Preparation method of proteoglycan protein conjugate carrying specified connection site

Abstract

The invention discloses a preparation method of a proteoglycan protein conjugate carrying a specified connection site. The method comprises the following steps: carrying out recombinant expression on a recombinant tetanus toxin heavy chain C fragment rTTHc containing a Sortase A 7M ligase recognition sequence, introducing O, O '-(ethane-1, 2-diyl) bis (hydroxylamine) as a nucleophilic reagent into the C tail end of the rTTHc under the action of ligase Sortase A 7M, and carrying out specific condensation reaction on the C tail end of the rTTHc as a unique chemical reaction site and polysaccharide carrying an aldehyde group functional group to obtain the tetanus toxin heavy chain C fragment rTTHc. Therefore, polysaccharide protein conjugates with definite polysaccharide and protein connection sites are produced. According to the invention, the problem that the key antigen epitope of the carrier protein and the heterogeneity of the product are possibly damaged by the traditional random modification strategy is solved, the antigen epitope of the carrier protein is prevented from being damaged to a great extent, and a reliable technical scheme is provided for preparing the polysaccharide conjugate vaccine with high quality and high immunogenicity.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing a polysaccharide-protein conjugate carrying a specified linker site. Background Technology

[0002] In the global public health field, invasive diseases such as bacterial pneumonia and meningitis continue to pose a serious threat to human health. Streptococcus pneumoniae, in particular, is a major pathogen causing pneumonia, sepsis, and meningitis in infants and young children. These pathogens possess abundant capsular polysaccharides on their surface, which are responsible for mediating receptor binding to host cells for initial adhesion and biological invasion, and are key virulence factors. Based on their unique biological characteristics, capsular polysaccharides have been selected as a core component of vaccines.

[0003] However, polysaccharide vaccines have significant limitations. As T-cell-independent antigens, they cannot effectively activate helper T cells, resulting in weak immunogenicity in infants and young children whose immune systems are not yet fully developed. Furthermore, they cannot induce immune memory or antibody class switching, and their protective effect is short-lived. To overcome these shortcomings, polysaccharide conjugate vaccines have emerged and achieved revolutionary success. The core design of this type of vaccine lies in the covalent linking of bacterial capsular polysaccharides to a highly immunogenic protein carrier using chemical conjugation technology. This design transforms the polysaccharide from a T-cell-independent antigen into a T-cell-dependent antigen. When B cells recognize the polysaccharide portion through their receptors, the carrier protein can be processed by antigen-presenting cells and presented as a T-cell epitope, thereby activating helper T cells. The activated T cells then provide a strong activation signal to B cells, ultimately producing high-quality, high-affinity antibodies, durable immune memory, and a significant enhanced immune response. This allows polysaccharide conjugate vaccines to provide strong and long-lasting protection for all age groups, including infants and young children.

[0004] Currently, the mainstream method for producing polysaccharide conjugate vaccines mainly relies on random chemical coupling based on the side chains of amino acid residues on the protein surface. This involves first activating the polysaccharide to introduce reactive groups, which then react with the abundant natural amino acid residue side chain groups (such as amino, thiol, and carboxyl groups) on the carrier protein surface, thereby achieving the connection between the polysaccharide and the protein. Existing technology reports a polysaccharide-protein coupling strategy based on reductive amination. First, sodium periodate is used to oxidize the vicinal diol structure on the polysaccharide chain to an aldehyde group, generating randomly distributed aldehyde groups on the sugar ring. Then, a reductive amination reaction occurs with the lysine side chain amino group on the carrier protein surface, thereby achieving the connection between the polysaccharide and the protein. Although this technology has been successfully applied to several marketed vaccines, its inherent random modification characteristics lead to some unavoidable core drawbacks. Specific amino acid sequences on the carrier protein constitute its key T-cell and B-cell antigenic epitopes. Random coupling means that the polysaccharide chain may directly link to or sterically obscure these key epitope regions. Once these epitopes are disrupted, the carrier protein's ability to activate T cells is significantly reduced, thereby directly weakening the core function of the polysaccharide conjugate vaccine in eliciting a highly efficient immune response. Furthermore, while the number and spatial location of lysine residues on the carrier protein surface are fixed, their accessibility varies, and the aldehyde sites of oxidized polysaccharides are also randomly distributed. This results in a highly heterogeneous mixture of linking sites, linker numbers, and molecular weights in the final product. This heterogeneity makes it extremely difficult to control the quality reproducibility between different production batches, posing significant challenges to the validation and standardization of the production process and the quality control of the final product. Moreover, random coupling makes it impossible to accurately determine the linking sites between the polysaccharide and the protein, hindering research on their structure-activity relationship and impeding a deeper understanding of the vaccine's immune mechanism and the rational optimization of subsequent products.

[0005] CN111050794B discloses an improved method for conjugating capsular polysaccharides from Streptococcus pneumoniae to a carrier protein. The polysaccharide-protein conjugate prepared using this method can be included in a multivalent pneumococcal conjugate vaccine. However, this technique involves random conjugation, resulting in poor product uniformity and potentially disrupting underlying antigenic epitopes.

[0006] CN120459289A discloses a polysaccharide-protein conjugate vaccine for swine streptococcal disease based on biotin and streptavidin linkage, its preparation method, and its application. However, this technology is a non-covalent linkage method, and the biotinylation efficiency needs to be controlled to ensure that the number of biotin molecules linked to each polysaccharide molecule is moderate; too many or too few will affect the coupling efficiency and quality uniformity.

[0007] CN120000775A discloses a multivalent pneumococcal polysaccharide-protein conjugate vaccine, its preparation method, and its application, which can simultaneously prevent pneumococcal infection and respiratory syncytial virus infection. This patent application's conjugate vaccine is an innovative vaccine that can simultaneously prevent two common diseases with a single vaccine product. However, the complex chemical coupling reaction conditions used in this patent application are highly susceptible to disrupting the fine conformation of the Pre-F protein, causing it to lose its ability to induce highly effective neutralizing antibodies.

[0008] Current methods for producing polysaccharide conjugate vaccines still have certain limitations. In recent years, with the continuous deepening of research on polysaccharide conjugate vaccines, scientists' demand for polysaccharide-protein conjugation methods has been increasing. This invention, as a site-selective conjugation method, can effectively solve the problem of uncertain polysaccharide-protein conjugation sites in existing polysaccharide-protein coupling methods, thereby greatly avoiding the destruction of carrier protein antigenic epitopes. Summary of the Invention

[0009] To address the above problems, the present invention aims to provide a method for preparing a polysaccharide-protein conjugate carrying a specified linker site.

[0010] To achieve this objective, the specific technical solution of this invention is as follows: In a first aspect, the present invention provides a method for preparing a polysaccharide-protein conjugate carrying a specified linker site, wherein the polysaccharide-protein conjugate comprises a covalently bonded hydroxylamined carrier protein (rTTHc-ONH2) and oxidized dextran (Glucan-CHO), and the amino acid sequence of rTTHc is shown in SEQ ID NO.1.

[0011] rTTHc is the non-toxic functional region of tetanus toxoid, completely eliminating neurotoxicity while retaining key T-cell epitopes, fundamentally eliminating the potential risks associated with incomplete detoxification of traditional tetanus toxoids. This fragment possesses highly efficient antigen presentation and immune activation capabilities. Its structure contains numerous universally recognized T-cell epitopes from tetanus toxoid, enhancing the immune response to bound polysaccharide antigens and inducing high-affinity antibodies and long-lasting immune memory. As a recombinant protein, rTTHc can achieve stable expression with high yield and purity, fundamentally ensuring batch-to-batch consistency of vaccine raw materials. Therefore, polysaccharide-protein conjugates prepared using rTTHc as a carrier exhibit better stability.

[0012] In the development of polysaccharide-protein conjugation methodologies, dextran was chosen as the validation tool due to its superior controllability, analyzability, and cost-effectiveness. Commercially available glutas have a well-defined and uniform molecular weight distribution and a regular glucose repeating unit structure. This structural determinism greatly simplifies variable control in the early stages of method development, allowing any successful conjugation or analytical results to be directly attributed to the method itself, rather than the heterogeneity of the polysaccharide raw material. The glutamate backbone is rich in highly activatable hydroxyl groups, facilitating the introduction of active groups through mature and controllable chemical methods, enabling specific conjugation with amino groups and other groups of the carrier protein. Furthermore, high-purity, standardized glutas are significantly cheaper than capsular polysaccharides extracted and purified from pathogen cultures, and are more readily available. This facilitates extensive reproducible process exploration and condition optimization experiments in the early stages of method development without concern for the consumption of precious samples, thereby accelerating the research and development process and reducing costs.

[0013] In this invention, the carrier protein is not limited to rTTHc. Other carrier proteins that can be used in polysaccharide conjugate vaccines can be used in the polysaccharide-protein conjugation method provided by this invention, such as tetanus toxoid (TT), diphtheria toxoid (DT), non-toxic diphtheria mutant (CRM197), group B meningococcal epimeningoid protein complex (OMP), and Haemophilus protein D, etc.

[0014] In this invention, polysaccharides are not limited to Glucan; other polysaccharides with aldehyde groups or activated polysaccharides with aldehyde groups can be used in the polysaccharide-protein coupling method provided by this invention.

[0015] Secondly, the present invention provides a method for site-specific hydroxylamine modification at the full-length protein level. The hydroxylamined protein is not limited to rTTHc, but can also be any recombinantly expressed protein carrying the Sortase A enzyme recognition sequence LPXTG, where X represents any amino acid, such as green fluorescent protein, antibody, nanobody, cytokine, etc., all of which are applicable to the protein site-specific hydroxylamine modification method provided by the present invention.

[0016] The specific steps are as follows: (1) Expression and hydroxylamineation of recombinant carrier protein: The carrier protein containing the recognition sequence of Sortase A 7M ligase was recombinantly expressed. Under the action of Sortase A 7M ligase, O,O'-(ethane-1,2-diyl)bis(hydroxylamine) dihydrochloride was used as a nucleophile to specifically introduce a hydroxylamine group at the C-terminus of the carrier protein to obtain the hydroxylamined carrier protein. (2) Aldehydeation of polysaccharides: Aldehyde groups are introduced by oxidizing the vicinal diol structure on the polysaccharide chain with sodium periodate to obtain aldehyde-modified polysaccharides; (3) Coupling reaction: The hydroxylamined carrier protein obtained in step (1) and the aldehyde polysaccharide obtained in step (2) are mixed in a buffer solution. Stable oxime bonds are formed through the specific condensation reaction of hydroxylamine groups and aldehyde groups, resulting in a polysaccharide-protein conjugate with a specific linking site, thus realizing the site-specific polysaccharide modification of the carrier protein.

[0017] Further, the amino acid sequence of the Sortase A 7M enzyme in step (1) is shown in SEQ ID NO.2, and the molar ratio of the carrier protein to the Sortase A 7M enzyme is 1:0.1-0.5, preferably 1:0.1.

[0018] Further, the final concentration of O,O'-(ethane-1,2-diyl)bis(hydroxylamine) dihydrochloride in step (1) is 0.08-0.2 M, preferably 0.1 M, and the reaction is carried out in PBS buffer at pH 7.0-7.5 with a concentration of 10-30 mmol / L, a reaction temperature of 37 °C, and a reaction time of 8-12 h.

[0019] Further, the expression of the carrier protein in step (1) includes: culturing the recombinant strain in LB medium containing ampicillin resistance, the composition of which is: 2 g tryptone, 2 g NaCl, 1 g yeast extract, 200 mL deionized water, and ampicillin to a final concentration of 10 mg / mL, the culture temperature is 37 ℃, and the culture time is 12-18 h; induction expression is performed using IPTG at a final concentration of 1 mM, the induction expression temperature is 30 ℃, and the induction expression time is 12-18 h; after induction expression, the protein is purified by ultrasonic disruption and Ni-NTA affinity chromatography.

[0020] Furthermore, the ultrasonic fragmentation method is as follows: insert the probe 1 cm below the liquid surface, start for 2 seconds, stop for 2 seconds, and sonicate for 20-40 minutes.

[0021] Furthermore, in the polysaccharide aldehyde reaction described in step (2), the molar ratio of sodium periodate to polysaccharide is 1:2, the reaction is carried out at room temperature for 20-40 min, and the reaction is terminated by adding glycerol.

[0022] Further, the coupling reaction described in step (3) is carried out in PBS buffer at pH 7.4, with a molar ratio of hydroxylated carrier protein to aldehyde polysaccharide of 1:3-5, a reaction temperature of 37 °C, and a reaction time of 8-12 h.

[0023] In this invention, the nucleophile O,O'-(ethane-1,2-diyl)bis(hydroxylamine) dihydrochloride has the following structure: .

[0024] Compared with the prior art, the present invention has the following beneficial effects: Compared to random conjugation methods, this invention achieves true site-specific modification, maximizing the protection of the antigenic epitopes of the carrier protein and yielding polysaccharide-protein conjugates with clearly defined linker sites. It also offers advantages such as low cost, ease of operation, and mild reaction conditions. The method for preparing polysaccharide-protein conjugates described in this invention can be widely applied to the development of polysaccharide conjugate vaccines and other research on protein chemical modification. Attached Figure Description

[0025] Figure 1 For experimental design diagrams; Figure 2 Results of rTTHc expression of the recombinant tetanus toxin heavy chain C fragment; Figure 3 It is a fluorescent probe structure; Figure 4 To identify the hydroxylation results of rTTHc using fluorescent probes; Figure 5 It has a dextran structure; Figure 6 Schiff reagent was used to detect the aldehyde esterification results of dextran; Figure 7 The results are from a polysaccharide-protein coupling reaction. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments. Although rTTHc is used as a specific embodiment, the scope of protection of the present invention is not limited thereto. Other proteins with similar sequences, including diphtheria toxin mutant (CRM197), tetanus toxoid (TT), diphtheria toxoid (DT), Haemophilus influenzae type b outer membrane protein (OMP), and other polysaccharides with aldehyde groups or activated polysaccharides with aldehyde groups, can be used in the polysaccharide-protein coupling method provided by the present invention.

[0027] The steps of the method of the present invention are as follows Figure 1 As shown, the recombinant tetanus toxin heavy chain C fragment rTTHc containing the Sortase A 7M ligase recognition sequence LPETG was recombinantly expressed. Using O,O'-(ethane-1,2-diyl)bis(hydroxylamine) as a nucleophile, it was introduced into the C-terminus of rTTHc under the action of the ligase Sortase A 7M. This nucleophile served as a unique chemical reaction site for a specific condensation reaction with a polysaccharide carrying an aldehyde functional group, thereby generating a polysaccharide-protein conjugate with a clearly defined polysaccharide-protein linkage site.

[0028] Example 1: Expression of rTTHc 1) Pick single colonies of the recombinant tetanus toxin heavy chain C fragment rTTHc-LPETGG-His6, whose amino acid sequence is shown in SEQ ID NO.1, and incubate them in 10 mL of LB medium containing ampicillin resistance at 37 ℃ and 220 rpm for 12 h. The formulation of the LB medium containing ampicillin resistance is as follows: 2 g tryptone, 2 g NaCl, 1 g yeast extract, 200 mL deionized water, and 200 μL 50 mg / mL ampicillin (Amp). 2) Take the bacterial culture from step 1) and scale it up to 1 L of LB medium containing ampicillin resistance at a volume ratio of 1:100. Continue to culture at 37 ℃ and 220 rpm. When the OD 600 absorbance of the bacterial culture reaches 0.8, add 400 μL of 1.0 M IPTG to induce the bacterial culture. Continue to culture at 30 ℃ and 200 rpm for 18 h. 3) Centrifuge to collect the bacterial culture from step 2) (4 ℃, 8000 rpm, 10 min), discard the supernatant, and then fully resuspend the obtained bacterial cells in lysis buffer (50 mM Tris・HCl, 150 mM NaCl, pH=7.4) (resuspend the bacterial cells obtained from 1 L LB medium in 20 mL of lysis buffer), and use an ultrasonic disruptor to lyse the bacterial cells (insert the probe of the ultrasonic disruptor 1 cm below the liquid surface, start for 2 s, stop for 2 s, and sonicate for 20 min). 4) Collect the supernatant using a centrifuge (4 ℃, 8000 rpm, 1 h); 5) Proteins were purified from the collected supernatant using Ni-NTA Beads via gravity column chromatography. The protein eluent was concentrated using ultrafiltration. The expression results of the recombinant tetanus toxin heavy chain C fragment rTTHc are as follows: Figure 2 As shown, the protein purity is above 90%, and the theoretical molecular weight of rTTHc is 54 kDa.

[0029] Example 2 rTTHc hydroxylation and product purification 1) Add the protein stock solution obtained in step 5) to a buffer solution (PBS, pH = 7.4) to a final concentration of 100 μM. Add 0.1 eq (molar equivalent relative to the protein substrate) of Sortase A7M with the sequence shown in SEQ ID NO.2. Add O,O'-(ethane-1,2-diyl)bis(hydroxylamine) dihydrochloride compound to a final concentration of 0.1 M. Incubate at 37 °C for 12 h. 2) The reaction solution was purified by gravity column method using Ni-NTA Beads. The eluent was collected, and small molecules were removed and the protein solution was concentrated using ultrafiltration tube.

[0030] Example 3: Identification of rTTHc hydroxylation using fluorescent probes To verify the rTTHc hydroxylation reaction, a previously validated peptide probe was synthesized using solid-phase synthesis. The probe's sequence is Thz-GK(FITC)G, where the K side chain carries the fluorescein FITC, and the N-terminus contains a Thz group. Its structure is as follows: Figure 3 As shown. The principle is that Thz undergoes a ring-opening reaction under copper ion catalysis, re-exposing the aldehyde group, which can then react specifically with the hydroxylamine group to generate a stable linker product. Therefore, by detecting changes in the fluorescence signal of the modified product, it can be determined whether the above protein has undergone hydroxylamine modification.

[0031] For the product rTTHc-ONH2, different experimental and control groups were designed according to Table 1, with groups 1 and 3 being replicate experiments. After preparation, the mixture was incubated at 37 ℃ for 12 h. After the reaction, 10 μL of the reaction solution was taken and subjected to SDS-PAGE electrophoresis using a 10% protein gel. The experimental results are shown below. Figure 4 As shown, images 5, 6, 7, and 8 are the fluorescence images of images 1, 2, 3, and 4, respectively. Only experimental groups 1 and 3 show a clear band under fluorescence conditions because Thz undergoes a ring-opening reaction under copper ion catalysis, re-exposing the aldehyde group. This necessitates a specific reaction with the hydroxylamine group to generate a stable linker product. This indicates that the probe successfully labeled rTTHc containing the hydroxylamine group, thus confirming the successful hydroxylation of rTTHc.

[0032] Table 1. Identification of rTTHc hydroxylation by fluorescent probes. .

[0033] Example 4: Sodium periodate oxidized polysaccharide Structure as Figure 5 The 5 kDa, 10 kDa, and 20 kDa dextran shown were dissolved in PBS buffer at pH 7.4, and sodium periodate was added to achieve a sodium periodate:dextran molar ratio of 1:2. After gentle stirring at room temperature for 40 min, glycerol was added to bring the final concentration to 2.5%, and the reaction was terminated by gentle stirring for 15 min. The reaction system was dialyzed to remove small molecules and then lyophilized. Groups 1 and 5 served as the Schiff reagent control group; groups 2-4 were the Schiff reagent experimental groups with oxidized 5 kDa, 10 kDa, and 20 kDa dextran, respectively; and groups 6-8 were the Schiff reagent experimental groups with unoxidized 5 kDa, 10 kDa, and 20 kDa dextran. The experimental results are shown below. Figure 6 As shown, only experimental groups 2, 3, and 4 changed color, proving that the dextran oxidized by sodium periodate contains aldehyde groups.

[0034] Example 5 Polysaccharide-protein coupling reaction The rTTHc-ONH2 obtained in Example 2 and the aldehyde polysaccharide obtained in Example 4 were dissolved in PBS at pH 7.4. Different experimental and control groups were designed according to Table 2. After preparation, the mixture was incubated at 37 ℃ for 12 h. After the reaction, 20 μL of the reaction solution was taken and subjected to SDS-PAGE electrophoresis using a 10% protein gel. The experimental results are shown below. Figure 7 As shown, diffuse polysaccharide-protein conjugate bands appeared only in lanes 1, 3, and 5 of the experimental groups, and the degree of diffusion varied depending on the size of the polysaccharide, thus confirming the successful conjugation of rTTHc-ONH2 with the polysaccharide. Table 2 Polysaccharide-protein conjugation reaction system .

Claims

1. A process for the preparation of a polysaccharide protein conjugate carrying specified attachment sites, characterized in that, The method comprises the following steps: (1) Expression and hydroxylamine modification of recombinant carrier protein: by recombinantly expressing a carrier protein containing a Sortase A 7M ligase recognition sequence, under the action of the Sortase A 7M ligase, and using O, O'- (ethane-1, 2-diyl) bis (hydroxylamine) dihydrochloride as a nucleophile, a hydroxylamine group is specifically introduced at the C-terminal end of the carrier protein, thereby obtaining a hydroxylamine-modified carrier protein; (2) Polysaccharide aldehyde modification: by oxidizing the vicinal diol structure on the polysaccharide chain with sodium periodate, an aldehyde group is introduced, thereby obtaining an aldehyde-modified polysaccharide; (3) Coupling reaction: the hydroxylamine-modified carrier protein obtained in step (1) is mixed with the aldehyde-modified polysaccharide obtained in step (2) in a buffer, and a stable oxime bond is formed through specific condensation reaction between the hydroxylamine group and the aldehyde group, thereby obtaining a polysaccharide-protein conjugate with specific coupling sites, and realizing site-specific polysaccharide modification of the carrier protein.

2. The production method according to claim 1, wherein The carrier protein in step (1) is selected from tetanus toxoid, diphtheria toxoid, nontoxic diphtheria mutant CRM197, group B meningococcal outer membrane protein complex or hemophilus protein D, and the polysaccharide in step (2) is selected from polysaccharides capable of introducing an aldehyde group by oxidation.

3. The production method according to claim 1, wherein The carrier protein in step (1) is recombinant tetanus toxin heavy chain C fragment rTTHc-LPETGG-His6, and the amino acid sequence is shown as SEQ ID NO. 1, and the polysaccharide in step (2) is dextran.

4. The production method according to claim 1, wherein The amino acid sequence of the Sortase A 7M ligase in step (1) is shown as SEQ ID NO. 2, and the molar concentration ratio of the carrier protein to the Sortase A 7M ligase is 1:0.1-0.

5.

5. The production method according to claim 1, wherein The reaction final concentration of O, O'- (ethane-1, 2-diyl) bis (hydroxylamine) dihydrochloride in step (1) is 0.08-0.2 M, and the reaction is carried out in PBS buffer with pH 7.0-7.5, the reaction temperature is 37 ℃, and the reaction time is 8-12 h.

6. The production method according to claim 1, wherein The expression of the carrier protein in step (1) comprises: culturing the recombinant strain in LB medium containing ampicillin resistance, purifying the protein through ultrasonic disruption and Ni-NTA affinity chromatography after induction of expression.

7. The production method according to claim 1, wherein The composition of the LB medium is tryptone 2 g, NaCl 2 g, yeast powder 1 g, deionized water 200 mL, and ampicillin to a final concentration of 10 mg / mL; the culture temperature is 37 ℃, the culture time is 12-18 h, IPTG is used for induction of expression, the final concentration is 1 mM, the induction of expression temperature is 30 ℃, and the induction of expression time is 12-18 h.

8. The production method according to claim 1, wherein The site-specific hydroxylamine modification in step (1) is applicable to other proteins that can be recombinantly expressed and carry the Sortase A enzyme recognition sequence LPXTG, wherein X represents any amino acid, including green fluorescent protein, antibody, nanobody or cytokine.

9. The production method according to claim 1, wherein In the polysaccharide aldehyde modification reaction in step (2), the molar concentration ratio of sodium periodate to polysaccharide is 1:2, the reaction is carried out at room temperature, and the reaction is terminated by adding glycerol.

10. The production method according to claim 1, wherein The coupling reaction in step (3) is carried out in PBS buffer at pH 7.4, and the molar concentration ratio of the hydroxylamine carrier protein to the aldehyde-based polysaccharide is 1:3-5, the reaction temperature is 37 ℃, and the reaction time is 8-12 h.

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