for preparing a bovine serum albumin-cyclic β-1,2-glucan conjugate, comprising the following steps:

[0070] (1) Fermentation preparation and purification of cyclic β-1,2-glucan (CβG);

[0071] refer to Figures 1-6 The fermentation preparation and purification of cyclic β-1,2-glucan (CβG) includes the following steps:

[0072] 1) Activation and Fermentation of the Strains: The *Rhizobium radiobacter* strain (*R. radiobacter* ATCC13333) preserved in glycerol tubes was inoculated onto a solid culture medium (10.0 g / L tryptone, 2.0 g / L yeast extract, 15.0 g / L agar powder, 1.0 g / L magnesium sulfate, pH 7.0) and cultured at 30°C for 48 h to activate and obtain single colonies. These single colonies were then inoculated into 200 mL of seed culture medium (10.0 g / L tryptone, 2.0 g / L yeast extract, 1.0 g / L magnesium sulfate, pH 7.0) and cultured at 30°C and 200 rpm for 22 h. Transfer the culture medium (mannitol 20.0 g·L / L, glutamic acid 1.0 g·L / L, sodium chloride 0.2 g·L / L, dipotassium hydrogen phosphate 1.0 g·L / L, magnesium sulfate 0.2 g·L / L, calcium chloride 0.04 g·L / L, trace elements 0.1 L·L / L) to a 10% inoculum and incubate at 30℃ and 200 rpm for 6 days.

[0073] During fermentation in a 5L fermenter, the mannitol content in the culture medium was increased to 30.0 g / L, glutamic acid to 5.0 g / L, the aeration rate was 1 vvm, and the stirring rate was 400 r·min⁻¹. The temperature was maintained at 30℃ for the first 24 h, then adjusted to 33℃. 2 mol· / L HCl and 20% NH₄⁺ were used. 3▪ H2O automatically adjusts the pH. During the 24-60 h phase, mannitol is added every 12 h to a final concentration of 5.0 g / L, and the culture is continued for a total of 6 days. 2) Crude extraction and purification: (e.g., ...) Figure 1 The process flow diagram shown illustrates the extraction and purification of CβG from the fermentation broth, including crude extraction and purification. A schematic diagram of the structure of CβG is also shown. Figure 2As shown. The fermentation broth was boiled for 30 min, centrifuged, and the supernatant was collected and concentrated to 1 / 4 of the original volume by rotary evaporation. Three volumes of anhydrous ethanol were added, and the mixture was incubated overnight at 4°C. The precipitate was then discarded by centrifugation, and the process was repeated once. Ten volumes of anhydrous ethanol were added again to precipitate the precipitate, which was then filtered and dried. The precipitate was desalted by dialysis using a 1 kDa dialysis bag and lyophilized to obtain crude oligosaccharide (yield 2.3 g / L). The crude oligosaccharide was prepared into a 20 g / L solution and repeatedly treated with Sevag reagent (n-butanol:chloroform = 1:4, v / v) until no protein precipitation was observed. Subsequently, a graphitized carbon column (SPE) was activated with 16 mL of acetonitrile, equilibrated with ultrapure water, and loaded onto the column. Salts and pigments were eluted with ultrapure water, followed by a gradient elution with 5%–30% acetonitrile. The CβG-containing fraction (15%–25% acetonitrile) was collected by TLC and concentrated and lyophilized. Finally, purification was performed using a DEAE-Sepharose Fast-Flow column (19 × 200 mm), eluted with ultrapure water and a gradient of 0.1–0.3 mol·L⁻¹ NaCl. The neutral sugar fraction was collected, dialyzed, and lyophilized, with a final yield of 0.3 g·L⁻¹. TLC results of the collected solutions after purification on graphitized carbon columns and DEAE columns are shown below. Figures 3-4 As shown. 3) The purified product CβG was analyzed by MALDI-TOF-MS and 1H NMR, and the FTIR chromatogram was obtained. The results are shown in the figure. Figure 5 and Figure 6 As shown. Figure 5 As shown, the m / z values ​​were detected using MALDI-TOF-MS (reflection cation mode, DHB matrix containing Na⁺) and were 2777.999, 2940.024, 3102.077, 3264.147, 3426.198, and 3588.284. The difference values ​​were within the range of 162 glucose repeating units, indicating that the degree of polymerization (DP) of the obtained CβG was 17-22, with DP=19 being the most common (m / z 3102.077). 1 The anomeric hydrogen chemical shift δ < 5.0 in 1H NMR confirmed that it is a β-type conformation.

[0074] like Figure 6 As shown, in the FTIR spectrum at 900 cm⁻¹ -1 The absorption peak at that point confirmed the presence of the β-glycosidic bond.

[0075] (2) Synthesis of CβG derivatives: Chemical derivatization of CβG to synthesize CβG derivatives with reactive functional groups;

[0076] The synthesis of the CβG derivative in this embodiment is based on Figure 7 The synthetic route diagram of reaction route a for the CβG derivative shown above synthesized a bromoamide type CβG (CβG-Br).

[0077] refer to Figures 1-6The synthesis method of bromoamide type CβG (CβG-Br) in this embodiment (intermediate of route a) includes the following steps:

[0078] 1) Synthesis of CβG-CDI:

[0079] CβG (200 mg, 60 μmol) was added to a reaction flask, followed by 5 mL of pyridine. The reaction was carried out at room temperature under N2 for 15 min. Then, CDI (200 mg, 1200 μmol) dissolved in 10 mL of DMSO was added, and the reaction was carried out at room temperature under N2 for 16 h. After the reaction, 10 times the volume of diethyl ether was added to precipitate the product. The product was washed twice with diethyl ether and dried to obtain CβG-CDI. 2) Synthesis of CβG-HDA:

[0080] Solid HDA (120 mg, 1000 μmol) was added to a reaction flask and crushed. CβG-CDI (200 mg, 60 μmol) dissolved in 15 mL DMSO was added, and the mixture was reacted at room temperature under N2 for 18 h. After the reaction, undissolved HDA was removed by centrifugation, and 10 volumes of diethyl ether were added to precipitate the precipitate. The precipitate was washed with diethyl ether, ethanol, and diethyl ether, respectively, and dried to obtain CβG-HDA. 3) Synthesis of CβG-Br:

[0081] CβG-HDA (200 mg, 60 μmol) was dissolved in 20 mL of DMSO, and triethylamine (156 μL, 960 μmol) was added as an acid-binding agent. The mixture was stirred at room temperature for 15 min, and then bromoacetyl bromide (84 μL, 960 μmol) was added. The mixture was reacted at room temperature in the dark for 12 h. After the reaction, 10 times its volume of acetone was added to precipitate the product. The precipitate was washed with acetone and then with diethyl ether, and dried to obtain CβG-Br.

[0082] The CβG derivatives prepared above were analyzed using MALDI-TOF-MS, FTIR, and other methods. 1 H NMR and 13 C NMR analysis, results are as follows Figures 8-10 As shown in Tables 1-3.

[0083] Table 1 shows the measured molecular weight (theoretical molecular weight) of the MALDI-TOF-MS spectrum of CβG-CDI of the present invention.

[0084]

[0085] in, a DP: Degree of aggregation; b TNS: Total number of imidazole ester substituents.

[0086] Table 2 shows the measured molecular weight (theoretical molecular weight) of the MALDI-TOF-MS spectrum of CβG-HDA of the present invention.

[0087]

[0088] in, a DP: Degree of aggregation; b TNS: Total number of substitutions in the amination reaction;

[0089] Table 3 shows the measured molecular weight (theoretical molecular weight) of the MALDI-TOF-MS spectrum of CβG-Br in this invention.

[0090]

[0091] in, a DP: Degree of aggregation; b TNS: Total number of substitutions in bromination reactions;

[0092] The primary hydroxyl group at the 6-position outside the cavity of CβG is highly reactive and can introduce a bromoacetamide group (-NHCO-CH2-Br). First, an N,N-carbonyldiimidazole (CDI) undergoes a nucleophilic substitution reaction with the 6-hydroxyl group of CβG to form a reactive imidazole ester intermediate (CβG-CDI). Then, the amino group at one end of the HDA (1,6-hexanediamine) molecule undergoes a nucleophilic substitution reaction with the imidazole ester group, covalently attaching a long-chain alkylamine to the surface of the CβG molecule, forming CβG-HDA with a primary amino group. Finally, a short-chain brominated CβG (CβG-Br) is synthesized via bromination, with an overall yield of 26.7%. Figure 8 As shown in Tables A-C and 1-3, MALDI-TOF-MS analysis indicates that the molecular weights of the CβG derivatives are consistent with the calculated values, mainly consisting of monosubstituted products in the DP range of 17-22, with a small amount of 2-4 substituted products. Figure 10 As shown in the figure, the FTIR spectrum contains a characteristic peak at 1748 cm⁻¹ in CβG-CDI. -1 1646 cm -1 and 1530 cm -1 These correspond to the stretching vibrations of the newly formed carbonyl C=O group, and the C=N and C=C stretching vibrations on the imidazole ring, respectively. The 1694 cm⁻¹ and 1534 cm⁻¹ values ​​are present in CβG-HDA. -1 The characteristic peaks correspond to the C=O stretching vibration and NH bending vibration of the amide bond, respectively, at 1643 cm⁻¹. -1 This corresponds to the NH bending vibration of the primary amine at the end of HDA. CβG-Br also exhibits a peak at 1694 cm⁻¹ corresponding to the C=O stretching vibration and NH bending vibration of the amide bond. -1 and 1538 cm -1 The primary amino group NH at the end of the HDA undergoes bending vibration at 1643 cm⁻¹. -1 disappear.

[0093] like Figure 9 As shown, the structure of the CβG derivative was further confirmed by 1H NMR and 13C NMR analyses.

[0094] like Figure 9 As shown in -A, CβG-CDI exhibits signals at δ 8.28, 7.88, and 7.12 in the low-field region, corresponding to the active hydrogen at position 1 of the imidazole substituent and two aryl hydrogens. CβG-HDA shows signals at δ 1.29–1.52 corresponding to the alkyl group of HDA in the high-field region, and at δ 7.03 corresponding to the secondary amine group signal. CβG-Br shows signals at δ 0.98–1.54 in the high-field region, corresponding to the alkyl group signal within the substituent, and at δ 6.94 and 7.64 corresponding to the secondary amine signal within the substituent.

[0095] like Figure 9 As shown in Figure -B, CβG-CDI exhibits signal peaks at δ 101.7, 121.4, and 135.1 for the carbon atom of the imidazole substituent group. CβG-HDA shows signal peaks at δ 25.6 and 30.7 for the carbon atom of hexamethylenediamine. CβG-Br shows signal peaks at δ 25.4 and 30.8 for the carbon atom of hexamethylenediamine, and at δ 29.6 for the carbon atom of acetyl bromide. Therefore, MALDI TOF-MS mass spectrometry, FT-IR spectra, and... 1 HNMR and 13 CNMR analysis confirmed the successful synthesis of the CβG derivative.

[0096] (3) Coupling reaction: The CβG derivative obtained in step (2) was coupled with bovine serum albumin (BSA) and purified to obtain bovine serum albumin-cyclic β-1,2-glucan conjugate.

[0097] refer to Figure 11 and 13 The preparation method of the BSA-CβG conjugate in this embodiment (reaction route a) includes the following steps:

[0098] like Figure 11 The diagram shows the synthesis of the BSA-CβG conjugate from CβG-Br and BSA (reaction route a). In a 0.1 M sodium bicarbonate solution at pH 8.3, the reaction proceeded according to the following ratio: BSA:cyclic sugar (CβG-Br) = 1:8.6 (equivalent ratio) and a protein concentration of 10.0 mg / mL. -1The following preparation ratio was used: BSA (25 mg, 0.375 μmol) was added to the reaction flask and dissolved in 2.5 mL of 0.1 M NaHCO3 buffer under ice bath conditions. TCEP·HCl (0.3 mg, 1.125 μmol) was added, and the mixture was reacted under N2 conditions in an ice bath for 10 min. Solid CβG-Br (10 mg, 3.225 μmol) was then added, and the mixture was gently stirred under N2 protection in an ice bath and in the dark for 16 h. The completeness of the reaction was monitored using Ellman's reagent. The reacted BSA-CβG conjugate needs to be completely desalted by ultrafiltration with Tris-HCl buffer (20 mM Tris-HCl, 50 mM NaCl, pH 7.5) to obtain the purified BSA-CβG conjugate.

[0099] First, 0.45 mL of BSA-CβG conjugate was added to a 30 kDa ultrafiltration tube and centrifuged once (1800 x g × 10 min, with subsequent centrifugation speed and time consistent). Tris-HCl buffer was then added, and the centrifugation was repeated three times. The tube was washed three times with pure water. Finally, 0.45 mL of pure water was added, and the tube was centrifuged upside down for 5 min. The filtrate was collected as the completely desalted BSA-CβG conjugate. Structural characterization showed: Figure 13 As shown in -A, the MALDI-TOF-MS detection m / z is 70.7 kDa; as Figure 13 As shown in -B, a characteristic peak appears at 1651 cm⁻¹ in FTIR detection. -1 and 1537 cm -1 The characteristic peak corresponds to the C=O stretching vibration and NH bending vibration of the amide bond, respectively, at 832 cm⁻¹. -1 The successful grafting of the BSA-CβG conjugate was confirmed by the SC group corresponding to the thiol reaction. In this route, BSA can specifically graft one CβG group.

[0100] Example 2

[0101] A bovine serum albumin-cyclic β-1,2-glucan conjugate is formed by coupling a cyclic β-1,2-glucan derivative with the thiol or amino groups of bovine serum albumin.

[0102] Specifically, the cyclic β-1,2-glucan derivative is an activated CβG-DSG, prepared by chemical coupling reaction between the cyclic β-1,2-glucan derivative and the amino group of bovine serum albumin (BSA).

[0103] This embodiment describes a method for preparing a bovine serum albumin-cyclic β-1,2-glucan conjugate, comprising the following steps:

[0104] (1) Fermentation preparation and purification of cyclic β-1,2-glucan (CβG);

[0105] The fermentation preparation and purification of cyclic β-1,2-glucan (CβG) were the same as in Example 1;

[0106] (2) Synthesis of CβG derivatives: Chemical derivatization of CβG to synthesize CβG derivatives with reactive functional groups;

[0107] The synthesis method of succinimide-activated CβG (CβG-DSG) in this embodiment (route b intermediate) includes the following steps:

[0108] according to Figure 7 The synthetic route diagram of reaction route b for the CβG derivative shown synthesizes a succinimide-activated CβG (CβG-DSG); wherein, the synthesis of CβG-CDI and CβG-HDA is the same as steps 1) and 2) of (2) in Example 1. 3) Synthesis of CβG-DSG:

[0109] CβG-HDA (200 mg, 60 μmol) and DSG (294 mg, 900 μmol) were dissolved in 20 mL of DMSO solution. 5 mL of PBS buffer (pH 7.4) was added dropwise under vigorous stirring, and the reaction was allowed to proceed at room temperature for 4 h. After the reaction, 10 volumes of acetone were added to precipitate the product, which was then washed with acetone, ethanol, and diethyl ether, respectively, and dried to obtain CβG-DSG. The overall yield was 35.3%. Figure 8 As shown in Table 4, the MALDI-TOF-MS detection results show that the molecular weight of the product is basically consistent with the theoretical calculation value, confirming the accuracy of the CβG-DSG structure.

[0110] Table 4 shows the measured molecular weight (theoretical molecular weight) of the MALDI-TOF-MS spectrum of CβG-DSG of the present invention.

[0111]

[0112] in, a DP: Degree of aggregation; b TNS: Total number of substitutions in amide condensation reaction. (3) Coupling reaction: The CβG derivative obtained in step (2) is coupled with bovine serum albumin (BSA) and purified to obtain bovine serum albumin-cyclic β-1,2-glucan conjugate.

[0113] The reaction diagram for the synthesis of BSA-CβG-DSG conjugates from CβG-DSG and BSA (reaction route b) is shown below. Figure 12 As shown.

[0114] In a 0.1 M sodium bicarbonate solution at pH 8.3, BSA was prepared at a ratio of BSA:cyclic sugar (CβG-DSG) = 1:30 (equivalent ratio) with a protein concentration of 3.0 mg·mL⁻¹. BSA (25 mg, 0.375 μmol) was dissolved in 8.3 mL of 0.1 M NaHCO₃ and added to the reaction flask. CβG-DSG (35 mg, 11.25 μmol) was then added, and the mixture was gently stirred at room temperature for 12 h. After the reaction, 0.45 mL of the BSA-CβG-DSG conjugate was added through a 30 kDa ultrafiltration tube, centrifuged once (1800 x g × 10 min, subsequent centrifugation speed and time were consistent), Tris-HCl buffer was added, and the mixture was centrifuged three more times. The mixture was washed three times with pure water, and finally 0.45 mL of pure water was added. The mixture was then centrifuged upside down for 5 min, and the filtrate was collected as the completely desalted BSA-CβG-DSG conjugate. Figure 14 As shown, MALDI-TOF-MS detected m / z 79.9–86.6 kDa (corresponding to 4–6 CβG grafts onto BSA). Compared to route a, this route significantly increases the amount of CβG grafted onto BSA, and is more likely to significantly improve the immunogenicity of BSA-CβG conjugates, making it more advantageous in the preparation of polyclonal and monoclonal antibodies.

[0115] Protein grafting amount assessment: The molecular weight of the products was determined by MALDI-TOF-MS. In Example 1, the BSA-CβG conjugate obtained using route a had a molecular weight of 70.7 kDa (specifically grafted with one CβG molecule); in Example 2, the BSA-CβG-DSG conjugate obtained using route b had a molecular weight of 79.9-86.6 kDa (corresponding to 4-6 grafted CβG molecules). The results show that both methods of this invention can successfully prepare the target conjugates, and the grafting efficiency of route b is significantly higher than that of route a.

[0116] In summary, this invention provides a novel and efficient method for preparing BSA-CβG conjugates. Route a utilizes a click reaction between a bromoamide-type cyclic oligosaccharide and the thiol group of reduced BSA, while route b utilizes a DSG bifunctional cross-linking agent to achieve high-density modification of BSA amino groups. Both methods effectively solve the technical challenge of chemically grafting CβG onto carrier proteins, laying the foundation for the large-scale preparation of brucellosis diagnostic antigens.

[0117] Example 3

[0118] Application of bovine serum albumin-cyclic β-1,2-glucan conjugate in the preparation of Brucella diagnostic antibodies or immunoassay reagents.

[0119] The above detailed description of the bovine serum albumin-cyclic β-1,2-glucan conjugate, its preparation method, and its application, with reference to the embodiments, is illustrative rather than limiting. Several embodiments can be listed within the defined scope; therefore, variations and modifications without departing from the overall concept of the invention should fall within the protection scope of the invention. Those skilled in the art will recognize that other variations of the specific structure in this invention are possible.

Claims

1. A bovine serum albumin-cyclic β-1,2-glucan conjugate, characterized in that, The bovine serum albumin-cyclic β-1,2-glucan conjugate is formed by coupling a cyclic β-1,2-glucan derivative with the thiol or amino groups of bovine serum albumin.

2. The bovine serum albumin-cyclic β-1,2-glucan conjugate as described in claim 1, characterized in that, The cyclic β-1,2-glucan derivative is a bromoamide-type cyclic β-1,2-glucan (CβG-Br) or activated CβG-DSG, and is prepared by chemical coupling reaction between the cyclic β-1,2-glucan derivative and bovine serum albumin (BSA).

3. A method for preparing the bovine serum albumin-cyclic β-1,2-glucan conjugate as described in claim 1 or 2, characterized in that, The preparation method includes the following steps: (1) Fermentation preparation and purification of cyclic β-1,2-glucan (CβG); (2) Synthesis of CβG derivatives: Chemical derivatization of CβG to synthesize CβG derivatives with reactive functional groups; (3) Coupling reaction: The CβG derivative obtained in step (2) was coupled with bovine serum albumin (BSA) and purified to obtain bovine serum albumin-cyclic β-1,2-glucan conjugate.

4. The method for preparing the bovine serum albumin-cyclic β-1,2-glucan conjugate as described in claim 3, characterized in that, The specific operation of step (1) includes: using Rhizobium radiophyllum ( R. radiobacter ATCC13333 The production strain was fermented; the fermentation broth was boiled, centrifuged, and subjected to three-step alcohol precipitation to obtain crude oligosaccharides; then, high-purity CβG was obtained by deproteinization with Sevag reagent, desalting and depigmentation by solid phase extraction (SPE), and purification by DEAE-agarose gel column chromatography.

5. The method for preparing the bovine serum albumin-cyclic β-1,2-glucan conjugate as described in claim 3 or 4, characterized in that, The CβG derivative in step (2) is a bromoamide type CβG (CβG-Br). The synthesis method of CβG-Br is as follows: N,N'-carbonyldiimidazole (CDI) is used to activate the 6-hydroxyl group of CβG, reacts with 1,6-hexanediamine (HDA) to introduce an amino group, and then bromination reaction is carried out with bromoacetyl bromide to obtain CβG-Br.

6. The method for preparing the bovine serum albumin-cyclic β-1,2-glucan conjugate as described in claim 5, characterized in that, The coupling reaction in step (3) is as follows: the disulfide bond of BSA is opened by using tris(2-carboxyethyl)phosphine (TCEP) reducing agent to expose the thiol group; under weakly alkaline conditions, the bromine group on CβG-Br prepared in step (2) undergoes a nucleophilic substitution reaction with the thiol group on BSA to obtain the BSA-CβG coupling compound.

7. The method for preparing the bovine serum albumin-cyclic β-1,2-glucan conjugate as described in claim 3 or 4, characterized in that, The CβG derivative in step (2) is an activated CβG-DSG. The method for synthesizing activated CβG-DSG is as follows: CDI is used to activate the 6-hydroxyl group of CβG, which is then reacted with HDA to introduce an amino group. The amino group of CβG is then amide condensed with the NHS active ester of disuccinimide glutarate (DSG) to obtain activated CβG-DSG.

8. The method for preparing the bovine serum albumin-cyclic β-1,2-glucan conjugate as described in claim 7, characterized in that, The coupling reaction in step (3) is as follows: under weakly alkaline conditions, the NHS active ester on the activated CβG-DSG directly undergoes an amide condensation reaction with the amino group on BSA to obtain the BSA-CβG-DSG conjugate.

9. The method for preparing the bovine serum albumin-cyclic β-1,2-glucan conjugate as described in claim 3, characterized in that, The degree of polymerization (DP) of the CβG is 17-22, with DP=19 being the main component.

10. The use of the bovine serum albumin-cyclic β-1,2-glucan conjugate as described in claim 1 or 2 in the preparation of Brucella diagnostic antibodies or immunoassay reagents.

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