A carrier protein with adjuvant function and its application

Abstract

This application discloses a carrier protein with adjuvant function and its application. The carrier protein with adjuvant function in this application has the sequence shown in SEQ ID NO.1. The carrier protein with adjuvant function in this application is a partial sequence extracted from the flagellin FliC of Burkholderia cepacia, which retains the adjuvant activity of flagellin while reducing its immunogenicity and antigenicity; when applied to fusion proteins or small molecule drugs, it can enhance their immunogenicity, which is of great significance for subunit vaccines, peptide vaccines, or small molecule drug carriers.

Description

Technical Field

[0001] This application relates to the field of carrier protein technology, and in particular to a carrier protein with adjuvant function and its application. Background Technology

[0002] Flagellin is a major structural protein of bacterial flagella and a ligand for pattern recognition receptors (PRRs). Its mechanism of action as an adjuvant is primarily based on the specific activation of Toll-like receptor 5 (TLR5), thereby initiating innate immune signaling pathways and enhancing vaccine immunogenicity. Flagellin activation of the TLR5 pathway can significantly increase antibody titers (IgG / IgA) and T cell responses (especially Th1), while simultaneously enhancing humoral and cellular immunity, showing significant effects against viruses, intracellular bacteria, and tumor antigens.

[0003] A carrier protein is a transmembrane protein that binds to the molecule being transported and moves it to the other side of the membrane; therefore, it is called a carrier or transporter.

[0004] Intact flagellin proteins have potential inflammatory side effects; systemic TLR5 activation may lead to inflammatory responses such as fever and localized redness and swelling. Repeated use may induce anti-flagellate antibodies, reducing adjuvant efficacy, and long-term exposure may induce immune tolerance. Therefore, developing low-antigenic flagellin proteins that retain their adjuvant function while reducing antigenicity is of great significance for the development of both preventative and therapeutic vaccines. Summary of the Invention

[0005] The purpose of this application is to provide a novel carrier protein with adjuvant function and its application.

[0006] The following technical solution is adopted in this application:

[0007] The first aspect of this application discloses a carrier protein with adjuvant function, which is the sequence shown in SEQ ID NO.1.

[0008] It should be noted that the inventive research of this application has found that after the hypervariable region of the flagellate protein FliC of Burkholderia cepacia is truncated, that is, the truncated hypervariable region is amino acids 171 to 285, the resulting protein, namely the sequence shown in SEQ ID NO.1, can be used as a carrier protein and has adjuvant function. It can effectively overcome the problems of traditional carrier proteins that require immune adjuvant stimulation, have low immunogenicity, and are prone to immune tolerance.

[0009] The second aspect of this application discloses a carrier protein with adjuvant function, which is obtained by mutating the sequence shown in SEQ ID NO.1 at amino acid positions 18, 131, 137, 139, and 238.

[0010] In one implementation of this application, the mutation occurs in at least one of the following cases;

[0011] (1) Asparagine at positions 18, 131, and 238 is mutated to alanine;

[0012] (2) Asparagine at position 137 is mutated to aspartic acid and asparagine at position 139 is mutated to serine.

[0013] In one implementation of this application, the carrier protein is the sequence shown in SEQ ID NO.2.

[0014] It should be noted that one of the key aspects of this application is the discovery that the protein truncated from amino acids 171 to 285 of the flagellate protein FliC from Burkholderia cepacia can be used as a carrier protein and has adjuvant function. Further research revealed that this truncated, artificially synthesized carrier protein has undergone mutations at amino acids 18, 131, 137, 139, and 238. In particular, the asparagine at positions 18, 131, and 238 has been mutated to alanine, the asparagine at position 137 to aspartic acid, and the asparagine at position 139 to serine, resulting in better carrier performance.

[0015] The third aspect of this application discloses a fusion protein, which is obtained by inserting a foreign protein sequence between the 170th and 171st amino acids of the carrier protein of this application.

[0016] It should be noted that the key to this application is the discovery of a new carrier protein. As for the specific exogenous protein that is delivered, existing technologies can be referenced, including but not limited to human CD24 protein and human CD47 protein.

[0017] In one implementation of this application, the exogenous protein sequence is the human CD24 protein sequence.

[0018] In one implementation of this application, when the exogenous protein sequence is a human CD24 protein sequence, the fusion protein is as shown in SEQ ID NO.3.

[0019] In one implementation of this application, the exogenous protein sequence is the human CD47 protein sequence.

[0020] In one implementation of this application, when the exogenous protein sequence is a human CD47 protein sequence, the fusion protein is as shown in SEQ ID NO.4.

[0021] The fourth aspect of this application discloses the nucleic acid of the vector protein of this application, or the fusion protein of this application.

[0022] The fifth aspect of this application discloses a recombinant plasmid containing the nucleic acid of this application.

[0023] The sixth aspect of this application discloses cells transformed with the recombinant plasmid of this application.

[0024] It should be noted that, in one implementation of this application, the vector protein of this application is obtained using genetic engineering technology. It can be understood that, based on this, the coding sequence of the vector protein can be artificially synthesized according to its sequence, and then expressed through cloning to obtain the vector protein of this application. The specific nucleic acid sequence can be optimized according to the preferences of the cloning expression microorganism or cell used. The specific cloning vector and host microorganism or cell can also refer to existing protein expression technologies, and are not specifically limited here.

[0025] The seventh aspect of this application discloses the use of the vector protein, fusion protein, nucleic acid, recombinant plasmid, or cell in the preparation of subunit vaccines, peptide vaccines, or small molecule drug carriers.

[0026] It is understood that the carrier protein or fusion protein of this application can be directly used as a subunit vaccine, peptide vaccine or small molecule drug carrier; as for the nucleic acid, recombinant plasmid and host cell of this application, they can be used as raw materials for preparing the carrier protein or fusion protein of this application. For example, by culturing the host cell of this application to express the carrier protein or fusion protein of this application, and then by protein recovery and purification, the carrier protein or fusion protein of this application can be obtained.

[0027] The eighth aspect of this application discloses an immunogenic small molecule drug, which is formed by conjugating the carrier protein of this application or the fusion protein of this application with the small molecule drug.

[0028] It should be noted that this application relates to immunogenic small molecule drugs, the key being the conjugation of the carrier protein or fusion protein of this application with small molecule drugs to obtain complete antigens of immunogenic small molecule drugs. This utilizes the adjuvant function of the carrier protein and its function of enhancing the immunogenicity of small molecule drugs. It is understood that the carrier protein of this application can be conjugated with different small molecule drugs to obtain complete antigens of small molecule drugs with different immunogenicities. The small molecule drugs of this application include, but are not limited to, therapeutic small molecule drugs, pesticide small molecule drugs, and veterinary small molecule drugs; among them, therapeutic small molecule drugs include: carbamazepine, valproic acid, phenytoin, olanzapine, clozapine, risperidone, primidone, bromide, phenobarbital, etc. Pesticide small molecules include: imidacloprid, thiamethoxam, cypermethrin, deltamethrin, carbendazim, chlorothalonil, azoxystrobin, acetochlor, etc.; veterinary small molecules include antibiotics, hormones, etc.

[0029] The beneficial effects of this application are as follows:

[0030] The adjuvant-functional carrier protein of this application is extracted from a partial sequence of the flagellate protein FliC from Burkholderia cepacia. It retains the adjuvant activity of the flagellate protein while reducing its immunogenicity and antigenicity. When applied to fusion proteins or small molecule drugs, it can enhance their immunogenicity, which is of great significance for subunit vaccines, peptide vaccines or small molecule drug carriers. Attached Figure Description

[0031] Figure 1 These are 3D structural diagrams of the adjuvant-functional carrier protein and fusion protein in the embodiments of this application; wherein, Figure A is the 3D structural diagram of the adjuvant-functional carrier protein, Figure B is the 3D structural diagram of the adjuvant-functional carrier protein after amino acid mutation, Figure C is the 3D structural diagram of the fusion protein of the adjuvant-functional carrier protein and human CD24, and Figure D is the 3D structural diagram of the fusion protein of the adjuvant-functional carrier protein and human CD47.

[0032] Figure 2 This is an SDS-PAGE electrophoresis image of the recombinant carrier protein in the embodiments of this application; wherein, lane M is the protein marker and lane 1 is the recombinant carrier protein;

[0033] Figure 3 This is an SDS-PAGE electrophoresis image of the recombinant vector protein and human CD24 fusion protein in the embodiments of this application; wherein, lane M is the protein marker and lane 1 is the recombinant vector protein and human CD24 fusion protein.

[0034] Figure 4This is an SDS-PAGE electrophoresis image of the recombinant vector protein and human CD47 fusion protein in the embodiments of this application; wherein, lane M is the protein marker and lane 1 is the recombinant vector protein and human CD47 fusion protein.

[0035] Figure 5 These are the statistical results of the in vitro TLR5 receptor activity assay of the recombinant carrier protein in the embodiments of this application;

[0036] Figure 6 This is a graph showing the antibody levels of human CD24 protein and carrier protein F in the serum of immunized mice detected by ELISA in the embodiments of this application;

[0037] Figure 7 This is a graph showing the antibody levels of human CD47 protein in the serum of immunized mice detected by ELISA in the embodiments of this application;

[0038] Figure 8 This is a graph showing the antibody levels of small molecule drugs in the serum of immunized mice detected by ELISA in an embodiment of this application. Detailed Implementation

[0039] Developing low-antigenicity flagellin variants that retain their adjuvant function while reducing antigenicity remains an important direction in flagellin research. In our research on flagellins, this application discovered that the bacterial flagellin FliC, with its hypervariable region truncated (amino acids 171-285), has a truncated amino acid sequence as shown in SEQ ID NO. 1. This truncated variant can function as a carrier protein, possessing adjuvant function, and exhibiting reduced immunogenicity and antigenicity compared to traditional carrier proteins.

[0040] Furthermore, by mutating asparagine at positions 18, 131, and 238 of the sequence shown in SEQ ID NO.1 to alanine, asparagine at position 137 to aspartic acid, and asparagine at position 139 to serine, a carrier protein with better performance can be obtained, namely the sequence shown in SEQ ID NO.2.

[0041] This application presents a carrier protein with adjuvant function. By inserting a long protein sequence between amino acids 170 and 171, a fusion protein with immunogenicity can be obtained; alternatively, by inserting a short protein sequence between amino acids 170 and 171, a fusion protein with immunogenicity can be obtained; furthermore, it can be conjugated with a small molecule drug to obtain a complete antigen of the small molecule drug with immunogenicity. Based on the adjuvant-functional carrier protein of this application, while retaining the adjuvant activity of flagellin, its immunogenicity and antigenicity are reduced. It can be applied to low molecular weight fusion proteins, peptide fusion proteins, and small molecule drugs, enhancing the immunogenicity of the latter. This has significant implications for the preparation of subunit vaccines, peptide vaccines, and small molecule drug carriers.

[0042] For example, inserting a human CD24 protein sequence between amino acids 170 and 171 yields a fusion protein, as shown in SEQ ID NO.3. Inserting a human CD47 protein sequence between amino acids 170 and 171 yields a fusion protein, as shown in SEQ ID NO.4.

[0043] SEQ ID NO.1:

[0044] MLGINSNINSLVAQQNLNGSQNALSQAITRLSSGKRINSAADDAAGLAISTRMQTQINGLNQGVSNANDGVSMIQTASSALSSLTNSLQRIRQLAVQASTGTMSSTDQAALQQEVAQQIQEVNRIASQTTY NGTNILNGNAGIVSFQVGANVGQTISLDLSQSMSAAKIGLDISTVSGANVAMVSIDNALQTVNNVQAQLGAAQNRFTAIATAQQAESTDLSSAQSQITDANFAQETANMSKNQVLQQAGISVLAQANSLPQQ

[0045] SEQ ID NO.2:

[0046] MLGINSNINSLVAQQNLAGSQNALSQAITRLSSGKRINSAADDAAGLAISTRMQTQINGLNQGVSNANDGVSMIQTASSALSSLTNSLQRIRQLAVQASTGTMSTTDQAALQQEVSQQIQEVNRIASQTTYAGTNILDGSAGIVSFQVGANVGQTISLDLSQSMSAAKIGLDISTVSGANVAMVSIDNALQTVNNVQAALGAAQNRFTAIATSQQAESTDLSSAQSQITDANFAQETAAMSKNQVLQQAGISVLAQANSLPQQ

[0047] SEQ ID NO.3:

[0048] MLGINSNINSLVAQQNLAGSQNALSQAITRLSSGKRINSAADDAAGLAISTRMQTQINGLNQGVSNANDGVSMIQTASSALSSLTNSLQRIRQLAVQASTGTMSTTDQAALQQEVSQQIQEVNRIASQTTYAGTNILDGSAGIVSFQV GANVGQTISLDLSQSMSAAKIGSETTTGTSSNSSQSTSNSGLAPNPTNATTKAAGLDISTVSGANVAMVSIDNALQTVNNVQAALGAAQNRFTAIATSQQAESTDLSSAQSQITDANFAQETAAMSKNQVLQQAGISVLAQANSLPQQ

[0049] SEQ ID NO.4:

[0050] MLGINSNINSLVAQQNLAGSQNALSQAITRLSSGKRINSAADDAAGLAISTRMQTQINGLNQGVSNANDGVSMIQTASSALSSLTNSLQRIRQLAVQASTGTMSTTDQAALQQEVSQQIQEVNRIASQTTYAGTNILDGSAGIVSFQVGANVGQTISLDLSQSMSAAKIGQLLFNKTKSVEFTFCN DTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIILDISTVSGANVAMVSIDNALQTVNNVQAALGAAQNRFTAIATSQQAESTDLSSAQSQITDANFAQETAAMSKNQVLQQAGISVLAQANSLPQQ.

[0051] The present application will be further described in detail below through specific embodiments. The following embodiments are only for further illustration of the present application and should not be construed as limiting the present application.

[0052] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Balb / c mice: Guangdong Provincial Experimental Animal Center, 5-6 weeks old. Example

[0053] I. Construction and Protein Expression of Carrier Protein Particles with Adjuvant Function

[0054] 1. Construction and 3D structural simulation of carrier proteins and fusion proteins

[0055] The simulated predicted protein structures were obtained by importing the amino acid sequences of truncated carrier proteins, amino acid-mutated carrier proteins, fusion proteins of carrier proteins and human CD24 peptides (UniProt accession number: P25063, amino acid sequence 27-59), and fusion proteins of carrier proteins and human CD47 fragments (UniProt accession number: Q08722, amino acid sequence 19-127) into the Phyre2 protein structure prediction server. The results are as follows: Figure 1As shown. Compared to the full-length FliC protein (UniProt accession number: A0AAE8T591), the truncated carrier protein, the amino acid-mutated carrier protein, the carrier protein fusion protein with human CD24 peptide, and the carrier protein fusion protein with human CD47 all maintain their native and relatively independent spatial conformations. The amino acid-mutated carrier protein reduces the number of glycosylation sites in the eukaryotic expression system, allowing it to be expressed both in prokaryotes and after fusion with other proteins in the eukaryotic expression system.

[0056] 2. Construction of expression vector

[0057] Using the *Burkholderia cepacia* flagella FliC protein (GenBank, QCY01824.1) as a template, a truncated prokaryotic expression vector pET-32a(+)-F was constructed using a whole-genome synthesis method. A TrxA tag was fused to the N-terminus, and a 6xHis tag was added to the C-terminus for affinity purification. Using the same whole-genome synthesis method, a human CD24 peptide sequence (UniProt accession number: P25063, amino acid sequence 27-59) was inserted between amino acids 170 and 171 of the mutated vector protein to construct the eukaryotic expression vector pcDNA3.1(+)-CD24-F, with a 6xHis tag added to the C-terminus for affinity purification.

[0058] Using a whole-genome protein synthesis sequence method, a human CD47 fragment (UniProt accession number: Q08722, amino acid sequence 19-127) was inserted between amino acids 170 and 171 of the mutated vector protein to construct the eukaryotic expression vector pcDNA3.1(+)-CD47-F of the fusion protein, and a 6xHis tag was added to the C-terminus for affinity purification.

[0059] After transfection of Escherichia coli (BL21) with the prokaryotic expression vector, recombinant vector protein F was expressed by IPTG-induced low-temperature culture. After transient transfection of HEK293 cells with the eukaryotic expression vector, the recombinant vector fusion proteins CD24-F and CD47-F were expressed.

[0060] 3. Prokaryotic expression and purification of carrier protein F

[0061] Transfect *Escherichia coli* (BL21) competent cells with pET-32a(+)-F plasmid. Spread 100 μL of the plasmid onto AMP-resistant TSA plates and incubate overnight at 37°C. The next day, pick a single colony with an inoculation loop and inoculate it into 10 mL of AMP-resistant TSB liquid medium. Incubate at 37°C with shaking for 8 hours. Dilute the bacterial culture 1:50 and transfer it to AMP-resistant TSB liquid medium. Incubate overnight at 37°C with shaking until OD (out of control). 600=Approximately 0.8. At 16℃, IPTG was added to a final concentration of 0.5mM for 12 hours to induce culture. The cells were collected by centrifugation at 9000 RPM for 5 minutes. The cell pellet was resuspended in lysis buffer, followed by sonication and centrifugation. The supernatant was filtered through a 0.45μm filter membrane and purified using cobalt beads. The purified protein was then purified by removing lipopolysaccharide (LPS) using an endotoxin removal kit. The purified sample was analyzed by SDS-PAGE. The results are as follows: Figure 2 As shown, the theoretical molecular weight of the protein is approximately 45 kDa.

[0062] Figure 2 The results shown are consistent with the expected theoretical molecular weight of the protein, indicating that the carrier protein F was successfully cloned and expressed in this case.

[0063] 4. Eukaryotic expression and purification of the vector fusion protein CD24-F

[0064] HEK293 cells in logarithmic growth phase were diluted with culture medium to a concentration of 2.0 × 10⁻⁶. 6 The concentration of cells / mL was determined. In a 15 mL sterile centrifuge tube, 5 mL of KPM and 100 μg of sterile plasmid pcDNA3.1(+)-CD24-F were added and gently mixed. In another centrifuge tube, 5 mL of KPM and 500 μL of TA-293 transfection reagent were added and gently mixed. This mixture was then added to the centrifuge tube containing the plasmid and gently mixed. The mixture was allowed to stand for 10 min to obtain the pcDNA3.1(+)-CD24-F plasmid-vector complex. This plasmid-vector complex was added to 100 mL of HEK293 cells and the cells were placed back into a CO2 incubator with shaking. After 5-6 days of culture, cells were collected by centrifugation. The cell pellet was resuspended in lysis buffer and lysed. After centrifugation, the lysate was filtered through a 0.45 μm filter and purified using cobalt beads. The purified sample was analyzed by SDS-PAGE. The results are shown below. Figure 3 As shown, the theoretical molecular weight of the protein is approximately 33.4 kDa.

[0065] Figure 3 The results shown are consistent with the expected theoretical molecular weight of the protein, indicating that the fusion protein CD24-F was successfully cloned and expressed in this case.

[0066] 5. Eukaryotic expression and purification of the vector fusion protein CD47-F

[0067] HEK293 cells in logarithmic growth phase were diluted with culture medium to a concentration of 2.0 × 10⁻⁶. 6The concentration of cells / mL was determined. In a 15 mL sterile centrifuge tube, 5 mL of KPM and 100 μg of sterile plasmid pcDNA3.1(+)-CD47-F were added and gently mixed. In another centrifuge tube, 5 mL of KPM and 500 μL of TA-293 transfection reagent were added and gently mixed. This mixture was then added to the centrifuge tube containing the plasmid and gently mixed. The mixture was allowed to stand for 10 min to obtain the pcDNA3.1(+)-CD47-F plasmid-vector complex. This plasmid-vector complex was added to 100 mL of HEK293 cells and the cells were placed back into a CO2 incubator with shaking. After 5-6 days of culture, cells were collected by centrifugation. The cell pellet was resuspended in lysis buffer and lysed. After centrifugation, the supernatant was filtered through a 0.45 μm filter and purified using cobalt beads. The purified sample was analyzed by SDS-PAGE. The results are shown below. Figure 4 As shown, the theoretical molecular weight of the protein is approximately 42.5 kDa.

[0068] Figure 4 The results shown are consistent with the expected theoretical molecular weight of the protein, indicating that the fusion protein CD47-F was successfully cloned and expressed in this case.

[0069] II. Conjugation of carrier proteins with small molecule drugs

[0070] The small molecule drug carbamazepine (CMZP) was conjugated with purified carrier protein F. 75 μg of carbamazepine hapten (S. Zhou, et al., 2020, Journal of Chromatography) was dissolved in 100 μL of DMF, and EDC / NHS (molar ratio 1:1) was added for activation reaction for 1 hour. Then, the activated carbamazepine hapten was added dropwise to 1 mg of carrier protein F and reacted for 2 hours. The mixture was dialyzed against 0.02 M PBS buffer for 48 hours to obtain the small molecule drug carbamazepine conjugated with carrier protein CMZP-F. Carbamazepine conjugated with OVA antigen and carbamazepine conjugated with BSA antigen were prepared using the same method.

[0071] III. Assessment of the Immunoadjuvant Effect of Carrier Proteins

[0072] 1. Detection of in vitro TLR5 receptor activity of carrier proteins

[0073] Using human colorectal adenocarcinoma cells (Caco2) expressing the TLR5 receptor as an in vitro cell model, the TLR5 receptor activity of the recombinant carrier protein was detected. After Caco2 cells were grown in confluent T75 cell culture flasks, they were divided into groups of 1.5 × 10⁶ cells per well. 5Caco2 cells were seeded into 96-well cell culture plates and cultured for 20 hours. Purified carrier protein F, fusion protein CD24-F, and fusion protein CD47-F were added to a final concentration of 1 μg / mL to stimulate the Caco2 cells, which were then cultured at 37°C for 12 hours. Untreated Caco2 cells served as a negative control. The cell culture supernatant was collected, and the concentration of IL-8 in the cell supernatant was detected using a human IL-8 ELISA kit (Sinosure, catalog number: SEK10098). The results are shown below. Figure 5 As shown.

[0074] Figure 5 The results showed that the carrier protein F and the recombinant fusion proteins CD24-F and CD47-F could all stimulate Caco2 to secrete IL-8, indicating that the carrier protein F and the recombinant fusion proteins CD24-F and CD47-F all have good TLR5 receptor activity.

[0075] 2. Detection of anti-CD24 antibody levels in immunized mouse serum

[0076] The eukaryotically expressed and purified fusion protein CD24-F was diluted with physiological saline, and 5 μg was injected intramuscularly into 6-8 week old female Balb / c mice as a control. The prokaryotically expressed and purified carrier protein F was diluted with physiological saline, and 5 μg was injected intramuscularly into 6-8 week old female Balb / c mice as a control. After the initial immunization, booster immunizations were administered every two weeks for a total of three immunizations. Seven days after the final immunization, blood was collected from the tail of the mice, serum was separated, and the titers of specific IgG antibodies against CD24 protein and carrier protein F in the mouse serum were detected using an indirect ELISA method. Indirect ELISA titer detection method:

[0077] (1) Antigen coating: The purified recombinant vector protein F and CD24 protein (Yiqiao Shenzhou, catalog number: 11030-H02H) were diluted with pH 9.6, 0.05M carbonate buffer to a protein concentration of 2 μg / mL, and 100 μL / well was added to the microplate and coated overnight at 4℃.

[0078] (2) After completing step (1), take the microplate, wash it 4 times with PBST solution, then add blocking buffer (200 μL / well) and incubate at 37°C for 2 hours. Blocking buffer: PBST solution containing 1 g / 100 mL BSA.

[0079] (3) After completing step (2), take the ELISA plate, wash it 4 times with PBST solution, and then add mouse serum antibody dilution buffer (100 μL / well) and incubate at 37°C for 1 h. Mouse serum antibody dilution: Take the serum of immunized mice, dilute it 100 times with 0.02M PBS buffer at pH 7.4, and then perform 10-fold serial dilution.

[0080] (4) After completing step (3), take the ELISA plate, wash it 4 times with PBST solution, then add HRP-labeled secondary antibody working solution and incubate at 37°C for 1 hour. HRP-labeled secondary antibody working solution: 1:4000 dilution of HRP-labeled goat anti-mouse secondary antibody.

[0081] (5) After completing step (4), take the ELISA plate, wash it 4 times with PBST solution, then add TMB substrate reaction solution for color development for 10 minutes, then add 2M sulfuric acid solution to stop the color development, and then measure the absorbance at 450nm using an ELISA reader. The results are as follows. Figure 6 As shown.

[0082] Figure 6 Figure A shows that the fusion protein CD24-F immunization group can produce a high titer of specific IgG antibodies against CD24 protein, while the control carrier protein F immunization group does not produce specific IgG antibodies against CD24 protein; Figure B shows that the specific IgG antibody titers against carrier protein F produced by the fusion protein CD24-F immunization group and the control carrier protein F immunization group are extremely low. Figure 6 The results showed that the carrier protein F had low self-antigenicity while possessing adjuvant function.

[0083] 3. Detection of anti-CD47 antibody levels in immunized mouse serum

[0084] The eukaryotically expressed and purified fusion protein CD47-F was diluted with physiological saline, and 5 μg was injected intramuscularly into 6-8 week old female Balb / c mice. CD47 protein (Sinochem, catalog number: 12283-H02H) was emulsified with Freund's complete adjuvant (Sigma, catalog number: F5881) or Freund's incomplete adjuvant (Sigma, catalog number: F5506) at a 1:1 volume ratio, and 50 μg was injected subcutaneously into 6-8 week old female Balb / c mice. Mice injected with only 50 μg of CD47 protein served as controls. After the initial immunization, booster immunizations were administered every two weeks for a total of three immunizations. Seven days after the final immunization, blood was collected from the tail of the mice, serum was separated, and the titer of specific IgG antibodies against CD47 protein in the mouse serum was detected using an indirect ELISA method. Indirect ELISA titer detection method:

[0085] (1) Antigen coating: CD47 protein (Yiqiao Shenzhou, catalog number: 12283-H02H) was diluted with pH 9.6, 0.05M carbonate buffer to a protein concentration of 2μg / mL, and 100μL / well was added to the microplate and coated overnight at 4℃.

[0086] (2) After completing step (1), take the microplate, wash it 4 times with PBST solution, then add blocking buffer (200 μL / well) and incubate at 37°C for 2 hours. Blocking buffer: PBST solution containing 1 g / 100 mL BSA.

[0087] (3) After completing step (2), take the ELISA plate, wash it 4 times with PBST solution, and then add mouse serum antibody dilution buffer (100 μL / well) and incubate at 37°C for 1 h. Mouse serum antibody dilution: Take the serum of immunized mice, dilute it 100 times with 0.02M PBS buffer at pH 7.4, and then perform 10-fold serial dilution.

[0088] (4) After completing step (3), take the ELISA plate, wash it 4 times with PBST solution, then add HRP-labeled secondary antibody working solution and incubate at 37°C for 1 hour. HRP-labeled secondary antibody working solution: 1:4000 dilution of HRP-labeled goat anti-mouse secondary antibody.

[0089] (5) After completing step (4), take the ELISA plate, wash it 4 times with PBST solution, then add TMB substrate reaction solution for color development for 10 minutes, then add 2M sulfuric acid solution to stop the color development, and then measure the absorbance at 450nm using an ELISA reader. The results are as follows. Figure 7 As shown.

[0090] Figure 7 The results showed that the antibody titer against CD47 protein produced by the CD47-F fusion protein immunization group was higher than that of the CD47 protein immunization group with adjuvant, while the CD47 protein control group without adjuvant did not produce specific IgG antibodies against CD47 protein. This result indicates that the carrier protein F, as a fusion protein, possesses superior adjuvant function compared to traditional adjuvants.

[0091] 4. Detection of serum anti-small molecule drug antibody levels in immunized mice

[0092] The small molecule drug-conjugated protein CMZP-F was diluted with physiological saline, and 10 μg was injected intramuscularly into 6-8 week old female Balb / c mice. OVA-conjugated small molecule drug protein CMZP-OVA was emulsified with Freund's complete adjuvant (Sigma, catalog number: F5881) or Freund's incomplete adjuvant (Sigma, catalog number: F5506) at a 1:1 volume ratio, and 50 μg was injected subcutaneously into 6-8 week old female Balb / c mice. Mice that received only 50 μg of CMZP-OVA served as the control group. After the initial immunization, booster immunizations were administered every two weeks for a total of three immunizations. Seven days after the final immunization, blood was collected from the tail of the mice, serum was separated, and the titer of specific IgG antibodies against CMZP in the mouse serum was detected using an indirect ELISA method. Indirect ELISA titer detection method:

[0093] (1) Antigen coating: Take CMZP-BSA conjugate antigen and dilute it with pH 9.6, 0.05M carbonate buffer to a protein concentration of 2μg / mL. Add 100μL / well to the microplate and coat overnight at 4℃.

[0094] (2) After completing step (1), take the microplate, wash it 4 times with PBST solution, then add blocking buffer (200 μL / well) and incubate at 37°C for 2 hours. Blocking buffer: PBST solution containing 1 g / 100 mL BSA.

[0095] (3) After completing step (2), take the ELISA plate, wash it 4 times with PBST solution, and then add mouse serum antibody dilution buffer (100 μL / well) and incubate at 37°C for 1 h. Mouse serum antibody dilution: Take the serum of immunized mice, dilute it 100 times with 0.02M PBS buffer at pH 7.4, and then perform 10-fold serial dilution.

[0096] (4) After completing step (3), take the ELISA plate, wash it 4 times with PBST solution, then add HRP-labeled secondary antibody working solution and incubate at 37°C for 1 hour. HRP-labeled secondary antibody working solution: 1:4000 dilution of HRP-labeled goat anti-mouse secondary antibody.

[0097] (5) After completing step (4), take the ELISA plate, wash it 4 times with PBST solution, then add TMB substrate reaction solution for color development for 10 minutes, then add 2M sulfuric acid solution to stop the color development, and then measure the absorbance at 450nm using an ELISA reader. The results are as follows. Figure 8 As shown.

[0098] Figure 8 The results showed that the antibody titer against CMZP produced by the small molecule drug conjugate protein CMZP-F immunization group was higher than that of the CMZP-OVA adjuvant immunization group, while the CMZP-OVA control group without adjuvant did not produce specific IgG antibodies against CMZP. This result indicates that the carrier protein F, as a small molecule drug carrier protein, is superior to traditional carrier proteins while possessing adjuvant functions.

[0099] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. Those skilled in the art to which this application pertains can make several simple deductions or substitutions without departing from the concept of this application.

Claims

1. A carrier protein with adjuvant function, characterized in that: The sequence is shown in SEQ ID NO.

1.

2. A carrier protein with adjuvant function, characterized in that: The sequence shown in SEQ ID NO.1 was obtained by mutating amino acids at positions 18, 131, 137, 139, and 238. The specific mutations are (1) the asparagine at positions 18, 131, and 238 is mutated to alanine; (2) the asparagine at position 137 is mutated to aspartic acid and the asparagine at position 139 is mutated to serine.

3. The carrier protein according to claim 2, characterized in that: The carrier protein is the sequence shown in SEQ ID NO.

2.

4. A fusion protein, characterized in that: The fusion protein is obtained by inserting a foreign protein sequence between the 170th and 171st amino acids of the carrier protein according to any one of claims 1-3.

5. The fusion protein according to claim 4, characterized in that: The exogenous protein sequence is either the human CD24 protein sequence or the human CD47 protein sequence.

6. The fusion protein according to claim 5, characterized in that: When the exogenous protein sequence is a human CD24 protein sequence, the fusion protein is the sequence shown in SEQ ID NO.3; when the exogenous protein sequence is a human CD47 protein sequence, the fusion protein is the sequence shown in SEQ ID NO.

4.

7. A nucleic acid encoding the carrier protein of any one of claims 1-3 or the fusion protein of any one of claims 4-6.

8. A recombinant plasmid containing the nucleic acid of claim 7.

9. Transforming cells containing the recombinant plasmid of claim 8.

10. The use of the carrier protein according to any one of claims 1-3, the fusion protein according to any one of claims 4-6, the nucleic acid according to claim 7, the recombinant plasmid according to claim 8, or the cell according to claim 9 in the preparation of subunit vaccines, peptide vaccines, or small molecule drug carriers.

11. An immunogenic small molecule drug, characterized in that: It is formed by conjugating a small molecule drug with the carrier protein as described in any one of claims 1-3 or the fusion protein as described in any one of claims 4-6.

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