Carrier protein with adjuvant function and application thereof

Abstract

The invention discloses a carrier protein with an adjuvant function and application thereof. The carrier protein with the adjuvant function has a sequence as shown in SEQ ID NO. 1. According to the carrier protein with the adjuvant function, a part of sequence is intercepted from the flagellin FliC of Burkholderia cepacia, so that the adjuvant activity of the flagellin is reserved, and the immunogenicity and antigenicity of the flagellin are reduced; when being applied to a fusion protein or a small-molecule drug, the fusion protein or the small-molecule drug can enhance the immunogenicity, so that the fusion protein or the small-molecule drug has important significance on subunit vaccines, polypeptide vaccines or small-molecule drug carriers.

Description

Technical Field

[0001] The present application relates to the technical field of carrier proteins, and in particular to a carrier protein with adjuvant function and its application. Background Art

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

[0003] A carrier protein is a transmembrane protein that can bind to the molecules being transferred and move them to the other side of the membrane; therefore, it is called a carrier or transporter.

[0004] Intact flagellin has potential inflammatory side effects. Systemic TLR5 activation may lead to inflammatory reactions such as fever and local redness and swelling. Repeated use may induce anti-flagellin antibodies, reducing the adjuvant's effectiveness, and long-term exposure may induce immune tolerance. Therefore, developing low-antigenic flagellin, which retains its adjuvant function while reducing antigenicity, is of great significance for the development of both preventive and therapeutic vaccines. Summary of the Invention

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

[0006] This application adopts the following technical solutions: The first aspect of the present application discloses a carrier protein with adjuvant function, which is the sequence shown in SEQ ID NO.1.

[0007] It should be noted that the creative research of this application found that after the hypervariable region of the Burkholderia cepacia flagellin FliC is truncated, that is, the truncated hypervariable region is from amino acid 171 to amino acid 285, the protein formed thereby, namely the sequence shown in SEQ ID NO.1, can be used as a carrier protein and has adjuvant function itself, which can effectively overcome the problems of traditional carrier proteins requiring immune adjuvant stimulation, low immunogenicity, and easy to produce immune tolerance.

[0008] The second aspect of the present application discloses a carrier protein with adjuvant function, which is obtained by mutating the amino acids 18, 131, 137, 139 and 238 of the sequence shown in SEQ ID NO.1.

[0009] In one implementation of the present application, the mutation is at least one of the following: (1) Asparagine at positions 18, 131, and 238 were mutated to alanine; (2) Asparagine at position 137 mutated to aspartic acid, and asparagine at position 139 mutated to serine.

[0010] In one implementation of the present application, the carrier protein is the sequence shown in SEQ ID NO.2.

[0011] It should be noted that one of the key points of this application is that the study found that the protein extracted from amino acids 171 to 285 of the flagellin FliC of Burkholderia cepacia can be used as a carrier protein and has adjuvant function itself; further research found that the artificially synthesized carrier protein after truncation has mutated amino acids at positions 18, 131, 137, 139, and 238, especially mutating asparagine at positions 18, 131, and 238 to alanine, mutating asparagine at position 137 to aspartic acid, and mutating asparagine at position 139 to serine, which can obtain better carrier performance.

[0012] The third aspect of the present 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 the present application.

[0013] It should be noted that the key to this application is the research and discovery of a new carrier protein. As for the specific exogenous protein to be delivered, reference can be made to the existing technology, including but not limited to human CD24 protein and human CD47 protein.

[0014] In one implementation of the present application, the exogenous protein sequence is a human CD24 protein sequence.

[0015] In one implementation of the present application, when the exogenous protein sequence is a human CD24 protein sequence, the fusion protein is shown as SEQ ID NO.3.

[0016] In one implementation of the present application, the exogenous protein sequence is a human CD47 protein sequence.

[0017] In one implementation of the present application, when the exogenous protein sequence is a human CD47 protein sequence, the fusion protein is shown as SEQ ID NO.4.

[0018] The fourth aspect of the present application discloses the carrier protein of the present application, or the nucleic acid of the fusion protein of the present application.

[0019] The fifth aspect of the present application discloses a recombinant plasmid containing the nucleic acid of the present application.

[0020] The sixth aspect of the present application discloses a cell transformed with the recombinant plasmid of the present application.

[0021] It should be noted that in one implementation of the present application, the vector protein of the present application is obtained using genetic engineering technology. It is understood that, based on this, the coding sequence of the vector protein of the present application can be artificially synthesized based on its sequence, and then expressed by cloning to obtain the vector protein of the present application. The specific nucleic acid sequence can be optimized based on the preference of the cloned 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.

[0022] The seventh aspect of the present application discloses the use of the carrier protein of the present application, the fusion protein of the present application, the nucleic acid of the present application, the recombinant plasmid of the present application or the cell of the present application in the preparation of subunit vaccines, polypeptide vaccines or small molecule drug carriers.

[0023] It can be understood that the carrier protein or fusion protein of the present application can be directly used as a subunit vaccine, polypeptide vaccine or small molecule drug carrier; as for the nucleic acid, recombinant plasmid and host cell of the present application, they can be used as raw materials for preparing the carrier protein or fusion protein of the present application, for example, by culturing the host cell of the present application to express the carrier protein or fusion protein of the present application, and then recovering and purifying the protein to obtain the carrier protein or fusion protein of the present application.

[0024] The eighth aspect of the present application discloses an immunogenic small molecule drug, which is formed by coupling the carrier protein of the present application or the fusion protein of the present application with the small molecule drug.

[0025] It should be noted that the key to the immunogenic small molecule drugs of the present application lies in the use of the carrier protein or fusion protein of the present application to couple with the small molecule drug to obtain a complete antigen of the immunogenic small molecule drug. It utilizes the adjuvant function of the carrier protein of the present application and the function of improving the immunogenicity of the small molecule drug. It is understandable that the carrier protein of the present application can be coupled with different small molecule drugs to obtain complete antigens of small molecule drugs with different immunogenicities. The small molecule drugs of the present 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, bromperidol, phenobarbital, etc. Small molecules of veterinary drugs, small molecules of pesticides, etc.; small molecules of pesticides such as imidacloprid, thiamethoxam, cypermethrin, deltamethrin, carbendazim, chlorothalonil, azoxystrobin, kresoxim-methyl, acetochlor, etc.; small molecules of veterinary drugs such as antibiotics, hormones, etc.

[0026] The beneficial effects of this application are: The carrier protein with adjuvant function in the present application is extracted from a partial sequence of the flagellin FliC of Burkholderia cepacia. It retains the adjuvant activity of the 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, polypeptide vaccines or small molecule drug carriers. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Figures 3D structural diagrams of carrier proteins and fusion proteins with adjuvant function in the examples of the present application; Figure A is a 3D structural diagram of the carrier protein with adjuvant function, Figure B is a 3D structural diagram of the carrier protein with adjuvant function after amino acid mutation, Figure C is a 3D structural diagram of the carrier protein with adjuvant function and human CD24 fusion protein, and Figure D is a 3D structural diagram of the carrier protein with adjuvant function and human CD47 fusion protein; Figure 2 This is an SDS-PAGE electrophoresis diagram of the recombinant carrier protein in the examples of the present application; wherein, lane M is the protein marker, and lane 1 is the recombinant carrier protein; Figure 3 : is an SDS-PAGE electrophoresis diagram of the recombinant carrier protein and the human CD24 fusion protein in the examples of the present application; wherein, lane M is the protein marker, and lane 1 is the recombinant carrier protein and the human CD24 fusion protein; Figure 4 : is an SDS-PAGE electrophoresis diagram of the recombinant carrier protein and the human CD47 fusion protein in the examples of the present application; wherein, lane M is the protein marker, and lane 1 is the recombinant carrier protein and the human CD47 fusion protein; Figure 5 This is the statistical result of the in vitro TLR5 receptor activity test of the recombinant carrier protein in the examples of this application; 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 examples of the present application; 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 examples of the present application; Figure 8 This is a graph showing the antibody levels of small molecule drugs in the serum of immunized mice detected by ELISA in the examples of the present application. DETAILED DESCRIPTION

[0028] It is still an important direction of the research on flagellin to develop low antigenicity flagellin variants which retain the adjuvant function while reducing the antigenicity. In the research on flagellin, the present application found that the truncated flagellin FliC with the truncated hypervariable region from the 171st amino acid to the 285th amino acid, the amino acid sequence after the truncation is shown in SEQ ID NO. 1, can be used as a carrier protein and has the adjuvant function, and reduces the immunogenicity and antigenicity of the carrier protein.

[0029] Further, the 18th, 131st, 238th asparagines in the sequence shown in SEQ ID NO. 1 are mutated to alanine, the 137th asparagine is mutated to aspartic acid, and the 139th asparagine is mutated to serine, to obtain a carrier protein with better performance, i.e. the sequence shown in SEQ ID NO. 2.

[0030] The carrier protein with the adjuvant function of the present application is inserted with a long protein sequence between the 170th amino acid and the 171st amino acid to obtain a fusion protein, which can obtain a fusion protein with immunogenicity; or a short protein sequence is inserted between the 170th amino acid and the 171st amino acid to obtain a fusion protein, which can obtain a fusion protein with immunogenicity; or coupled with a small molecule drug to obtain a small molecule drug complete antigen with immunogenicity. Based on the carrier protein with the adjuvant function of the present application, the adjuvant activity of flagellin is retained while the immunogenicity and antigenicity are reduced, which can be applied to low molecular weight fusion proteins, polypeptide fusion proteins, small molecule drugs, and can enhance the immunogenicity of the latter, and has important significance in the application of subunit vaccine, polypeptide vaccine, and small molecule drug carrier preparation.

[0031] For example, a human CD24 protein sequence is inserted between the 170th amino acid and the 171st amino acid to obtain a fusion protein, i.e. the sequence shown in SEQ ID NO. 3. A human CD47 protein sequence is inserted between the 170th amino acid and the 171st amino acid to obtain a fusion protein, i.e. the sequence shown in SEQ ID NO. 4.

[0032] SEQ ID NO. 1: MLGINSNINSLVAQQNLNGSQNALSQAITRLSSGKRINSAADDAAGLAISTRMQTQINGLNQGVSNANDGVSMIQTASSALSSLTNSLQRIRQLAVQASTGTMSSTDQAALQQEVAQQIQEVNRIASQTTYNGTNILNGNAGIVSFQVGANVGQTISLDLSQSMSAAKIGLDISTVSGANVAMVSIDNALQTVNNVQAQLGAAQNRFTAIATAQQAESTDLSSAQSQITDANFAQETANMSKNQVLQAGISVLAQANSLPQQ SEQ ID NO.2: MLGINSNINSLVAQQNLAGSQNALSQAITRLSSGKRINSAADDAAGLAISTRMQTQINGLNQGVSNANDGVSMIQTASSALSSLTNSLQRIRQLAVQASTGTMSTTDQAALQQEVSQQIQEVNRIASQTTYAGTNILDGSAGIVSFQVGANVGQTISLDLSQSMSAAKIGLDISTVSGANVAMVSIDNALQTVNNVQAALGAAQNRFTAIATSQQAESTDLSSAQSQITDANFAQETAAMSKNQVLQQAGISVLAQANSLPQQ SEQ ID NO.3: MLGINSNINSLVAQQNLAGSQNALSQAITRLSSGKRINSAADDAAGLAISTRMQTQINGLNQGVSNANDGVSMIQTASSALSSLTNSLQRIRQLAVQASTGTMSTTDQAALQQEVSQQIQEVNRIASQTTYAGTNILDGSAGIVSFQV GANVGQTISLDLSQSMSAAKIGSETTTGTSSNSSQSTSNSGLAPNPTNATTKAAGLDISTVSGANVAMVSIDNALQTVNNVQAALGAAQNRFTAIATSQQAESTDLSSAQSQITDANFAQETAAMSKNQVLQQAGISVLAQANSLPQQ SEQ ID NO.4: MLGINSNINSLVAQQNLAGSQNALSQAITRLSSGKRINSAADDAAGLAISTRMQTQINGLNQGVSNANDGVSMIQTASSALSSLTNSLQRIRQLAVQASTGTMSTTDQAALQQEVSQQIQEVNRIASQTTYAGTNILDGSAGIVSFQVGANVGQTISLDLSQSMSAAKIGQLLFNKTKSVEFTFCN DTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIILDISTVSGANVAMVSIDNALQTVNNVQAALGAAQNRFTAIATSQQAESTDLSSAQSQITDANFAQETAAMSKNQVLQQAGISVLAQANSLPQQ.

[0033] The present invention is further described in detail below through specific examples. The following examples are only used to further illustrate the present invention and should not be construed as limiting the present invention.

[0034] Unless otherwise noted, the experimental methods in the following examples are conventional methods, performed according to the techniques and conditions described in literature in the field or according to product specifications. Materials and reagents used in the following examples were commercially available unless otherwise noted. Balb / c mice: Guangdong Provincial Laboratory Animal Center, 5-6 weeks old. Example

[0035] 1. Construction of carrier protein plasmids with adjuvant function and protein expression 1. Construction and 3D structure simulation of carrier protein and fusion protein The protein structure prediction server Phyre2 was used to obtain the simulated predicted protein structure by importing the amino acid sequences of the truncated carrier protein, the carrier protein after amino acid mutation, the carrier protein fused with the human CD24 peptide (UniProt accession number: P25063, amino acid sequence 27-59), and the carrier protein fused with the human CD47 fragment (UniProt accession number: Q08722, amino acid sequence 19-127). The results are shown in Figure 2. Figure 1The truncated carrier protein, the carrier protein after amino acid mutation, the carrier protein fused with human CD24 peptide segment, and the carrier protein fused with human CD47 all maintain the natural and relatively independent spatial conformation compared with the full-length FliC protein (UniProt accession number: A0AAE8T591). The carrier protein after amino acid mutation reduces the glycosylation site in the eukaryotic expression system, and can be expressed by prokaryotic expression or fused with other proteins and expressed by eukaryotic expression system.

[0036] 2. Expression vector construction The truncated carrier protein prokaryotic expression vector pET-32a(+)-F was constructed by using the protein full gene synthesis sequence method with the Burkholderia cenocepacia flagellum FliC protein (GenBank, QCY01824.1) as the template, the TrxA tag was fused at the N-terminus, and the 6xHis tag was used for affinity purification at the C-terminus. The eukaryotic expression vector pcDNA3.1(+)-CD24-F of the fusion protein was constructed by using the protein full gene synthesis sequence method to insert the human CD24 peptide segment (UniProt accession number: P25063, amino acid sequence 27-59) protein sequence between the 170th amino acid and the 171st amino acid of the carrier protein after amino acid mutation, and the 6xHis tag was used for affinity purification at the C-terminus.

[0037] The eukaryotic expression vector pcDNA3.1(+)-CD47-F of the fusion protein was constructed by using the protein full gene synthesis sequence method to insert the human CD47 fragment (UniProt accession number: Q08722, amino acid sequence 19-127) protein sequence between the 170th amino acid and the 171st amino acid of the carrier protein after amino acid mutation, and the 6xHis tag was used for affinity purification at the C-terminus.

[0038] After the prokaryotic expression vector was transfected into E. coli (BL21) and cultured, the recombinant carrier protein F was induced to express by IPTG at low temperature. After the eukaryotic expression vector was transiently transfected into HEK293 cells, the recombinant carrier fusion proteins CD24-F and CD47-F were expressed.

[0039] 3. Prokaryotic expression and purification of carrier protein F The pET-32a(+)-F plasmid was transfected into E. coli (BL21) competent cells, 100 μL of which was inoculated and cultured on an AMP-resistant TSA plate, and incubated at 37°C overnight. The next day, a single colony was picked up with a inoculation loop and inoculated into 10 mL of AMP-resistant TSB liquid medium, and incubated at 37°C for 8 hours. The bacterial solution was diluted at a ratio of 1:50 and transferred to AMP-resistant TSB liquid medium, and incubated at 37°C overnight until the OD600 reached 0.6-0.8. 600= about 0.8. Under the temperature condition of 16℃, IPTG was added to a final concentration of 0.5mM to induce culture for 12 hours. The bacteria were collected by centrifugation at 9000 RPM for 5 minutes. The bacterial pellet was added to the lysis buffer and resuspended, then ultrasonically lysed and centrifuged. The lysate supernatant was filtered through a 0.45μm filter membrane and purified by adding cobalt beads. The purified protein was removed from the sample by endotoxin removal kit. The purified sample was detected and analyzed by SDS-PAGE. The results are shown as follows. Figure 2 As shown, the theoretical molecular weight of the protein is approximately 45 kDa.

[0040] Figure 2 The results shown are consistent with the expected results of the theoretical molecular weight of the protein, indicating that the carrier protein F was successfully cloned and expressed in this example.

[0041] 4. Eukaryotic Expression and Purification of Vector Fusion Protein CD24-F HEK293 cells in logarithmic growth phase were diluted to 2.0 × 10 6 cells / mL concentration. Add 5mL of KPM and 100μg of sterile plasmid pcDNA3.1(+)-CD24-F to a 15mL sterile centrifuge tube, and gently blow to mix; take another centrifuge tube, add 5mL of KPM and 500μL of TA-293 transfection reagent, gently blow to mix, then add to the centrifuge tube containing the plasmid, gently blow to mix, and let it stand for 10 minutes to obtain the pcDNA3.1(+)-CD24-F plasmid-vector complex; add the plasmid-vector complex to 100mL of HEK293 cells, put it back in the CO2 constant temperature shaker for shaking culture. After 5-6 days of culture, collect the cells by centrifugation, resuspend the cell pellet in lysis buffer, lyse, centrifuge, filter the lysate supernatant with a 0.45μm filter membrane, add cobalt beads for purification, and analyze the purified sample by SDS-PAGE. The results are as follows Figure 3 As shown, the theoretical molecular weight of the protein is approximately 33.4 kDa.

[0042] Figure 3 The results shown are consistent with the expected results of the theoretical molecular weight of the protein, indicating that the fusion protein CD24-F was successfully cloned and expressed in this example.

[0043] 5. Eukaryotic Expression and Purification of Vector Fusion Protein CD47-F HEK293 cells in logarithmic growth phase were diluted to 2.0 × 10 6cells / mL concentration. Add 5mL of KPM and 100μg of sterile plasmid pcDNA3.1(+)-CD47-F to a 15mL sterile centrifuge tube, and gently blow to mix; take another centrifuge tube, add 5mL of KPM and 500μL of TA-293 transfection reagent, gently blow to mix, then add to the centrifuge tube containing the plasmid, gently blow to mix, and let it stand for 10 minutes to obtain the pcDNA3.1(+)-CD47-F plasmid-vector complex; add the plasmid-vector complex to 100mL of HEK293 cells, put it back in the CO2 constant temperature shaker for shaking culture. After 5-6 days of culture, collect the cells by centrifugation, resuspend the cell pellet in lysis buffer, lyse, centrifuge, filter the lysate supernatant with a 0.45μm filter membrane, add cobalt beads for purification, and analyze the purified sample by SDS-PAGE. The results are as follows Figure 4 As shown, the theoretical molecular weight of the protein is approximately 42.5 kDa.

[0044] Figure 4 The results shown are consistent with the expected results of the protein's theoretical molecular weight, indicating that the fusion protein CD47-F was successfully cloned and expressed in this case.

[0045] 2. Conjugation of carrier proteins and small molecule drugs The small molecule drug carbamazepine (CMZP) was conjugated to the purified carrier protein F. 75 μg of carboxylated CMZP hapten (S. Zhou, et al., 2020, Journal of Chromatography) was dissolved in 100 μL of DMF and activated with EDC / NHS (1:1 molar ratio) for 1 hour. The activated CMZP hapten was then added dropwise to 1 mg of carrier protein F for 2 hours. The reaction was then dialyzed against 0.02 M PBS buffer for 48 hours to obtain the small molecule drug CMZP-F conjugated to the carrier protein. CMZP-F conjugated to OVA and BSA antigens were prepared using the same method.

[0046] 3. Evaluation of the immune adjuvant effect of carrier proteins 1. In vitro TLR5 receptor activity assay of carrier protein The TLR5 receptor activity of the recombinant vector protein was tested using human colorectal adenocarcinoma cells (Caco2) that can express TLR5 receptor as an in vitro cell model. Caco2 cells were grown to confluence in T75 cell culture flasks and plated at 1.5×10 cells per well. 5Cells 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 stimulate Caco2 cells at a final concentration of 1 μg / mL and cultured at 37°C for 12 hours. Untreated Caco2 cells served as negative controls. The cell culture supernatant was aspirated and the IL-8 concentration in the cell supernatant was detected using a human IL-8 ELISA kit (Sino Biological, Cat. No. SEK10098). The results were as follows: Figure 5 shown.

[0047] Figure 5 The results showed that carrier protein F and recombinant fusion proteins CD24-F and CD47-F could stimulate Caco2 to secrete IL-8, indicating that carrier protein F and recombinant fusion proteins CD24-F and CD47-F had good TLR5 receptor activity.

[0048] 2. Detection of anti-CD24 antibody levels in sera of immunized mice The eukaryotic expression and purification of the fusion protein CD24-F was diluted with normal saline, and 5 μg was injected intramuscularly to immunize 6-8 week old Balb / c female mice. The prokaryotic expression and purification of the carrier protein F was diluted with normal saline, and 5 μg was injected intramuscularly to immunize 6-8 week old Balb / c female mice as a control. After the first immunization, booster immunization was performed every two weeks for a total of 3 immunizations. On the 7th day after the last immunization, blood was collected from the tail of the mouse, and the serum was separated. The indirect ELISA method was used to detect the specific IgG antibody titer against CD24 protein and carrier protein F in the mouse serum. Indirect ELISA titer detection method: (1) Antigen coating: Purified recombinant carrier protein F and CD24 protein (Sino Biological, Cat. No. 11030-H02H) were diluted with 0.05 M carbonate buffer at pH 9.6 to a protein concentration of 2 μg / mL. 100 μL / well was added to the ELISA plate and coated overnight at 4°C.

[0049] (2) After completing step (1), take the ELISA plate, wash it four times with PBST solution, then add blocking solution (200 μL / well) and incubate at 37°C for 2 hours. Blocking solution: PBST solution containing 1g / 100mL BSA.

[0050] (3) After completing step (2), take the ELISA plate, wash it four times with PBST solution, then add mouse serum antibody dilution solution (100 μL / well) and incubate at 37°C for 1 hour. Mouse serum antibody dilution: Take the immune mouse serum, dilute it 100 times with pH 7.4, 0.02M PBS buffer, and then perform a 10-fold gradient dilution.

[0051] (4) After completing step (3), take the ELISA plate, wash it four 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.

[0052] (5) After completing step (4), take the ELISA plate, wash it with PBST solution 4 times, then add TMB substrate reaction solution to develop color for 10 minutes, then add 2M sulfuric acid solution to stop the color development, and then use an ELISA reader to measure the absorbance at 450nm. The results are as follows Figure 6 shown.

[0053] Figure 6 Figure A shows that the fusion protein CD24-F immunization group can produce high titer specific IgG antibodies against CD24 protein, while the control carrier protein F immunization group did not produce specific IgG antibodies against CD24 protein; Figure B shows that the specific IgG antibody titer against carrier protein F produced by the fusion protein CD24-F immunization group and the control carrier protein F immunization group is extremely low. Figure 6 The results showed that carrier protein F has low self-antigenicity while having adjuvant function.

[0054] 3. Detection of anti-CD47 antibody levels in sera of immunized mice The eukaryotic expressed and purified fusion protein CD47-F was diluted with physiological saline, and 5 μg was injected intramuscularly to immunize 6-8 week old Balb / c female mice. CD47 protein (Sino Biologics, Catalog No.: 12283-H02H) was fully emulsified with Freund's complete adjuvant (Sigma, Catalog No.: F5881) or Freund's incomplete adjuvant (Sigma, Catalog No.: F5506) at a volume ratio of 1:1, and 50 μg was injected subcutaneously to immunize 6-8 week old BALB / c female mice. Mice injected with only 50 μg of CD47 protein served as controls. After the first immunization, booster immunizations were performed every two weeks for a total of 3 immunizations. On the 7th day after the last immunization, blood was collected from the tail of the mouse, and the serum was separated. The titer of specific IgG antibodies against CD47 protein in the mouse serum was detected by indirect ELISA. Indirect ELISA titer detection method: (1) Antigen coating: Dilute CD47 protein (Sino Biological, Cat. No. 12283-H02H) with 0.05 M carbonate buffer (pH 9.6) to a protein concentration of 2 μg / mL. Add 100 μL / well to the ELISA plate and coat overnight at 4°C.

[0055] (2) After completing step (1), take the ELISA plate, wash it four times with PBST solution, then add blocking solution (200 μL / well) and incubate at 37°C for 2 hours. Blocking solution: PBST solution containing 1g / 100mL BSA.

[0056] (3) After completing step (2), take the ELISA plate, wash it four times with PBST solution, then add mouse serum antibody dilution solution (100 μL / well) and incubate at 37°C for 1 hour. Mouse serum antibody dilution: Take the immune mouse serum, dilute it 100 times with pH 7.4, 0.02M PBS buffer, and then perform a 10-fold gradient dilution.

[0057] (4) After completing step (3), take the ELISA plate, wash it four 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.

[0058] (5) After completing step (4), take the ELISA plate, wash it with PBST solution 4 times, then add TMB substrate reaction solution to develop color for 10 minutes, then add 2M sulfuric acid solution to stop the color development, and then use an ELISA reader to measure the absorbance at 450nm. The results are as follows Figure 7 shown.

[0059] Figure 7 The results showed that the antibody titers against CD47 protein were higher in the group immunized with the fusion protein CD47-F than in the group immunized with the CD47 protein plus adjuvant, while the control group of CD47 protein without adjuvant produced no specific IgG antibodies against CD47 protein. This result indicates that the adjuvant function of carrier protein F as a fusion protein is superior to that of traditional adjuvants.

[0060] 4. Detection of Anti-Small Molecule Drug Antibody Levels in Sera of Immunized Mice The small molecule drug-coupled protein CMZP-F was diluted with normal saline, and 10 μg was injected intramuscularly to immunize 6-8 week old Balb / c female mice. The OVA-coupled small molecule drug protein CMZP-OVA was fully emulsified with Freund's complete adjuvant (Sigma, Catalog No.: F5881) or Freund's incomplete adjuvant (Sigma, Catalog No.: F5506) at a volume ratio of 1:1, and 50 μg was injected subcutaneously to immunize 6-8 week old BALB / c female mice. The mice in the group injected with only 50 μg of CMZP-OVA served as the control group. After the first immunization, booster immunizations were performed every two weeks for a total of 3 immunizations. On the 7th day after the last immunization, blood was collected from the tail of the mouse, and the serum was separated. The indirect ELISA method was used to detect the specific IgG antibody titer against CMZP in the mouse serum. Indirect ELISA titer detection method: (1) Antigen coating: Take the CMZP-BSA conjugated antigen and dilute it with pH 9.6, 0.05M carbonate buffer to a protein concentration of 2 μg / mL. Add 100 μL / well to the ELISA plate and coat it at 4°C overnight.

[0061] (2) After completing step (1), take the ELISA plate, wash it four times with PBST solution, then add blocking solution (200 μL / well) and incubate at 37°C for 2 hours. Blocking solution: PBST solution containing 1g / 100mL BSA.

[0062] (3) After completing step (2), take the ELISA plate, wash it four times with PBST solution, then add mouse serum antibody dilution solution (100 μL / well) and incubate at 37°C for 1 hour. Mouse serum antibody dilution: Take the immune mouse serum, dilute it 100 times with pH 7.4, 0.02M PBS buffer, and then perform a 10-fold gradient dilution.

[0063] (4) After completing step (3), take the ELISA plate, wash it four 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.

[0064] (5) After completing step (4), take the ELISA plate, wash it with PBST solution 4 times, then add TMB substrate reaction solution to develop color for 10 minutes, then add 2M sulfuric acid solution to stop the color development, and then use an ELISA reader to measure the absorbance at 450nm. The results are as follows Figure 8 shown.

[0065] Figure 8 The results showed that the antibody titer against CMZP produced by the immunization group with small molecule drug coupling protein CMZP-F was higher than that of the immunization group with CMZP-OVA plus adjuvant, while the CMZP-OVA control group without adjuvant did not produce specific IgG antibodies against CMZP; this result indicates that carrier protein F, as a small molecule drug carrier protein, has the adjuvant function and is superior to traditional carrier proteins.

[0066] The above content is a further detailed description of the present application in conjunction with specific implementation methods, and the specific implementation of the present application cannot be considered to be limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present 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.

3. The carrier protein according to claim 2, characterized in that: The mutation is at least one of the following: (1) Asparagine at positions 18, 131, and 238 were mutated to alanine; (2) Asparagine at position 137 mutated to aspartic acid, and asparagine at position 139 mutated to serine.

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

2.

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

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

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

4.

8. A nucleic acid encoding the carrier protein according to any one of claims 1 to 4 or the fusion protein according to any one of claims 5 to 7.

9. A recombinant plasmid containing the nucleic acid according to claim 8.

10. A cell transformed with the recombinant plasmid according to claim 9.

11. Use of the carrier protein according to any one of claims 1 to 4, the fusion protein according to any one of claims 5 to 7, the nucleic acid according to claim 8, the recombinant plasmid according to claim 9, or the cell according to claim 10 in the preparation of a subunit vaccine, a polypeptide vaccine, or a small molecule drug carrier.

12. An immunogenic small molecule drug, characterized in that: It is formed by coupling the carrier protein according to any one of claims 1 to 4, or the fusion protein according to any one of claims 5 to 7, with a small molecule drug.

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