A helicobacter pylori cell wall oligosaccharide-carrier protein conjugate, its preparation method and use

Abstract

This invention discloses a Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate, its preparation method, and its application, relating to the field of organic synthesis. The preparation method of the Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate of this invention includes the following steps: S1, reacting oligosaccharides with sulfur phosgene at room temperature to obtain a hapten; S2, conjugating the hapten with a carrier protein to obtain the Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate. This invention combines Helicobacter pylori cell wall-specific oligosaccharides with a carrier protein (CRM). 197 Covalently coupled with bovine serum albumin (or bovine serum albumin), the resulting conjugate can effectively activate T cell-dependent immune responses, overcome the weakness of oligosaccharide haptens in autoimmunity, and induce the production of high-titer specific anti-oligosaccharide antibodies.

Description

Technical Field

[0001] This invention relates to the field of organic synthesis technology, specifically to a Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate, its preparation method, and its application. Background Technology

[0002] Helicobacter pylori (H. pylori) is a Gram-negative bacterium that colonizes the stomach and can cause chronic gastritis, peptic ulcers, and stomach cancer. In 1994, chronic H. pylori infection was classified as a Group 1 carcinogen by the World Health Organization; in 2021, the 15th edition of the U.S. Carcinogens Reports on Cancer added chronic H. pylori infection as a definite carcinogen. Stomach cancer is one of the most common malignant tumors worldwide and ranks third among cancer deaths. Summary of the Invention

[0003] To address the aforementioned technical problems, the present invention aims to provide a Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate, its preparation method, and its application, which combines Helicobacter pylori cell wall-specific oligosaccharides with carrier proteins (CRMs). 197 Covalently coupled with bovine serum albumin (or bovine serum albumin), the resulting conjugate can effectively activate T cell-dependent immune responses, overcome the weakness of oligosaccharide haptens in autoimmunity, and induce the production of high-titer specific anti-oligosaccharide antibodies.

[0004] The technical solution of this invention to solve the above-mentioned technical problems is as follows: A method for preparing Helicobacter pylori cell wall oligosaccharide-carrier protein conjugates is provided, comprising the following steps:

[0005] S1. React the oligosaccharides of Helicobacter pylori cell wall with sulfur phosgene at room temperature to obtain a hapten;

[0006] S2. The hapten is coupled to the carrier protein to obtain the Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate.

[0007] Furthermore, in step S1, the Helicobacter pylori cell wall oligosaccharide is... , or .

[0008] Furthermore, in step S1, the mass-to-volume ratio of the Helicobacter pylori cell wall oligosaccharide to phosgene is 8 mg: 5-6 μL.

[0009] Furthermore, in step S2, the carrier protein is CRM. 197 Or bovine serum albumin.

[0010] The present invention also discloses the method for preparing the above-mentioned Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate, and the Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate obtained therefrom.

[0011] This invention also discloses the application of the above-mentioned Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate in the preparation of immunomodulatory drugs.

[0012] The present invention has the following beneficial effects:

[0013] 1. This invention covalently couples Helicobacter pylori cell wall-specific oligosaccharides with carrier proteins (CRM197 or bovine serum albumin). The resulting conjugate can effectively activate T cell-dependent immune responses, overcome the defect of weak autoimmunogenicity of oligosaccharide haptens, and induce the production of high-titer specific anti-oligosaccharide antibodies.

[0014] 2. The oligosaccharide of Helicobacter pylori cell wall selected in this invention is its characteristic surface antigen structure. The prepared conjugate can specifically recognize Helicobacter pylori, avoid cross-reaction, and provide a candidate vaccine for precise prevention and control of Helicobacter pylori infection.

[0015] 2. This invention adopts a strategy of phosgene activation at room temperature, which can obtain the hapten intermediate in one step without complicated purification steps; the subsequent coupling reaction conditions are mild and the operation is simple, making it suitable for large-scale production.

[0016] 3. Verification by SDS-PAGE electrophoresis and MALDI-TOF mass spectrometry showed that the molecular weight of the conjugate relative to the carrier protein (CRM) was [data missing]. 197 The significant increase in the molecular weight of oligosaccharides (or BSA) and the fact that the increase in molecular weight was consistent with the molecular weight and number of couplings of the oligosaccharides confirmed that the oligosaccharides had been successfully covalently linked to the carrier protein. Attached Figure Description

[0017] Figure 1 The Helicobacter pylori cell wall oligosaccharide-CRM obtained in Examples 1-3 197 SDS-PAGE electrophoresis results of the conjugates;

[0018] Figure 2 The results of SDS-PAGE electrophoresis of the Helicobacter pylori cell wall oligosaccharide-BSA conjugates obtained in Examples 4-6 are shown.

[0019] Figure 3 The Helicobacter pylori cell wall oligosaccharide-CRM obtained in Examples 1-3 197 MALDI-TOF mass spectrometry analysis results of the conjugates;

[0020] Figure 4 The results of MALDI-TOF mass spectrometry analysis of the Helicobacter pylori cell wall oligosaccharide-BSA conjugates obtained in Examples 4-6 are shown. Detailed Implementation

[0021] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0022] Example 1:

[0023] A Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate, the preparation route of which is as follows:

[0024] ;

[0025] Specifically, the following steps are included:

[0026] S1. Dissolve Helicobacter pylori cell wall oligosaccharide (compound 1) (8 mg) in sodium bicarbonate aqueous solution (0.68 mL, 10 mg / mL), add phosgene (5.2 μL, 67.6 μmol) in chloroform (1.02 mL) at room temperature, stir the reaction at room temperature for 3 hours, dilute the reaction mixture with water (3.4 mL), extract the aqueous layer with chloroform (3.4 mL × 3), remove excess phosgene, collect the aqueous layer, freeze dry, and obtain hapten 4;

[0027] S2, Mix hapten 4 (5.0 mg) with 5.0 mg of CRM 197 Mix at a mass ratio of 1:1, heat in a phosphate buffer solution at pH 8.5 at 30 °C overnight, and then use a 10 kDa ultrafiltration tube to remove free, unconjugated excess hapten, to obtain Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate.

[0028] Example 2:

[0029] A Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate, the preparation route of which is as follows:

[0030] ;

[0031] Specifically, the following steps are included:

[0032] S1. Dissolve Helicobacter pylori cell wall oligosaccharide (compound 2) (8 mg) in sodium bicarbonate aqueous solution (0.65 mL, 10 mg / mL), add phosgene (4.9 μL, 64.9 μmol) in chloroform (0.98 mL) at room temperature, stir the reaction at room temperature for 3 hours, dilute the reaction mixture with water (3.3 mL), extract the aqueous layer with chloroform (3.3 mL × 3), remove excess phosgene, collect the aqueous layer, freeze dry, and obtain hapten 5;

[0033] S2, Mix hapten 5 (5.0 mg) with 5.0 mg of CRM 197 Mix at a mass ratio of 1:1, heat in a phosphate buffer solution at pH 8.5 at 30 °C overnight, and then use a 10 kDa ultrafiltration tube to remove free, unconjugated excess hapten, to obtain Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate.

[0034] Example 3:

[0035] A Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate, the preparation route of which is as follows:

[0036] ;

[0037] Specifically, the following steps are included:

[0038] S1. Dissolve Helicobacter pylori cell wall oligosaccharide (compound 3) (8 mg) in sodium bicarbonate aqueous solution (0.23 mL, 10 mg / mL), add 1.8 μL, 23.0 μmol of phosgene in chloroform (0.35 mL) at room temperature, stir the reaction at room temperature for 3 hours, dilute the reaction mixture with water (0.35 mL), extract the aqueous layer with chloroform (0.35 mL × 3), remove excess phosgene, collect the aqueous layer, freeze dry, and obtain hapten 6;

[0039] S2, Mix hapten 6 (8.0 mg) with 5.0 mg of CRM 197 Mix at a mass ratio of 1:1, heat in a phosphate buffer solution at pH 8.5 at 30 °C overnight, and then use a 10 kDa ultrafiltration tube to remove free, unconjugated excess hapten, to obtain Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate.

[0040] Example 4:

[0041] The difference between Example 4 and Example 1 is that the carrier protein CRM is used. 197 Replace with BSA.

[0042] Example 5:

[0043] The difference between Example 5 and Example 2 is that the carrier protein CRM is used. 197 Replace with BSA.

[0044] Example 6:

[0045] The difference between Example 6 and Example 3 is that the carrier protein CRM is used. 197 Replace with BSA.

[0046] Experimental example:

[0047] I. Conjugation of Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate and CRM 197 The carrier protein and BSA protein were detected by SDS-PAGE electrophoresis.

[0048] The specific detection method is as follows:

[0049] 1. SDS-PAGE gel preparation:

[0050] (1) Preparation of 12% separating gel: Using a pipette, pipette 1.72 mL of deionized water, 2 mL of acrylamide / methylenebisacrylamide (29:1) solution, and 1.25 mL of 4× separating gel buffer (pH 8.8), mix well, add 50 μL of 10% APS and 2 μL of LTEMED, mix well, and pour the solution into the gel plate. Add an appropriate amount of isopropanol or deionized water to remove air bubbles and level the liquid surface. Let it stand naturally until solidified, then rinse with isopropanol and blot dry.

[0051] (2) 5% stacking gel: Use a pipette to draw 1.7 mL of ultrapure water, add 0.43 mL of acrylamide / methylenebisacrylamide (29:1) solution and 0.33 mL of 4× stacking gel buffer, mix well, add 20 μL of 10% APS and 2.5 μL of TEMED, mix well, add the solution to the top layer of the separating gel, insert into the 10-well or 15-well comb as needed, and wait for solidification.

[0052] 2. Sample preparation: Centrifuge the Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate at 12,000 rpm, take 20 μL of the supernatant, add 5 μL of 5X laoding to each, treat at 100 ℃ for 5 min, and place the treated samples in a 4 ℃ refrigerator for later use.

[0053] 3. Electrophoresis: Add 10 μL of the sample to be tested to each lane of the SDS-PAGE gel, and set up a carrier protein control group. Add an appropriate amount of 1× SDS-PAGE electrophoresis buffer, run the electrophoresis apparatus at 90 V, and when the leading edge is flush, adjust the voltage to 150 V. Stop electrophoresis when the leading edge runs out of the separating gel.

[0054] 4. Staining: Use Coomassie Brilliant Blue R-250 staining solution to stain the gel after electrophoresis at room temperature for 30 minutes.

[0055] 5. Decolorization: Place the stained gel in a box containing decolorizing solution and decolorize overnight.

[0056] Helicobacter pylori cell wall oligosaccharide-CRM obtained in Examples 1-3 197 The conjugates (referred to as Helicobacter pylori cell wall oligosaccharide 1-CRM) 197 Conjugates, Helicobacter pylori cell wall oligosaccharides 2-CRM 197 Conjugates, Helicobacter pylori cell wall oligosaccharides 3-CRM 197 The electrophoresis results of the conjugate are as follows: Figure 1 As shown; where lane 1 is an uncoupled CRM 197 Proteins, lanes 2-4 are the Helicobacter pylori cell wall oligosaccharide-CRM from Examples 1-3, respectively. 197 Results of gel electrophoresis of the conjugate.

[0057] Depend on Figure 1 It can be seen that lane 1 is an uncoupled CRM. 197 The protein, with a molecular weight of 58.3 kDa, was consistent with the expected value on SDS-PAGE gel electrophoresis. Examples 1-3: Helicobacter pylori cell wall oligosaccharides-CRM 197 The results of gel electrophoresis of the conjugates (lane2-lane4) compared to the unmodified CRM in lane 1. 197 Lanes 2-4 all showed molecular weight migration on electrophoresis, located above 58.3 kDa, indicating that the oligosaccharides in the Helicobacter pylori cell wall have a positive effect on CRM. 197 The modification was successful.

[0058] The electrophoresis results of the Helicobacter pylori cell wall oligosaccharide-BSA conjugates obtained in Examples 4-6 (denoted as Helicobacter pylori cell wall oligosaccharide 1-BSA conjugate, Helicobacter pylori cell wall oligosaccharide 2-BSA conjugate, and Helicobacter pylori cell wall oligosaccharide 3-BSA conjugate, respectively) are as follows: Figure 2 As shown; where lane 1 is the uncoupled BSA protein, and lanes 2-4 are the gel electrophoresis results of the Helicobacter pylori cell wall oligosaccharide-BSA conjugates from Examples 4-6, respectively.

[0059] Depend on Figure 2It was found that lane 1 was the uncoupled BSA protein with a molecular weight of 66.4 kDa, which was consistent with the prediction by SDS-PAGE gel electrophoresis. The electrophoresis results of Helicobacter pylori cell wall oligosaccharide-BSA gel (lane 2-lane 4) showed that, compared to the unmodified BSA in lane 1, lane 2-lane 4 all exhibited molecular weight shifts on electrophoresis, located above 66.4 kDa, indicating that the Helicobacter pylori cell wall oligosaccharide successfully modified the BSA.

[0060] II. MALDI-TOF mass spectrometry analysis of Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate.

[0061] The specific detection method is as follows: MALDI-MS data were recorded using an Autoflex II MALDI-TOF system (BrukerDaltonics) with 2,5-dihydroxybenzoic acid (DHB) as the matrix.

[0062] Helicobacter pylori cell wall oligosaccharide-CRM obtained in Examples 1-3 197 The results of MALDI-TOF mass spectrometry analysis of the conjugate are shown in the figure. Figure 3 .

[0063] Depend on Figure 3 It can be seen that the Helicobacter pylori cell wall oligosaccharide 1-CRM in Example 1 197 The conjugate has a molecular weight of 60.2 kDa, and the Helicobacter pylori cell wall oligosaccharide 2-CRM from Example 2 is also mentioned. 197 The conjugate has a concentration of 60.5 kDa and is the Helicobacter pylori cell wall oligosaccharide 3-CRM from Example 3. 197 The coupling has a capacity of 60.3 kDa, which is higher than that of CRM. 197 The increase of approximately 1.9–2.2 kDa is consistent with the expected molecular weight of a single oligosaccharide (theoretically approximately 0.63–1.78 kDa) and the coupling of multiple oligosaccharides, further confirming the covalent coupling.

[0064] The MALDI-TOF mass spectrometry analysis results of the Helicobacter pylori cell wall oligosaccharide-BSA conjugates obtained in Examples 4-6 are shown in the figure. Figure 4 .

[0065] Depend on Figure 4It can be seen that the molecular weight of the Helicobacter pylori cell wall oligosaccharide 1-BSA conjugate in Example 4 is 69.6 kDa, the molecular weight of the Helicobacter pylori cell wall oligosaccharide 2-BSA conjugate in Example 2 is 68.6 kDa, and the molecular weight of the Helicobacter pylori cell wall oligosaccharide 3-BSA conjugate in Example 3 is 68.7 kDa. All of them are about 2.2-3.2 kDa higher than BSA, which is consistent with the expected molecular weight of a single oligosaccharide (theoretically about 0.63-1.78 kDa) and the conjugation of multiple oligosaccharides, further confirming the covalent conjugation.

[0066] III. The Helicobacter pylori cell wall oligosaccharide-carrier protein conjugates obtained in Examples 1-3 and CRM 197 Statistical analysis was performed on the molecular weight data of the carrier protein and the BSA carrier protein to calculate the coupling rate.

[0067] The specific detection method is as follows: Based on the average molecular weight measured by MALDI-TOF, the number of oligosaccharides coupled to each carrier protein is estimated according to the following formula:

[0068] .

[0069] Among them, M 寡糖 This corresponds to the molecular weight of oligosaccharides in the cell wall of Helicobacter pylori.

[0070] Calculate each CRM 197 The average number of oligosaccharides coupled on the molecule was 1.02-3.12, and the average number of oligosaccharides coupled on each BSA molecule was 1.25-4.95, confirming the success of covalent coupling.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate, characterized in that, Includes the following steps: S1. React the oligosaccharides of Helicobacter pylori cell wall with sulfur phosgene at room temperature to obtain a hapten; S2. The hapten is coupled to the carrier protein to obtain the Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate.

2. The method for preparing the Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate as described in claim 1, characterized in that, In step S1, the Helicobacter pylori cell wall oligosaccharide is , or .

3. The method for preparing the Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate as described in claim 1, characterized in that, In step S1, the mass-to-volume ratio of the Helicobacter pylori cell wall oligosaccharide to phosgene is 8 mg: 5-6 μL.

4. The method for preparing the Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate as described in claim 1, characterized in that, In step S2, the carrier protein is CRM. 197 Or bovine serum albumin.

5. The Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate prepared by the method of any one of claims 1-4.

6. The use of the Helicobacter pylori cell wall oligosaccharide-carrier protein conjugate according to claim 5 in the preparation of immunomodulatory drugs.

Images