A novel blood group antigen and its enzymatic synthesis method

Abstract

The present invention discloses a novel blood group antigen and an enzymatic synthesis method thereof. The invention adopts an azide-modified disaccharide substrate to connect with fucose by an α-1,2 glycosidic bond under the action of fucosyltransferase to obtain an H antigen. The H antigen is used as a substrate, and the azide forms of the A antigen and the B antigen are obtained respectively under the action of acetylgalactosamine transferase and galactosyltransferase. Then, the modified end is reduced to NH by a chemical method. 2 That is, new blood type antigens A and B are obtained; the method can realize one-pot multi-enzyme synthesis, the method is simple and efficient, and its preparation and purification scale-up can achieve the preparation of high-purity products above the gram level, and at the same time has the advantage of industrial scale-up production, providing strong support for the domestic supply of raw materials for purified antigen and antibody preparation media.

Description

Technical Field

[0001] The invention belongs to the technical field of blood type antigen synthesis, and in particular relates to a novel blood type antigen and an enzymatic synthesis method thereof. Background Art

[0002] Human tissue blood group antigens are widely present on the surface of red blood cells and are also distributed in epithelial cells, endothelial cells, secretions, etc. of human organs. Blood group antigens are divided into three categories: A, B, and H according to different determinants. Each category is further divided into six types according to different disaccharide precursors, of which five categories (Ⅰ, Ⅱ, Ⅲ, Ⅳ, Ⅵ) exist in nature.

[0003] Antigens and antibodies can be specifically recognized, and the pure antigen is obtained and combined with a suitable carrier to form a separation column product that can purify the antibody.

[0004] At present, the synthesis of blood type antigens is divided into chemical synthesis and enzymatic synthesis. A, B, and H antigens can be obtained by chemical synthesis in 5 to 7 steps, but due to the generation of by-products and the conversion rate of some steps being less than 50%, the yield is low and the cost is high; while the enzymatic reaction has a high conversion rate, a short reaction time, and is green and environmentally friendly. The existing enzymatic synthesis of A and B antigens mainly relies on a variety of glycosyltransferases to be synthesized step by step in different reaction systems. Although it has a higher conversion rate than chemical reactions, the preparation and purification of its intermediates are still cumbersome. Therefore, this patent provides a one-pot multi-enzyme method for synthesizing antigens A and B in one step, and finally a one-step chemical reduction will make it the final product. The method is simple and efficient, and its preparation and purification amplification can achieve the preparation of high-purity products above the gram level. At the same time, it has the advantage of industrial amplification production, and provides a powerful solution for the domestic supply of raw materials for purifying antigen antibody preparation media. Summary of the invention

[0005] The present invention aims to provide a method for synthesizing novel blood group antigens A and B by a one-pot multi-enzyme method, so as to solve at least one of the many problems in the prior art.

[0006] A new type of blood group antigen has the following structural formula:

[0007]

[0008] Wherein, R is OH or NHAc.

[0009] The enzymatic synthesis method of the novel blood group antigen comprises the following steps:

[0010] S1. linking the azide-modified disaccharide substrate to fucose via an α-1,2 glycosidic bond under the action of fucosyltransferase;

[0011] S2. The products are used as substrates and converted under the action of transferases;

[0012] S3. Reducing the azide-modified end to an amino group to obtain a novel blood group antigen;

[0013] When R in the blood group antigen is OH, the transferase is galactosyltransferase; when R is NHAc, the transferase is acetylgalactosamine transferase.

[0014] Preferably, the azide-modified disaccharide substrate is Lac-linker N3, having a structural formula as shown in Formula I:

[0015]

[0016] Furthermore, in the step S1, Lac-linker N3 reacts in the presence of GDP-Fuc and α-1,2-FucT.

[0017] Furthermore, in step S2, Mn is used under the action of transferase. 2+ As a catalyst.

[0018] Furthermore, in step S3, the antigen is obtained by catalytic reduction under the action of hydrogen.

[0019] Furthermore, the above synthesis method also includes the purification of blood type antigens: using solid phase extraction C18 and eluting with methanol aqueous solutions of different concentrations from low to high.

[0020] The present invention also provides the application of the above enzymatic synthesis method in the industrial scale-up production of novel blood group antigens, which can be applied to the synthesis of kilogram-level novel antigens A and B and can ensure a high yield and purity.

[0021] Compared with the prior art, the beneficial effects of the present invention are at least:

[0022] 1. The blood type antibodies obtained by the method provided by the present invention have an ideal conversion rate and few by-products; the method is simple and efficient, and its preparation and purification scale-up can achieve the preparation of high-purity products above the gram level, which can be used as an ideal solution for industrial scale-up.

[0023] 2. The novel blood type antibody of the present invention has a strong specific binding effect with various serum antibodies and can be used as a replacement for the blood type antibodies currently on the market, thus expanding the scope of blood type antibody selection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The synthetic route of the present invention is shown in FIG.

[0025] Figure 2 This is a diagram showing the TLC results of the H antigen preparation in the example of the present invention.

[0026] Figure 3This is a diagram showing the TLC results of preparing A antigen-N3 according to an example of the present invention.

[0027] Figure 4 This is a diagram showing the TLC results of the B antigen-N3 prepared in the example of the present invention.

[0028] Figure 5 This is a diagram showing the TLC results of reducing B antigen-N3 according to an example of the present invention.

[0029] Figure 6 This is a graph showing the HPLC results after antigen purification in Example A of the present invention.

[0030] Figure 7 This is a graph showing the HPLC results after antigen purification in Example B of the present invention.

[0031] Figure 8 This is the mass spectrum of the antigen in Example A of the present invention.

[0032] Fig. 9 This is the mass spectrum of the antigen in Example B of the present invention. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and beneficial technical effect of the present invention clearer, the present invention is further described in detail below in conjunction with specific implementation methods. It should be understood that the specific implementation methods described in this specification are only for explaining the present invention, not for limiting the present invention.

[0034] The present invention provides a novel blood type antigen having the following structural formula:

[0035]

[0036] Wherein, R is OH or NHAc.

[0037] The present invention provides a method for synthesizing a novel blood group antigen by a one-pot multi-enzyme method, comprising the following steps:

[0038] The disaccharide substrate is linked to fucose by α-1,2 glycosidic bond under the action of fucosyltransferase (FucT) to obtain H antigen. With H antigen as substrate, the N3 forms of A antigen and B antigen are obtained respectively under the action of acetylgalactosamine transferase (BgtA) and galactosyltransferase (GTB). Finally, the N3 end is reduced to NH2 group form by chemical method to obtain new blood group antigens A and B respectively. The flow chart of the synthesis method of the present invention is as follows: Figure 1 shown.

[0039] The disaccharide substrate is Lac-linker N3, and the structural formula is as shown in Formula I:

[0040]

[0041] The structural formula of H antigen is as shown in Formula II:

[0042]

[0043] A antigen-N3 has the structural formula III:

[0044]

[0045] B antigen-N3 has structural formula IV:

[0046]

[0047] The new blood group antigen A has the structural formula V:

[0048]

[0049] The novel blood group antigen B is shown in structural formula VI:

[0050]

[0051] Raw materials and intermediates; Lac-linker N3, GDP-Fuc, UDP-GalNAc, UDP-Gal.

[0052] Product preparation method:

[0053] 1) Obtaining H antigen from disaccharide substrate

[0054] The reaction temperature is 20-40°C, and the metal ion is 5-20 mM / L Mn 2+ , 10~200mM / L Lac-Linker, 10~200mM / L GDP-Fuc and 0.04~0.1g / mL α-1,2-FucT crude enzyme react under weak alkaline conditions for 10~20h to obtain H-Antigen linker N3. Note that the pH should be adjusted to weak alkaline with NaOH before adding the enzyme. During the reaction, attention should be paid to the change of pH. 50~200mM / L Tris-HCl with pH (6~9) can be added to stabilize the pH of the reaction system.

[0055] 2) Enzymatic method to obtain A and B antigens-N3

[0056] A antigen-N3: reaction temperature is 20-40°C, metal ion is 5-20mM / L Mn 2+, 10~200mM / L Lac-Linker, 10~200mM / L GDP-Fuc, 10~200mM / L UDP-GalNAc and 0.02~0.5g / mL BgtA and 0.4~1g / mL α-1,2-FucT two crude enzymes reacted under weak alkaline conditions for 10~20h to obtain A-Antigen linker N3. Note that before adding enzymes, the pH should be adjusted to weak alkaline with NaOH. During the reaction, attention should be paid to the change of pH. 50~100mM / L pH (6~10) Tris-HCl can be added to stabilize the pH of the reaction system.

[0057] B antigen-N3: reaction temperature is 20-40°C, metal ion is 5-20mM / L Mn 2+ , 10~200mM / L Lac-Linker, 10~200mM / L GDP-Fuc, 10~200mM / L UDP-Gal and 0.02~0.1g / mL GTB and 0.04~1g / mL α-1,2-FucT two crude enzymes reacted under weak alkaline conditions for 4~20h to obtain B-Antigen linker N3. Before adding enzymes, the pH should be adjusted to weak alkaline with NaOH. During the reaction, attention should be paid to the change of pH. 50~100mM / L Tris-HCl with pH (6~10) can be added to stabilize the pH of the reaction system.

[0058] 3) Reduction of antigen A, B linker N3

[0059] The obtained A Antigen linker N3 and B Antigen linker N3 are respectively added with a reducing agent and a catalyst to carry out a reduction reaction to obtain A Antigen linker NH2 and B Antigen linker NH2. The catalytic reduction can be carried out by using Pd / C under the action of hydrogen.

[0060] Purification: Solid phase extraction C18 was used for purification, and different concentrations of methanol aqueous solutions were used for elution from low to high to obtain A Antigen linker NH2 and B Antigen linker NH2 with a purity of >95%.

[0061] Detection method: HPLC-ELSD was used to detect the response of the product in evaporative light, evaluate the purity of the product, and perform mass spectrometry qualitative analysis and HNMR structural analysis verification.

[0062] Example 1 Preparation of H antigen

[0063] 85mM / L Lac-Linker, 90mM / L GDP-Fuc and 0.08g / mL α-1,2-FucT crude enzyme were reacted at pH=8, 27℃ for 10h, and the metal ion was 5mM / L Mn 2+ Before adding the enzyme, add 2mL 1M / L NaOH to adjust the pH to a weak alkaline state, and add 100mM / L Tris-HCl with a pH of 7.5 to stabilize the pH of the reaction system. After 0.5h of reaction, add 0.5mL 1M / L NaOH to keep the pH at 8.0. After 1h of reaction, add 0.5mL 1M / L NaOH again to keep the pH at 7.5. The reaction conversion rate can reach 100%. The reaction TLC results are as follows: Figure 2 As shown, the developing solvent is ethyl acetate: methanol: water = 50:33:17, the staining agent is anisaldehyde, wherein 1 is a mixture of GDP-Fucose and Lacose-Linker; 2 is GDP-Fucose; 3 is Fucose; 4 is Lacose-Linker; and 5 is H-Antigen reaction solution.

[0064] Example 2 Preparation of A antigen-N3 by one-pot multi-enzyme method

[0065] 60mM / L Lac-Linker, 60mM / L GDP-Fuc, 60mM / L UDP-GalNAc and 0.05g / mL BgtA and 0.08g / mL α-1,2-FucT were reacted at pH=8 and 27℃ for 6h. The metal ion was 10mM / L Mn 2+ Before adding the enzyme, add 3.5mL 1M / L NaOH to adjust the pH to a weak alkaline state, and add 100mM / L Tris-HCl with a pH of 8.5 to stabilize the pH of the reaction system. After 0.5h of reaction, add 1mL 1M / L NaOH to keep the pH at 8.5. After 1h of reaction, add 1mL 1M / L NaOH again to keep the pH at 8.5. The reaction conversion rate can reach 100%. The reaction TLC results are as follows: Figure 3 As shown, the developing agent is ethyl acetate:methanol:water=4:2:1, and the staining agent is anisaldehyde. Figure 3 In the figure, 1 is UDP-GalNAc; 2 is a mixture of H-Antigen and UDP-GalNAc; 3 is an A-Antigen reaction solution.

[0066] Example 3 Preparation of B antigen-N3 by one-pot multi-enzyme method

[0067] 80mM / L Lac-Linker, 80mM / L GDP-Fuc, 80mM / L UDP-Gal, 0.1g / mL α-1,2-FucT and 0.1g / mL GTB crude enzyme were reacted at pH=8, 37℃ for 12h, and the metal ion was 10mM / L Mn 2+ Before adding the enzyme, add 3.5mL 1M / L NaOH to adjust the pH to a weak alkaline state, and add 100mM / L Tris-HCl with a pH of 8.0 to stabilize the pH of the reaction system. After 0.5h of reaction, add 1mL 1M / L NaOH to keep the pH at 8.0. After 1h of reaction, add 1mL 1M / L NaOH again to keep the pH at 8.0. The reaction conversion rate can reach 100%. The reaction TLC results are as follows: Figure 4 As shown, the developing agent is ethyl acetate:methanol:water=4:2:1, and the staining agent is anisaldehyde. Figure 3 In the figure, 1 is H-Antigen; 2 is B-Antigen; 3 is the mixture of H-Antigen and UDP-Gal; 4 is the B-Antigen reaction solution.

[0068] Example 4 Reduction of antigens A and B

[0069] 4g of B Antigen linker N3 and 300mL of methanol were mixed and added to a three-necked flask (specification: 500mL). The three-necked flask was sealed with a balloon and a rubber stopper, and filled with N2 to replace O2. Subsequently, 200mg of Pd / C was added, and then N2 was replaced with H2. The mixture was stirred at room temperature for 24h to obtain B Antigen linker NH2. The reaction conversion rate under this condition could reach 100% as detected by TLC. The reaction TLC results are shown in Figure 1. Figure 5 As shown, the developing agent is ethyl acetate: methanol: water: acetic acid = 33:33:17:1, and the staining agent is phosphomolybdic acid. Figure 6 In the figure, 1 is B Antigen Linker N3; 2 is a mixture of B Antigen Linker N3 and B Antigen Linker NH2; and 3 is a reaction solution of B Antigen Linker NH2.

[0070] Example 5 Purification

[0071] Purification of novel antigen A: The reversed-phase silica gel packing was self-filled, and 10 g of the sample was loaded on 1 kg of packing at a time. The crude A Antigen linker NH2 was eluted with pure water for 4 column volumes and then with 10% methanol in water for 24 column volumes. The eluate from the 7th to 24th column volumes was collected to obtain A Antigen linker NH2 with a purity of >99% and a yield of more than 90%. The HPLC results are as follows: Figure 6 shown.

[0072] Purification of novel antigen B: The reversed-phase silica gel packing was self-filled, and 10 g of the sample was loaded on 1 kg of packing at a time. The crude B Antigen linker NH2 was eluted with pure water for 4 column volumes and then with 10% methanol in water for 24 column volumes. The eluate from the 10th to 20th column volumes was collected to obtain B Antigen linker NH2 with a purity of >99% and a yield of more than 90%. The HPLC results are as follows: Figure 7 shown.

[0073] Mass spectrometry was used for qualitative analysis and HNMR structural analysis was used to verify the purified novel antigens A and B. The mass spectra were as follows: Figure 8 , 9 shown.

[0074] Example 6 One-pot multi-enzyme method for preparing kilogram-level A, B Antigen linker N3 antigen

[0075] In a 20L enzyme reactor, 60mM / L Lac-Linker, 60mM / L GDP-Fuc, 60mM / L UDP-GalNAc, 0.05g / mL BgtA and 0.08g / mL α-1,2-FucT were added, and the metal ion was 10mM / L Mn 2+ The reaction pH was controlled at 8±0.5 by the enzyme reactor automatic flow addition and monitoring device, and the reaction time was 10 hours, with a conversion rate of 100%. The enzyme reactor achieved a single preparation of 945g of A antigen linker N3 in a one-pot multi-enzyme system, which can be further scaled up to industrial production.

[0076] In a 20L enzyme reactor, 80mM / L Lac-Linker, 80mM / L GDP-Fuc, 80mM / L UDP-Gal, 0.1g / mL α-1,2-FucT, and 0.1g / mL GTB crude enzyme were added, and the metal ion concentration was 10mM / L Mn 2+The reaction pH was controlled at 7.5±0.5 by the enzyme reactor automatic flow addition and monitoring device, and the reaction time was 16 hours, with a conversion rate of 100%. The enzyme reactor achieved a single preparation of 897g of B antigen linker N3 in a one-pot multi-enzyme system, which can be further scaled up to industrial production.

[0077] The A and B Antigen linker N3 antigens prepared above were scaled up according to the feed ratios of Examples 4 and 5 to obtain the reduced and purified novel blood group antigens A and B, respectively. The final yields were greater than 80% and the purities were greater than >99%.

[0078] Experimental Example 7 Verification of the performance of new blood group antigens A and B

[0079] The purified novel blood group antigens A and B (three parallel samples each) obtained in the above examples were tested according to the evaluation method given in Chinese patent document CN106802351B. Purified Roman snail HP lectin (L11271, Alexa Fluor) and GSI-B4 lectin (Shanghai Maokang) were used for evaluation, and the ability of IVIG additives to remove anti-A IgG and anti-B IgG antibodies was compared.

[0080] The blank group was used as a control, as shown in Table 1 and Table 2, respectively.

[0081] Table 1:

[0082]

[0083] Table 2:

[0084]

[0085] It is not difficult to see from Table 1-2 that the novel blood type antibodies A and B provided by the present invention have significant binding abilities to HP agglutinin and GSI-B4 agglutinin, respectively, and can significantly remove anti-A and anti-B antibodies.

[0086] The above-mentioned method for synthesizing the novel blood group antigens A and B is described in detail with reference to the specific embodiments, but the description of the implementation example is illustrative rather than restrictive, and several embodiments can be listed according to the limited scope. Therefore, changes and optimizations without departing from the essence and scope of the technology of the present invention should fall within the scope of protection of the present invention.

Claims

1. A novel blood group antigen, characterized in that: It has the following structural formula: ； Wherein, R is OH or NHAc; The novel blood group antigen is used to remove anti-A IgG, anti-B IgG antibodies, and / or to bind HP lectin and GSI-B4 lectin; The enzymatic synthesis method of the novel blood group antigen comprises the following steps: S1. linking the azide-modified disaccharide substrate to fucose via an α-1,2 glycosidic bond under the action of fucosyltransferase; S2. The products are used as substrates and converted under the action of transferases; S3. Reducing the azide-modified end to an amino group to obtain a novel blood group antigen; When R in the blood group antigen is OH, the transferase is galactosyltransferase; when R is NHAc, the transferase is acetylgalactosamine transferase; The application of the enzymatic synthesis method in industrial scale-up production of novel blood group antigens can reach kilogram level, with a yield greater than 80% and a purity greater than 99%.

2. The novel blood group antigen according to claim 1, characterized in that: The azide-modified disaccharide substrate is Laclinker N3, which has the structural formula of Formula I: .

3. The novel blood group antigen according to claim 2, characterized in that: In the step S1, Lac-linker N3 reacts in the presence of GDP-Fuc and α-1,2-FucT.

4. The novel blood group antigen according to claim 1, characterized in that: In step S2, Mn is used under the action of transferase. 2+ As a catalyst.

5. The novel blood group antigen according to claim 1, characterized in that: In step S3, the antigen is obtained by catalytic reduction under the action of hydrogen.

6. The novel blood group antigen according to claim 1, characterized in that: It also includes the purification of blood group antigens: using solid phase extraction C18 and eluting with methanol aqueous solutions of different concentrations from low to high.

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