Fatty acid-modified diphtheria toxin antigen and use thereof

Abstract

The present invention relates to the field of biomedicine and specifically to a fatty acid-modified diphtheria toxin antigen and a use thereof. In the diphtheria toxin antigen containing fatty acid modification, the fatty acid modification comprises a fatty acid-modified N-terminal cysteine (Cys), and the fatty acid-modified N-terminal Cys is Cys in which the sulfhydryl group is modified by diacylglycerol and / or the alpha-amino group is modified by a fatty acyl group. Compared with existing diphtheria toxin protein antigens, the fatty acid-modified diphtheria toxin antigen of the present invention not only achieves faster conventional intramuscular injection induction and a higher immune protection effect than the existing diphtheria toxin antigens, but can also induce respiratory mucosal immunity, providing a basis for a vaccine for immune prevention to block diphtheria infection and diffusion.

Description

A fatty acid-modified diphtheria toxin antigen and its application Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a fatty acid-modified diphtheria toxin antigen and its application. Background Technology

[0002] Vaccines are one of the most important inventions in the treatment of infectious diseases, significantly reducing morbidity and mortality. In many countries and regions, vaccines have reduced the mortality rate of infectious diseases by 99%, such as smallpox, polio, diphtheria, and measles. Currently available vaccines mainly include live attenuated vaccines, inactivated vaccines, and subunit vaccines. Due to the numerous safety concerns and ineffective immune responses associated with whole-pathogen-based live attenuated and inactivated vaccines, and with in-depth research into the life cycle, genetic characteristics, and pathogenic mechanisms of pathogens, the selection and design of vaccine antigens have shifted from whole-pathogen antigens to one or several clearly defined protective components, ushering in the era of subunit vaccines. Subunit vaccines offer excellent safety, but their immunogenicity is relatively weak, and the antigens are easily cleared. Therefore, selecting appropriate antigens and antigen design schemes, immunostimulants, and antigen delivery systems are key factors for the success of subunit vaccines.

[0003] Bacterial lipoproteins are a class of fatty acid-modified proteins that are bound to the membrane via fatty acid chains. In vivo synthesis of bacterial lipoproteins is guided by a signal peptide. The bacterial lipidation signal peptide consists of three regions: an N-terminal positively charged region, a mid-terminal hydrophobic region, and a C-terminal conserved lipidation sequence [LVI][ASTVI][GAS][C]. The C-terminal cysteine ​​residue is the lipidation modification site. Guided by the signal peptide, the target protein is secreted to the extracellular space via the Sec or TAT secretion pathway. Lgt enzymes use phosphatidylglycerol (PG) as a substrate to link diacylglycerol to the sulfhydryl group of cysteine ​​(Cys). LspA enzymes cleave the signal peptide except for the C-terminal Cys residue. Lnt enzymes can use phosphatidylethanolamine (PE) as a substrate to transfer another fatty acid chain to the amino group of Cys.

[0004] Diphtheria toxin is an exotoxin produced by Corynebacterium diphtheriae. It possesses high biological activity and toxicity. The toxin binds to ribosomes within host cells, inhibiting ribosome function and thus interfering with protein synthesis. This inhibitory effect significantly impacts cell growth, division, and repair, making it a crucial pathogenic factor in diphtheria. CRM197 is a non-toxic variant of diphtheria toxin isolated from Corynebacterium diphtheriae C7 (β197) cultures. CRM197 differs from wild-type diphtheria toxin in that the 52nd amino acid is mutated from glycine to glutamic acid, eliminating enzymatic activity and toxicity, and providing more lysine side chains for polysaccharide binding. CRM197 is a single-chain polypeptide composed of 535 residues with a relative molecular mass of 58,000 Da. Its protein crystal structure can be divided into three independent domains: the catalytic domain (C-domain), the transmembrane domain (T-domain), and the receptor-binding domain (R-domain). The C-domain possesses adenosine diphosphate (ADP) ribosyltransferase activity, making it a crucial catalyst for protein synthesis in eukaryotic cells. The T-domain comprises two hydrophilic A-helical structures and one hydrophobic A-helical structure, with Asp and Glu as the connecting regions between the helical layers. Under neutral conditions, the T-domain is hydrophilic; under acidic conditions, some polar groups in the T-domain protonate, allowing the nonpolar hydrophobic layer to insert into the membrane and further assisting the C-domain in translocation to the cytoplasm. The R-domain is used to bind cell receptors. These three domains do not overlap in their primary structure and are independent of each other in their tertiary structure. Summary of the Invention

[0005] In view of the technical problems existing in the prior art, the present invention provides a fatty acid-modified diphtheria toxin antigen and its application, particularly for the preparation of diphtheria toxin vaccines, especially for respiratory immunization vaccines.

[0006] According to a first aspect of the technical solution of the present invention, a diphtheria toxin antigen modified with fatty acid is provided.

[0007] Furthermore, the fatty acid modification includes fatty acid-modified N-terminal cysteine ​​(Cys).

[0008] Furthermore, the fatty acid modification is that the N-terminal cysteine ​​Cys thiol group is modified with diacylglycerol containing fatty acid chain residues and / or the fatty acid chain is acylated.

[0009] Preferably, the fatty acid chain is a long-chain fatty acid containing C16-C18 carbon atoms.

[0010] Preferably, the fatty acid-modified diphtheria toxin antigen is a non-toxic mutant CRM197.

[0011] More preferably, the fatty acid-modified diphtheria toxin antigen includes a variant that retains the same function as diphtheria toxin. The fatty acid-modified diphtheria toxin antigen comprises the 33-566 amino acid sequence shown in SEQ ID NO. 1.

[0012] Furthermore, the fatty acid-modified diphtheria toxin antigen is a polymer.

[0013] According to a second aspect of the present invention, a method for preparing the above-mentioned fatty acid-modified diphtheria toxin antigen is provided. The method includes expressing the diphtheria toxin protein gene containing a fatty acid-modified signal peptide in *Escherichia coli*, and purifying and preparing the fatty acid-modified diphtheria toxin protein. The signal peptide contains three regions: an N-terminal positively charged region, a mid-segment hydrophobic region, and a C-terminal conserved lipidation sequence [LVI][ASTVI][GAS][C]. Preferably, the signal peptide is the amino acid sequence of SEQ ID NO. 3. Further, the C-terminal cysteine ​​residue is the fatty acid modification site.

[0014] According to a third aspect of the present invention, the application of the fatty acid-modified diphtheria toxin antigen described above in the preparation of a product capable of inducing antigen-specific antibodies in animals is provided. The immune composition can be administered via intramuscular or subcutaneous injection, nasal drops, or nebulized immunization. Preferably, the product is a vaccine.

[0015] According to a fourth aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising the above-mentioned fatty acid-modified diphtheria toxin antigen.

[0016] According to a fifth aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising a nucleic acid encoding the aforementioned fatty acid-modified diphtheria toxin antigen.

[0017] According to a sixth aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising a carrier encoding the above-mentioned fatty acid-modified diphtheria toxin antigen.

[0018] According to a seventh aspect of the present invention, a biomaterial is provided, wherein the biomaterial is any one of the following:

[0019] C1) Encoding the nucleic acid molecule of the fatty acid-modified diphtheria toxin antigen as described in any one of claims 1-8;

[0020] C2) An expression cassette containing the nucleic acid molecule described in C1);

[0021] C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2);

[0022] C4) Recombinant microorganisms containing the nucleic acid molecules described in C1), or recombinant microorganisms containing the expression cassette described in C2), or recombinant microorganisms containing the recombinant vector described in C3);

[0023] C5) Recombinant cells containing the nucleic acid molecule described in C1), or recombinant cells containing the expression cassette described in C2), or recombinant cells containing the recombinant vector described in C3).

[0024] According to the eighth aspect of the technical solution of the present invention, a method for preparing a vaccine using the above-mentioned fatty acid modification of diphtheria toxin antigen is provided.

[0025] According to the ninth aspect of the technical solution of the present invention, a method for preparing a vaccine using the above-mentioned biological materials is provided.

[0026] Compared with the prior art, the present invention has at least the following beneficial effects:

[0027] 1. This invention provides a fatty acid-modified diphtheria toxin antigen, which can be polymerized into nanoparticles, significantly improving its immunogenicity. When used as the main component of an intramuscularly injected vaccine, it significantly improves the mucosal immune effect and can induce specific IgG antibodies, showing great promise for practical applications.

[0028] 2. The fatty acid-modified diphtheria toxin antigen disclosed in this invention, when administered intramuscularly to animals, produces high titers of IgG-binding antibodies after two immunizations; when administered respiratoryly to animals, diphtheria toxin-specific IgG-binding antibodies can be detected after three immunizations.

[0029] 3. Compared with existing diphtheria toxin protein antigens, this antigen not only has a faster and stronger immune protection effect induced by routine intramuscular injection, but also induces respiratory mucosal immunity, providing a basis for comprehensive immune protection to block the infection and spread of diphtheria toxin at the site of infection. Attached Figure Description

[0030] Figure 1 shows the expression results of the supernatant and precipitated protein after ultrasonic lysis of the fatty acid-modified CRM197 positive clone according to the present invention.

[0031] Figure 2 shows the SDS-PAGE and Western Blot images of the CRM197 purified sample modified with fatty acid according to the present invention.

[0032] Figures 3A, 3B, and 3C show the antibody detection in mice after immunization according to the present invention. Figure 3A shows the antibody titer against CRM197 protein in mouse serum two weeks after the first immunization, Figure 3B shows the antibody titer two weeks after the second immunization, and Figure 3C shows the antibody titer two weeks after the third immunization.

[0033] Figures 4A and 4B are particle size diagrams of the fatty acid-modified CRM197 protein according to the present invention.

[0034] Figure 5 shows the dynamic light scattering pattern of the fatty acid-modified CRM197 protein according to the present invention.

[0035] Figure 6 is a transmission electron microscope image of the fatty acid-modified CRM197 protein according to the present invention.

[0036] In the graphs of the quantitative results, **** indicates P < 0.0001. Detailed Implementation

[0037] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0038] This invention specifically relates to a fatty acid-modified diphtheria toxin antigen and its applications, belonging to the field of biomedicine. The fatty acid-modified diphtheria toxin antigen disclosed in this invention, administered intramuscularly to animals, produces high titers of IgG-binding antibodies after two immunizations; in animals immunized via the respiratory tract, diphtheria toxin-specific IgG-binding antibodies are detectable after three immunizations. Compared with existing diphtheria toxin protein antigens, the fatty acid-modified diphtheria toxin antigen of this invention not only provides faster and higher immune protection induced by conventional intramuscular injection, but also induces respiratory mucosal immunity, providing a foundation for comprehensive immune protection to block diphtheria toxin infection and spread at the infection site.

[0039] This invention provides a diphtheria toxin antigen containing fatty acid modification. Its innovation lies in the fatty acid modification of the diphtheria toxin antigen, which is a non-toxic mutant CRM197. Further, the diphtheria toxin antigen comprises an amino acid sequence of 33-566 as shown in SEQ ID NO.1, and a variant retaining the same function as diphtheria toxin, preferably a variant with greater than 90% homology. In one embodiment, the fatty acid-modified diphtheria toxin antigen contains an N-terminal cysteine ​​residue (Cys) modified with fatty acid, wherein the thiol group of Cys is modified with diacylglycerol and / or the alpha amino group is modified with an acyl group. Alternatively, the fatty acid modification involves modifying the N-terminal cysteine ​​Cys thiol group with diacylglycerol containing fatty acid chain residues and / or acylation of the fatty acid chain. The fatty acid chain is a long-chain fatty acid containing C16-C18 carbon atoms. The diphtheria toxin described in this invention is an exotoxin produced by Corynebacterium diphtheriae. It binds to ribosomes within host cells, inhibiting ribosome function and thus interfering with protein synthesis. This inhibitory effect significantly impacts cell growth, division, and repair, making it a crucial pathogenic factor in diphtheria. CRM197 is a non-toxic variant of diphtheria toxin isolated from Corynebacterium diphtheriae C7 (β197) cultures. CRM197 differs from wild-type diphtheria toxin in that the 52nd amino acid is mutated from glycine to glutamic acid. CRM197 is a single-chain polypeptide composed of 535 residues with a relative molecular mass of 58,000 Da. Its protein crystal structure can be divided into three independent domains: the catalytic domain (C-domain), the transmembrane domain (T-domain), and the receptor-binding domain (R-domain).

[0040] In one embodiment, the present invention provides a diphtheria toxin antigen containing fatty acid modification, wherein the fatty acid modification comprises a fatty acid-modified N-terminal cysteine ​​(Cys); the fatty acid modification is wherein the thiol group of the N-terminal cysteine ​​Cys is modified with a diacylglycerol containing fatty acid chain residues and / or acylated with the fatty acid chain. The fatty acid chain is a long-chain fatty acid containing C16-C18 carbon atoms. Preferably, the fatty acid-modified diphtheria toxin antigen is a non-toxic mutant CRM197. The fatty acid-modified diphtheria toxin antigen comprises the amino acid sequence 33-566 as shown in SEQ ID NO.1, and a variant retaining the same function as diphtheria toxin. More preferably, the fatty acid-modified diphtheria toxin antigen is a polymer.

[0041] Furthermore, a method for preparing the above-mentioned fatty acid-modified diphtheria toxin antigen is provided, the method comprising expressing the diphtheria toxin protein gene containing the fatty acid-modified signal peptide in *Escherichia coli*, and purifying and preparing the fatty acid-modified diphtheria toxin protein. In the method for preparing the above-mentioned fatty acid-modified diphtheria toxin antigen, the signal peptide contains three regions: an N-terminal positively charged region, a mid-segment hydrophobic region, and a C-terminal conserved lipidation sequence [LVI][ASTVI][GAS][C], wherein the C-terminal cysteine ​​residue is the lipidation modification site; the signal peptide is the amino acid sequence of SEQ ID NO.3.

[0042] Furthermore, the invention provides the use of the above-described fatty acid-modified diphtheria toxin in the preparation of a product capable of inducing antigen-specific antibodies in animals, wherein the immunizing composition can be administered via intramuscular or subcutaneous injection, nasal drops, or nebulized immunization. It also provides a method for preparing a vaccine using the above-described fatty acid-modified diphtheria toxin.

[0043] In another embodiment, the fatty acid-modified diphtheria toxin antigen is a polymer, preferably a particle larger than 10 nm. Compared to non-fatty acid-modified antigens, the fatty acid-modified diphtheria toxin antigen of this invention has the characteristics of increased molecular weight, expression on the cell membrane, and solubility in solutions containing detergents. Further, the fatty acid modification involves modifying the N-terminal cysteine ​​(Cys) thiol group with a diacylglycerol containing fatty acid chain residues, and / or further modifying the amino group with fatty acid chain acylation. The fatty acid chain is a long-chain fatty acid containing C16-C18 carbon atoms. The fatty acid-modified diphtheria toxin antigen can form polymers, preferably particles larger than 10 nm.

[0044] In one embodiment, a method for preparing the above-mentioned fatty acid-modified diphtheria toxin antigen is provided, comprising expressing the diphtheria toxin protein gene containing a fatty acid-modified signal peptide in *Escherichia coli* (the amino acid sequence encoded by the gene is shown in SEQ ID NO. 1), and purifying to prepare the fatty acid-modified diphtheria toxin protein. The signal peptide contains three regions: an N-terminal positively charged region, a mid-segment hydrophobic region, and a C-terminal conserved lipidation sequence [LVI][ASTVI][GAS][C], wherein the C-terminal cysteine ​​residue is the lipidation modification site. Preferably, the signal peptide is the amino acid sequence of SEQ ID NO. 3. Further, the protein may be a protein containing various diphtheria toxins and / or diphtheria toxoid antigens and fragments thereof. The bacteria are preferably *Escherichia coli* (expressing the diphtheria toxin protein gene containing the fatty acid-modified signal peptide, the amino acid sequence encoded by the gene is SEQ ID NO. 1). The bacterial fatty acid-modified signal peptide (the diphtheria toxin protein gene containing the fatty acid-modified signal peptide is SEQ ID NO.2) consists of three regions: an N-terminal positively charged region, a mid-segment hydrophobic region, and a C-terminal conserved lipid-modified sequence [LVI][ASTVI][GAS][C]. The C-terminal cysteine ​​residue is the lipid modification site, and amino acid sequences 1-21 of SEQ ID NO.3 constitute the signal peptide sequence. Purification of the fatty acid-modified protein can include various techniques such as bacterial cell isolation, disruption, dissolution, and purification.

[0045] In another embodiment, the present invention also provides the application of the above-mentioned fatty acid-modified diphtheria toxin in the preparation of products capable of inducing antigen-specific antibodies in animals, wherein the immune composition can be administered via nasal drops or nebulized immunization. The present invention also provides a method for preparing a vaccine using the above-mentioned fatty acid-modified diphtheria toxin. The present invention further includes expression vectors, bacterial strains, and fatty acid-modified proteins prepared according to the above-mentioned invention. The present invention also includes a method for preparing and preventing diphtheria toxin-induced diseases using the above-mentioned fatty acid-modified diphtheria toxin antigen, including the preparation of various single- or multi-dose vaccines, and the preparation of various vaccines by mixing with aluminum adjuvants, MF59, CPG, etc. The vaccines prepared by the present invention can be injected, including intramuscular injection, subcutaneous injection, etc.; they can also be administered via inhalation or other methods for respiratory mucosal immunization.

[0046] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the present invention in any way. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments are commercially available.

[0047] In the following embodiments, the diphtheria toxin antigen is specifically a fatty acid-modified diphtheria toxin, namely fatty acid-modified CRM197, which is recorded in the non-patent literature "NCBI: toxin CRM197 [synthetic construct]". The public may obtain this biological material from the applicant in accordance with relevant national biosafety regulations. This biological material is only for repeating the relevant experiments of this invention and may not be used for any other purpose.

[0048] In the following examples, the carrier PET30(a) is a product of Invitrogen.

[0049] In the following examples, nickel ion affinity chromatography (Chelating Fast Flow column) and gel size exclusion chromatography (Superdex-G200) were both products of GE Healthcare.

[0050] In the following examples, the goat anti-rabbit IgG secondary antibody (SAB3700885) was a product of Sigma.

[0051] In the following examples, kana is an Invitrogen product, catalog number ant-zn-1.

[0052] Unless otherwise specified, in the following examples, whole-genome synthesis, nucleotide synthesis, primer synthesis, sequencing, etc., were performed by Shanghai Sangon Biotech Co., Ltd. and Beijing Qingke Biotechnology Co., Ltd.

[0053] In the following examples, the BALB / c mice were products of Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0054] In the following examples, unless otherwise specified, the first position of each nucleotide sequence is the 5′ terminal nucleotide of the corresponding DNA, and the last position is the 3′ terminal nucleotide of the corresponding DNA.

[0055] SEQ ID NO.1 (Lipo-CRM197 amino acid sequence)

[0056] SEQ ID NO.2 (Lipo-CRM197 nucleotide sequence)

[0057] SEQ ID NO.3 (ML signal peptide amino acid sequence)

[0058] The present invention will be further illustrated below through specific embodiments.

[0059] Example 1: Construction of fatty acid-modified recombinant Escherichia coli strain CRM197

[0060] (1.1) Obtaining the fatty acid-modified CRM197 gene and constructing an expression vector for Escherichia coli

[0061] Based on the published sequence of CRM197 (NCBI: KU521393.1), the inventors selected the whole-genome amino acid sequence of CRM197, artificially designed a codon DNA sequence, and added a signal peptide and a CGS linker peptide at the N-terminus, and a GGS linker peptide and 6 his tags at the C-terminus, which is the DNA fragment with the sequence shown in SEQ ID No. 2. Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the relevant DNA fragment (from the 5' end to the 3' end, the NdeI restriction site, the codon DNA sequence shown in SEQ ID No. 2, and the NotI restriction site, respectively). The synthesized fragment was inserted between the NdeI and NotI restriction sites of the PET30(a) vector to obtain the recombinant expression vector PET30(a)-Lipo-CRM197, i.e., the Lipo-CRM197 expression vector.

[0062] The recombinant expression vector PET30(a)-Lipo-CRM197 is described as follows: a recombinant plasmid in which the DNA fragment between the NdeI and NotI recognition sequences of the PET30(a) vector is replaced with the DNA fragment shown in SEQ ID No. 2. SEQ ID No. 2 is the coding gene sequence obtained after artificial codon optimization based on Lipo-CRM197, encoding Lipo-CRM197 shown in SEQ ID No. 1, which carries 6 His tags and a GGS linker peptide. Positions 1-20 of SEQ ID No. 1 are lipidation signal peptides, and positions 33-566 are the CRM197 protein.

[0063] (1.2) Recombinant expression vector PET30(a)-Lipo-CRM197 was transformed into C43(DE3) competent cells.

[0064] Escherichia coli strain C43(DE3) was streaked onto LB agar plates for resuscitation, and single colonies were isolated. Resuscitated single colonies were picked and inoculated into LB liquid medium. After reaching the logarithmic growth phase, 1 mL was transferred to a 100 mL LB shake flask and cultured at 37°C and 200 rpm until the OD600 reached 0.4-0.8. The culture was centrifuged at 5000 rpm for 5 min at 4°C, the supernatant was discarded, and the cells were resuspended in 20 mL of pre-chilled distilled water and centrifuged at 5000 rpm for 5 min at 4°C. The supernatant was discarded, and this step was repeated 3 times. The cells were then resuspended in 20 mL of pre-chilled 10% glycerol and centrifuged at 5000 rpm for 5 min at 4°C. The supernatant was discarded, and this step was repeated 3 times. The bacterial pellets obtained after three cycles of distillation and 10% glycerol treatment were resuspended in 1 mL of 10% glycerol, and aliquoted into 100 μL aliquots into sterile centrifuge tubes and stored at -80°C to obtain Escherichia coli C43(DE3) electroporation competent cells.

[0065] Add 2 μL of the expression plasmid PET30(a)-Lipo-CRM197 constructed in step 1.1 to 100 μL of *E. coli* C43(DE3) electroporation-transformed competent cells, mix gently, and transfer to a pre-chilled 0.2 cm electroporation cuvette. Incubate on ice for 5 min. Following the *E. coli* electroporation manual, electroporate at 2 kV and immediately add 900 μL of pre-chilled antibiotic-free LB medium. Transfer to a clean test tube and incubate at 37°C and 200 rpm for 1 hour. Spread 100 μL of the bacterial culture obtained from the above shaking culture onto LB agar plates selected for KANA resistance and incubate upside down at 37°C for 8–16 hours.

[0066] (1.3) Screening of recombinant expression strains

[0067] After single colonies have grown on the plate, pick one colony and inoculate it into 3 mL of LB liquid medium containing 50 μg / mL Kana. Incubate at 37°C and 200 rpm on a shaker. Once the bacterial culture has thickened, transfer it to 3 mL of medium #1 at a 1% (volume percentage) inoculation rate. Incubate at 37°C and 200 rpm on a shaker until the OD600 reaches 0.4-0.8. Add 0.5 mM IPTG and induce incubation at 15°C and 200 rpm for 18 h. Collect the precipitate at 12000 rpm for 5 min. Resuspend the precipitate in an equal volume of water. Sonicate at 80V for 5 min (3 seconds on, 3 seconds off). Separate the supernatant and precipitate at 12000 rpm for 5 min. Resuspend the precipitate in an equal volume of water.

[0068] The supernatant and precipitate separated by ultrasonic disruption were screened by Western Blot (WB). The Western Blot steps were as follows: (1) Separate the sample with 10% SDS-PAGE gel; (2) Transfer the sample on the SDS-PAGE gel to a PVDF membrane; (3) Block the PVDF membrane with the target protein transferred with 5% milk blocking solution and block at room temperature for 1 h; (4) Transfer to anti-His tag antibody (Anti-his, SIGMA, A7058-1VL) diluted with 5% milk at a dilution of 1:2000 and incubate for 1 h; (5) Wash with PBST for 5 min and wash 5 times; (6) Develop color with Pro-light HRP Chemiluminescent chromogenic solution (Tiangen Biotech, PA112-02).

[0069] The results are shown in Figure 1. The positive clone can express the target protein Lipo-CRM197 at a high level. The positive clone was selected as the clone strain for the next step of the experiment and named PET30(a)-Lipo-CRM197.

[0070] Example 2: Expression and purification of recombinant Lipo-CRM197 protein

[0071] (2.1) Culture of recombinant strain PET30(a)-Lipo-CRM197

[0072] Positive clones identified in Example 1 (i.e., recombinant strain PET30(a)-Lipo-CRM197) were inoculated into LB / Kana liquid medium (containing 50 μg / mL Kana) and cultured at 37°C and 200 rpm until the OD600 reached 1.5-2.0. The clones were then transferred to medium #1 at an inoculum of 1% (V / V) and cultured at 37°C and 200 rpm until the OD600 reached 0.4-0.8. 0.5 mM IPTG was added and the clones were cultured at 15°C and 200 rpm for 18 h to induce the expression of Lipo-CRM197. The bacterial cells were then collected by centrifugation.

[0073] (2.2) Purification of Lipo-CRM197

[0074] 1. Nickel ion affinity chromatography

[0075] The bacterial cells induced for expression for 18 hours in step one were resuspended in water at a ratio of 1:20, homogenized (550-700 Bar), and 1% Triton X-100 was added. The mixture was stirred at room temperature for 2 hours, and 20 mM Tris-HCl was added to adjust the pH to 7.5. Then, 0.5 M NaCl + 20 mM imidazole was added to adjust the pH to 7.5. The supernatant was collected by centrifugation, and pre-equilibrated Ni column packing was added. The mixture was stirred and adsorbed for 5 hours. The mixture was then allowed to stand at 4°C overnight. The supernatant was discarded the next day, and the mixture was resuspended in solution A. After standing, the mixture was packed into a column.

[0076] Purification was performed using nickel ion chromatography medium, with the following mobile phase composition:

[0077] Solution A: 20mM Tris HCl 7.5 + 0.5M NaCl + 20mM imidazole + 1% Triton X-100;

[0078] Solution B: 20mM Tris HCl 7.5 + 0.5M NaCl + 1M imidazole + 1% Triton X-100;

[0079] After the column is packed, it is equilibrated with solution A, then eluted with solution B at concentrations of 15%-25%-50%-100%, and finally washed with NaOH.

[0080] 2. Gel size exclusion chromatography

[0081] The sample purified using a nickel column was purified using a Supdex G200 mobile phase with the following composition:

[0082] Solution C: 1×PBST (0.2% TWEEN-20) + 150mM NaCl;

[0083] After loading the sample, equilibrate with the mobile phase and collect the sample in segments.

[0084] The purified sample is shown in Figure 2. As can be seen from the figure, the Lipo-CRM197 protein is expressed correctly, consistent with the design.

[0085] Example 3: Mouse Immunization Experiment

[0086] The immunization method has been published in several papers, such as "Replication of Animal Models of Human Diseases," edited by Li Cai and published by People's Medical Publishing House. Specifically, 45 female BALB / c mice aged 6-8 weeks were randomly divided into the following 9 groups:

[0087] Immunization group 1: i.e. control group: 100μL PBST (0.2% TWEEN-20) and 150mM NaCl were injected intramuscularly on days 0, 14 and 28, respectively.

[0088] Group 2 immunization: 100 μL of vaccine was administered intramuscularly on days 0, 14, and 28. The vaccine used was 1 μg Lipo-CRM197. Lipo-CRM197 is the C43(DE3)-expressed Lipo-CRM197 protein prepared earlier, and the vaccine was prepared in 100 μL volume using 1×PBST (0.2% TWEEN-20) and 150 mM NaCl solution, containing 1 μg of Lipo-CRM197.

[0089] Group 3 immunizations: 100 μL of vaccine was administered intramuscularly on days 0, 14, and 28. The vaccine used was 5 μg Lipo-CRM197. Lipo-CRM197 is the C43(DE3)-expressed Lipo-CRM197 protein prepared earlier, and was prepared in 100 μL volume using 1×PBST (0.2% TWEEN-20) and 150 mM NaCl solution, containing 5 μg of Lipo-CRM197.

[0090] Group 4 was immunized: 50 μL of vaccine was delivered to the lungs on days 0, 14, and 28. The vaccine used was 5 μg Lipo-CRM197. Lipo-CRM197 is the C43(DE3)-expressed Lipo-CRM197 protein prepared earlier. The vaccine was prepared in 50 μL volumes containing 5 μg of Lipo-CRM197 using 1×PBST (0.2% TWEEN-20) and 150 mM NaCl solution.

[0091] Group 5 immunizations: 20 μL of vaccine was administered intranasally on days 0, 14, and 28. The vaccine used was 5 μg Lipo-CRM197. Lipo-CRM197 is the C43(DE3)-expressed Lipo-CRM197 protein prepared previously. The vaccine was prepared in 20 μL volumes containing 5 μg of Lipo-CRM197 using 1×PBST (0.2% TWEEN-20) and 150 mM NaCl solution.

[0092] Group 6 was immunized with 100 μL of vaccine via intramuscular injection on days 0, 14, and 28. The vaccine used was 1 μg CRM197 (CRM197 protein purchased from Sinovac Biotech). CRM197 refers to the CRM197 protein purchased from Sinovac Biotech, with 5 μg of CRM197 per 100 μL volume, prepared using 1×PBST (0.2% TWEEN-20) and 150 mM NaCl solution.

[0093] Group 7 was immunized with 100 μL of vaccine via intramuscular injection on days 0, 14, and 28. The vaccine used was 5 μg CRM197 (CRM197 protein purchased from Sinovac Biotech). CRM197 refers to the CRM197 protein purchased from Sinovac Biotech, with 5 μg of CRM197 per 100 μL volume, prepared using 1×PBST (0.2% TWEEN-20) and 150 mM NaCl solution.

[0094] Group 8 was immunized with 50 μL of vaccine via intramuscular injection on days 0, 14, and 28. The vaccine used was 5 μg CRM197 (CRM197 protein purchased from Sinovac Biotech). CRM197 refers to the CRM197 protein purchased from Sinovac Biotech, with each 50 μL volume containing 5 μg of CRM197 prepared using 1×PBST (0.2% TWEEN-20) and 150 mM NaCl solution.

[0095] Group 9 was immunized with 50 μL of vaccine via intramuscular injection on days 0, 14, and 28. The vaccine used was 5 μg CRM197 (CRM197 protein purchased from Sinovac Biotech). CRM197 refers to the CRM197 protein purchased from Sinovac Biotech, with 5 μg CRM197 per 20 μL volume, prepared using 1×PBST (0.2% TWEEN-20) and 150 mM NaCl solution.

[0096] Blood was collected from the eyes of immunized mice before immunization and two weeks after the first immunization, two weeks after the second immunization, and three weeks after the third immunization in each group.

[0097] The antibody titer against CRM197 in the serum of mice in each group was measured using ELISA. For the procedure, please refer to the "Concise Guide to Molecular Biology Experiments" [M]. Science Press, 2008.

[0098] Figures 3A, 3B, and 3C show the antibody detection in mice after immunization according to the present invention. Figure 3A shows the antibody titer against CRM197 protein in mouse serum two weeks after the first immunization, Figure 3B shows the antibody titer two weeks after the second immunization, and VC shows the antibody titer three weeks after the third immunization. The results are shown in Figures 3B and 3C: the antibody titer in the immunized group was significantly higher than that in the control group. In the intramuscular injection group, the IgG antibody titer of Lipo-CRM197 was significantly different from that of CRM197.

[0099] Example 4: Protein Particle Size Detection Experiment

[0100] (4.1) The protein particle size was detected by gel size exclusion chromatography using Supdex G200 and compared with the peak position of the standard to determine the protein particle size.

[0101] The sample purified using a nickel column was purified using a Supdex G200 mobile phase with the following composition:

[0102] Solution C: 1×PBST (0.2% TWEEN-20) + 150mM NaCl;

[0103] After loading the sample, equilibrate with the mobile phase and collect the sample in segments.

[0104] Figures 4A and 4B are particle size distributions of the fatty acid-modified CRM197 protein according to the present invention. The purified peak diagrams are shown in Figures 4A and 4B. As can be seen from the figures, the Lipo-CRM197 protein has the same peak position as the standard protein, indicating that the Lipo-CRM197 protein self-aggregates into particles larger than 10 nm.

[0105] (4.2) Protein particle size was detected using a dynamic light scattering instrument.

[0106] Figure 5 shows the particle size distribution of the fatty acid-modified CRM197 protein according to the present invention. As can be seen from the figure, the particle size of Lipo-CRM197 protein is approximately 27.77 nm, indicating that Lipo-CRM197 protein self-aggregates into particles larger than 10 nm.

[0107] (4.3) Protein particle size was detected by transmission electron microscopy (TEM).

[0108] Figure 6 is a transmission electron microscope image of the fatty acid-modified CRM197 protein according to the present invention. As can be seen from the figure, the Lipo-CRM197 protein self-aggregates into nanoparticles.

[0109] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fatty acid-modified diphtheria toxin antigen.

2. The diphtheria toxin antigen modified with fatty acid according to claim 1, characterized in that, It contains an N-terminal cysteine ​​(Cys) that has been fatty acid modified.

3. The diphtheria toxin antigen modified with fatty acid according to claim 2, characterized in that, The fatty acid modification is specifically the modification of the N-terminal cysteine ​​Cys group by diacylglycerol, or the modification of the sulfhydryl group of Cys by diacylglycerol and the modification of the α-amino group by an acyl group.

4. The fatty acid-modified diphtheria toxin antigen according to claim 3, characterized in that, The acyl carbon chain length is C16 to C18.

5. The diphtheria toxin antigen modified with fatty acid according to claim 1, characterized in that, The fatty acid-modified diphtheria toxin antigen is a non-toxic mutant, CRM197.

6. The fatty acid-modified diphtheria toxin antigen according to claim 1, characterized in that, The fatty acid-modified diphtheria toxin antigen includes variants that retain the same function as diphtheria toxin.

7. The diphtheria toxin antigen modified with fatty acid according to claim 1, characterized in that, The fatty acid-modified diphtheria toxin antigen comprises the amino acid sequence 33-566 as shown in SEQ ID NO.

1.

8. The diphtheria toxin antigen modified with fatty acid according to any one of claims 1-7, characterized in that, The fatty acid-modified diphtheria toxin antigen is a polymer.

9. A method for preparing the fatty acid-modified diphtheria toxin antigen according to any one of claims 1-7, characterized in that, The method includes the steps of expressing the diphtheria toxin protein gene containing a fatty acid-modified signal peptide in Escherichia coli and purifying the fatty acid-modified diphtheria toxin protein.

10. The method according to claim 9, characterized in that, The signal peptide contains three regions: an N-terminal positively charged region, a mid-terminal hydrophobic region, and a C-terminal lipid conserved sequence [LVI][ASTVI][GAS][C].

11. The method according to claim 9, characterized in that, The signal peptide has the amino acid sequence of SEQ ID NO.

3.

12. The method according to claim 9, characterized in that, The C-terminal cysteine ​​residue is a fatty acid modification site.

13. The use of the fatty acid-modified diphtheria toxin antigen as described in any one of claims 1-8 in the preparation of a product capable of inducing the production of antigen-specific antibodies in animals, wherein the immune composition can be administered by intramuscular or subcutaneous injection, nasal drops, or nebulized immunization.

14. The application according to claim 13, characterized in that, The product in question is a vaccine.

15. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the fatty acid-modified diphtheria toxin antigen as described in any one of claims 1-8.

16. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a nucleic acid encoding the fatty acid-modified diphtheria toxin antigen as described in any one of claims 1-8.

17. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a carrier encoding the fatty acid-modified diphtheria toxin antigen as described in any one of claims 1-8.

18. A biomaterial, characterized in that, The biomaterial is any one of the following: C1) Encoding the nucleic acid molecule of the fatty acid-modified diphtheria toxin antigen as described in any one of claims 1-8; C2) An expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2); C4) Recombinant microorganisms containing the nucleic acid molecules described in C1), or recombinant microorganisms containing the expression cassette described in C2), or recombinant microorganisms containing the recombinant vector described in C3); C5) Recombinant cells containing the nucleic acid molecule described in C1), or recombinant cells containing the expression cassette described in C2), or recombinant cells containing the recombinant vector described in C3).

19. A method for preparing a vaccine using the fatty acid-modified diphtheria toxin antigen according to any one of claims 1-8.

20. A method for preparing a vaccine using the biomaterials of claim 18.

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