Preparation and application of oligopeptide immune carrier protein

Abstract

The invention discloses preparation and application of oligopeptide immune carrier protein, and belongs to the technical field of biology. The method comprises the following steps: fusing a gene sequence for coding oligopeptide and a gene sequence for coding ferritin through a chemical method by utilizing the characteristics that oligopeptide molecules are small in mass and ferritin is self-assembled to form a polymer, and then cloning the fused gene into a prokaryotic biological carrier for expression; the recombinant protein is purified by using an expression system which is not limited to prokaryotes such as escherichia coli and eukaryotes. The protein has the advantages of low preparation cost, strong immunogenicity and the like, and lays a foundation for obtaining a high-affinity anti-oligopeptide nano antibody.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the preparation and application of a short peptide immune carrier protein. Background Technology

[0002] Short peptides, as antigenic epitopes, are difficult to directly induce the production of high-titer neutralizing antibodies due to their low molecular weight and weak immunogenicity. To improve their immunogenicity, existing technologies typically employ carrier protein conjugation strategies, such as keyhole lithocyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA), or virus-like particles (VLPs). These carriers can covalently conjugate short peptides onto their surfaces, thereby enhancing T-cell-dependent B-cell responses. However, such methods have significant limitations: proteins like KLH have limited sources and high costs, and their expression and purification processes are complex, resulting in low yields; while viral vectors possess excellent immunostimulatory properties, they may present issues with biosafety and batch-to-batch consistency. Furthermore, traditional carriers are insufficient in terms of the number, spatial conformation, and density of epitopes displayed, making it difficult to efficiently mimic the multivalent aggregation state of surface antigens from natural pathogens, thus limiting the efficient cross-linking and activation of B-cell receptors (BCRs).

[0003] Ferritin is a cage-like protein particle formed by the self-assembly of 24 subunits. These subunits can be directionally modified through genetic engineering to display exogenous short peptide antigenic epitopes at specific sites. After self-assembly, the protein forms a highly ordered, high-density epitope array on its outer surface, exhibiting a virus-like particle (VLP) structure. This not only effectively mimics the polymerized state of natural antigens on pathogen surfaces but also promotes the full exposure of protective epitopes. Furthermore, ferritin particles have a long circulating half-life in vivo, which prolongs antigen presentation time and significantly enhances the strength of humoral immune responses by promoting efficient cross-linking with multiple B cell receptors.

[0004] Although ferritin has shown significant advantages in antigen display and vaccine design, there are currently no reports of its systematic application in the polymerization display and immunogenicity enhancement of short peptide antigens. On the other hand, research on high-affinity nanobodies targeting linear epitopes of short peptides is still in its early stages, with the main limiting factors being the insufficient immunogenicity of short peptides and the lack of efficient and controllable multivalent antigen presentation systems. Summary of the Invention

[0005] To address the aforementioned technological gaps, the present invention aims to provide a short peptide antigen display strategy based on ferritin carriers: a gene sequence encoding a target short peptide is fused with a gene sequence encoding ferritin using a flexible linker; the fused gene is then cloned into a prokaryotic expression vector; purified protein is prepared using systems not limited to prokaryotes such as *E. coli*, eukaryotes, and yeast; the resulting recombinant protein is used to immunize camels, aiming to provide a reliable immunogenic basis for further screening and preparation of high-affinity nanobodies targeting linear epitopes of short peptides; simultaneously, a short peptide epitope titer detection protein is provided, and the application effect of the short peptide immunocarrier protein is detected by enzyme-linked immunosorbent assay (ELISA).

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a short peptide immune carrier protein, the short peptide immune carrier protein comprising ferritin and a short peptide antigenic epitope protein, which are linked by a flexible linker chain, and its amino acid sequence is shown in SEQ ID NO.1.

[0007] Furthermore, the short peptide immunocarrier protein self-assembles into a 24-tetrameric particle, and multiple copies of the short peptide antigen epitope are presented on its outer surface, which can be used to immunize camels.

[0008] Secondly, the present invention provides a short peptide epitope potency detection protein, wherein the short peptide epitope potency detection protein comprises a short peptide antigen epitope protein and a carrier protein containing a GST tag, and its amino acid sequence is shown in SEQ ID NO.2.

[0009] Furthermore, the short peptide epitope titer detection protein is used in enzyme-linked immunosorbent assay (ELISA) to detect serum antibody titers.

[0010] Thirdly, the present invention provides a recombinant expression plasmid that inserts a gene encoding the aforementioned short peptide immune vector protein or short peptide epitope titer detection protein.

[0011] Fourthly, the present invention provides a recombinant engineered bacterium carrying the above-mentioned recombinant plasmid.

[0012] Fifthly, the present invention provides a method for preparing the above-mentioned short peptide immunocarrier protein or short peptide epitope titer detection protein, comprising the following steps: Preparation of short peptide immunocarrier protein: The gene encoding the above-mentioned short peptide immunocarrier protein was inserted into the pET26b(+) expression vector to obtain a recombinant expression vector, which was transformed into Escherichia coli BL21(DE3). Positive clones were obtained by screening with kanamycin, and the obtained positive clones were inoculated into culture medium for culture. Protein expression was induced by IPTG, the bacterial cells were collected, and after disruption, the short peptide immunocarrier protein was obtained by nickel column affinity chromatography.

[0013] Preparation of short peptide epitope potency detection protein: The gene encoding the above-mentioned short peptide epitope potency detection protein was inserted into the pET42a(+) expression vector to obtain a recombinant expression vector, which was transformed into Escherichia coli BL21(DE3). Positive clones were obtained by screening with kanamycin, and the obtained positive clones were inoculated into culture medium for culture. Protein expression was induced by IPTG, the bacterial cells were collected, and after disruption, the short peptide epitope potency detection protein was obtained by nickel column affinity chromatography.

[0014] Furthermore, the induction conditions were as follows: IPTG at a final concentration of 0.5-1.5M was added to the bacterial culture for induction for 5-7 hours.

[0015] Further, the nickel column affinity chromatography purification process is as follows: equilibrate the nickel column to 3-7 column volumes with equilibration buffer, load the protein sample onto the nickel column affinity chromatography column, elute with PBS buffer containing 15-25 mM imidazole to 3-7 column volumes to elute impurities, perform gradient elution with PBS buffer containing 40-500 mM imidazole, collect the eluent to obtain the protein solution.

[0016] Sixthly, the present invention provides the application of the above-mentioned short peptide immunocarrier protein in the preparation of nanobodies.

[0017] Furthermore, the purified short peptide immunocarrier protein was desalted and sterilized, then emulsified with Freund's adjuvant, and subcutaneously immunized at multiple sites on one healthy 2-3 year old Bactrian camel with no underlying disease, no immune background, and a moderate body size.

[0018] Furthermore, the specific immunization procedure is as follows: immunize once a week for a total of 7 immunizations. Mix 1 mg of short peptide immunocarrier protein with Freund's adjuvant at a 1:1 ratio and inject. At the same time, collect 3-6 mL of camel peripheral blood for antibody titer testing.

[0019] In a seventh aspect, the present invention provides the application of the above-mentioned short peptide immunocarrier protein or short peptide epitope titer detection protein in antibody titer detection.

[0020] Further, detection was performed using an enzyme-linked immunosorbent assay (ELISA), with the following specific steps: (1) The short peptide immunocarrier protein and the short peptide epitope titer detection protein were coated on the ELISA plate, 90~110μL / well, and the control group was BSA; (2) Wash the ELISA plate and block it with blocking solution; (3) Wash the ELISA plate and perform primary antibody binding: Dilute the camel serum with 5% skim milk powder according to 7 dilution gradients: 1:1000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000. Mix the serum with a vortex mixer, 90~110μL / well, and let stand at 37℃ for 1h. (4) Wash the microplate and bind the secondary antibody: Use secondary antibody (Rabbit Anti-Camelid VHH Antibody [HRP], concentration 1:5000, diluted in 5% skim milk powder), 90~110μL / well, 37℃, stand for 1h; (5) Wash the microplate, add TMB chromogenic solution to the wells in the dark, 90~110μL / well, and let stand at 37℃ in the dark for 5min; (6) Terminate the reaction with 2M HCl, 48~52μL / well.

[0021] Further, the washing conditions were as follows: wash the plate 5 times with 0.1% PBST (1 mL Tween-20 dissolved in 1 L 1×PBS), 240~260 μL / well, and pat dry on absorbent paper to remove residual liquid from the wells.

[0022] Furthermore, the sealing solution is 3%~5% skim milk powder, 240~260μL / well, and the sealing treatment conditions are 37℃, standing for 1h~1.5h.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention uses a flexible linker to tandem multiple short peptide sequences at the N-terminus and C-terminus of ferritin, utilizing its self-assembly property of naturally forming multimers to construct a fusion protein, which significantly improves the immunogenicity of the short peptides.

[0024] 2. Through desalting and sterilization processes, the high purity and safety of the immunogen are ensured.

[0025] 3. Freund's adjuvant emulsification technology is used to enhance the antigenicity of the immunogen and improve antibody production efficiency.

[0026] 4. Healthy young adult camels are selected for immunization. They are large in size, have a sound immune system, and are more likely to produce high-affinity nanobodies.

[0027] 5. Use subcutaneous multi-site immunization, combined with booster immunizations every 1-2 weeks, to ensure a continuous increase in antibody titer.

[0028] 6. Serum antibody titers can be detected using short peptide immunocarrier proteins and short peptide epitope titer detection proteins, enabling efficient and accurate monitoring of immunization efficacy. Attached Figure Description

[0029] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0030] Figure 1 This is a schematic diagram of the gene structure design of a short peptide immunotransmitter protein.

[0031] Figure 2 A schematic diagram of the gene structure design for a short peptide epitope titer detection protein.

[0032] Figure 3 This is a plasmid gene map of a short peptide immunovector protein.

[0033] Figure 4 This is a plasmid gene map of a protein for detecting the titer of short peptide epitopes.

[0034] Figure 5 SDS-PAGE results of protein expression levels of whole protein, soluble protein, and insoluble protein after 2, 4, and 6 hours of IPTG induction, without induction.

[0035] Figure 6 The purification results of short peptide immunocarrier protein (A) and short peptide epitope titer detection protein (B) are shown in Figure A. In Figure A, M: pre-stained protein marker, supernatant: protein solution before nickel column affinity chromatography, flow-through: protein solution after nickel column affinity chromatography, and other bands represent protein solutions eluted with imidazole buffer at different concentrations from 20 to 120 mM. In Figure B, M: pre-stained protein marker, and other bands represent protein solutions eluted with imidazole buffer at different concentrations from 120 to 500 mM.

[0036] Figure 7 The results of ELISA serum titer assays for short peptide immunocarrier protein (A) and short peptide epitope titer detection protein (B) are presented. Detailed Implementation

[0037] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0038] LB liquid culture medium formula: tryptone: 4g; yeast extract: 2g; sodium chloride: 4g; RO water to a final volume of 400mL.

[0039] LB solid medium formula: Agar powder: 4-5g; Tryptone: 3g; Yeast extract: 1.5g; Sodium chloride (NaCl): 3g; RO water to a final volume of 300mL.

[0040] Example 1: Gene Synthesis – Design of Expression Vectors for Short Peptide Immunotransferases and Short Peptide Epitope Titer Detection Proteins A nucleotide sequence encoding a short peptide epitope protein was selected and linked with a nucleotide residue sequence encoding ferritin to form a fusion gene. Figure 1 The amino acid sequence of Myc is: EQKLISEEDL; the amino acid sequence of 6×His is: HHHHHH; ferritin is a carrier protein derived from Pyrococcus furiosus, and its amino acid sequence is: LSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYDRNGRVELDEIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEEASVKKILDKLFA; the amino acid sequence of the flexible linker peptide Linker1 is: GGGGS; the amino acid sequence of the short peptide immunocarrier protein is as follows (SEQ ID NO.1): GGGGSHHHHHHGGGGSEQKLISEEDLGGGGSHHHHHHGGGGSGGGGS LSERMLKALNDQLNRELYSAY LYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYDRNGRVELDEIPKPPKEWESPLKAFEAAYEHEKFIS KSIYELAALAEEEKDYSTRAFLEWFINEQVEEEASVKKILDKLFA GGGGSHHHHHHGGGGSEQKLISEEDLGGGGSHHHHHH; A fusion gene is formed by linking the nucleotide sequence encoding the short peptide epitope titer detection protein with the nucleotide sequence encoding the GST tag. Figure 2The amino acid sequence of GST is: MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKS; the amino acid sequence of 6×His is: HHHHHH; the amino acid sequence of the flexible linker peptide Linker2 is: SDGSTSGSG; the amino acid sequence of Myc is: EQKLISEEDL; the amino acid sequence of the flexible linker peptide Linker3 is: SGGGG; the amino acid sequence of the short peptide epitope titer detection protein is as follows (SEQ ID NO.2): MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQS MAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLN GDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKS SDGSTSGSGHHHHHHSGGGGEQKLISEEDLSGGGGHHHHHH; The aforementioned genes were synthesized by Nanjing Genscript Biotech Co., Ltd. and Jiutian Gene Technology (Tianjin) Co., Ltd.

[0041] The fusion gene was cloned into the restriction sites of pET26b(+) and pET42a(+) respectively to construct expression vectors, which were named short peptide immunovector protein recombinant plasmids Immunity protein_pET26b(+). Figure 3 ) and short peptide epitope titer detection protein recombinant plasmid Peptide test protein_pET42a(+) Figure 4 ).

[0042] The recombinant plasmids Immunity protein_pET26b(+) and Peptide test protein_pET42a(+) were transformed into E. coli BL21(DE3): 1 µL of plasmid with a concentration of 100 ng / µL was taken, and 50 µL of BL21(DE3) competent cells (purchased from Shenzhen Kangti Life Pharmaceutical Technology Co., Ltd.) was added to the bottom of the tube with the pipette tip. The mixture was gently mixed and incubated on ice for 20 min. The centrifuge tube was then placed in a 42℃ water bath for 80 s heat shock, followed by 5 min on ice. 200 µL of warm SOC medium was added, and the tube was incubated at 37℃ for 30 min. The tube was then transferred to a shaker and incubated at 37℃ and 220 rpm for 1 h. 100 µL of the cultured bacterial solution was spread on an LB-Kan plate and incubated upside down overnight at 37℃.

[0043] Example 2: Detection and purification of short peptide immunocarrier proteins and short peptide epitope titers; protein expression and purification. (1) IPTG-induced expression Positive monoclonal colonies from the above culture plate were picked and inoculated into 10 mL of LB medium containing a final kanamycin concentration of 50 μg / mL. The culture was incubated overnight at 37°C with shaking at 220 rpm. OD values ​​were collected the following day. 600 =1 bacterial culture was used as the pre-induction whole protein sample, and then inoculated at 1:100 into 400 mL LB medium containing kanamycin at a final concentration of 50 μg / mL, and cultured at 37°C and 220 rpm until OD. 600 When the concentration reached 0.6-0.8, IPTG was added to a final concentration of 1M, and OD values ​​were collected at 2h, 4h, and 6h after induction. 600 =2 bacterial culture, store at 4℃.

[0044] SDS-PAGE results showed that protein expression was optimal after 6 hours of induction. Therefore, 400 ml of bacterial culture after 6 hours of induction was centrifuged at 8000 rpm for 10 min at 4°C. The bacteria were collected in two white 250 mL collection bottles. The bacteria were resuspended and dissolved in 0.2 mol / L salt-free PBS according to the bacterial weight (1 g of bacteria corresponds to 5-10 ml of buffer). The bottles were stored at -80°C for purification.

[0045] (2) Protein sample preparation I. OD values ​​of bacterial cultures were measured before induction and at 2, 4, and 6 hours after induction. 600 Value, take OD 600 =2 bacterial suspension samples were retained; II. Collect bacterial cells by centrifugation at 12000 rpm for 10 min at 4℃; III. Resuspend the bacterial cells in 160 μL of 0.02M salt-free PBS (80 μL of salt-free PBS corresponds to 1OD of bacterial cells), wash the bacterial cells, centrifuge at 12000 rpm for 2 min at 4℃, and discard the supernatant. IV. The bacterial pellet was resuspended in 160 μL of 0.02 M salt-free PBS (1 OD of bacteria corresponds to 80 μL of salt-free PBS), and the bacterial cells were sonicated using a handheld sonicator (sonication for 2 seconds, pause for 3 seconds, power 30%). Separate soluble and insoluble components by centrifugation at 4℃, 12000rpm for 10min. VI. Collect the supernatant into a new 1.5 mL EP tube as a soluble protein sample; VII. Resuspend the precipitate in 0.02M salt-free PBS, wash the bacterial cells, centrifuge at 12000 rpm for 10 min at 4℃, discard the supernatant, and then resuspend the precipitate in 160 μL of 0.02M salt-free PBS. This is an insoluble protein sample.

[0046] VIII. Sample Preparation: Take 80 μL of each protein sample, add 20 μL of 5*loading buffer, and lyse at 99℃ for 10 min.

[0047] IX. SDS-PAGE electrophoresis.

[0048] like Figure 5 As shown, the SDS-PAGE electrophoresis lanes represent the protein expression levels of whole protein, soluble protein, and insoluble protein after uninduced protein and IPTG induction for 2 h, 4 h, and 6 h, respectively. The results indicate that the protein electrophoresis bands were thickest and the protein expression levels were highest at 6 h of induction, indicating the best expression effect.

[0049] (3) Purification of recombinant protein by nickel column affinity chromatography I. Repeat the freeze-thaw cycle of the -80℃ bacterial solution at 37℃ and -80℃ three times to completely dissolve the bacteria; II. Add lysozyme to a final concentration of 50 μg / mL, and incubate in a 37°C water bath for 30 min to allow the lysozyme to fully exert its effect of breaking down bacterial cell walls. III. After an ice bath for a period of time, the ice bath is ultrasonically broken up for 10-15 minutes (conditions: 2 seconds of ultrasound, 3 seconds of pause, 15% power). IV. Place in an ice bath for 10 min, then centrifuge at 12000 rpm for 10 min in a 4℃ low-temperature centrifuge. Discard the precipitate and transfer the supernatant into a new sterile 50 mL conical centrifuge tube. V. Repeat step IV multiple times until no obvious precipitate is found after centrifugation; VI. Add an equal volume of 40 mM imidazole to the supernatant to bring the final concentration to 20 mM, then add 3% Tween-20 by mass and pass it through a nickel column. VII. Equilibrate the nickel column to 5 column volumes with equilibration buffer, load the sample onto the column, and after sample loading, elute with 5 column volumes of 20 mM imidazole-containing saline PBS solution to remove contaminating proteins. Then, perform gradient elution with imidazole at concentrations of 40, 80, 120, 240, and 500 mM. Collect the eluent, and separate and purify the recombinant protein, which is the short peptide immunogenic carrier protein and the short peptide epitope titer detection protein. The results are as follows: Figure 6 As shown.

[0050] Example 3: Preparation and titer detection of nanobodies (1) Desalting and sterilizing recombinant protein The purified recombinant protein was desalted and deimidazole-free using a PD-10 desalting column with medical saline as the protein solvent. The desalted recombinant protein was then filtered through a 0.22 μm filter membrane for sterilization. Finally, the protein concentration was determined using a BCA quantitative detection kit for subsequent camel immunization.

[0051] (2) Camel immunization strategy One healthy male Bactrian camel aged 3 years with no underlying diseases, no immune background, and moderate body size was selected. After emulsification, it was injected subcutaneously at multiple sites (1 mg short peptide immunocarrier protein / dose, mixed with Freund's adjuvant in a 1:1 ratio, once a week). 5 mL of peripheral blood was collected after each subcutaneous injection, and a total of 7 immunizations were performed.

[0052] (3) Antibody titer detection The antibody serum titer was detected using the prepared short peptide immunocarrier protein and short peptide epitope titer detection protein, respectively, and the detection was performed by enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows: I. Coating: The short peptide immunocarrier protein and the short peptide epitope titer detection protein were diluted to 2.5 μg / mL with coating buffer and coated with 96-well plates, 100 μL / well. The control group was coated with BSA. The 96-well microplates were sealed with sealing film to prevent liquid evaporation and incubated overnight at 4°C. II. Washing: The next day, discard the antigen coating solution, then wash the plate 3 times with 0.05% PBST (500 μL between 20 dissolved in 1 L 1×PBS), 250 μL / well, and pat dry on absorbent paper to remove residual liquid from the wells. III. Sealing: Use 3%~5% skim milk powder as the sealing solution, 250μL / well, 37℃, and let stand for 1~1.5h; IV. Washing: Discard the blocking solution, then wash the plate three times with 0.05% PBST, 250 μL / well, and pat dry on absorbent paper to remove any residual liquid from the wells.

[0053] V. Primary antibody binding: Camel serum was diluted with 5% skim milk powder at 1:1000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, and 1:128000, for a total of 7 dilution gradients. The solutions were mixed using a vortex mixer, 100 μL / well, and incubated at 37°C for 1 h. VI. Washing: Discard the diluted serum in the wells, then wash the plate 5 times with 0.1% PBST (1 mL Tween 20 dissolved in 1 L 1×PBS), 250 μL / well. Pat the plate dry on absorbent paper to remove any residual liquid in the wells. VII. Secondary antibody conjugation: Add secondary antibody (purchased from GenScript, Rabbit Anti-Camelid VHHAntibody [HRP], concentration 1:5000, diluted in 5% skim milk powder), 100 μL / well, incubate at 37℃ for 1 h; VIII. Washing: Discard the secondary antibody in the wells, then wash the plate 5 times with 0.1% PBST, 250 μL / well, and pat dry on absorbent paper to remove any residual liquid in the wells; IX. Color development: Add 100 μL of TMB color development solution to the wells in the dark, and let stand at 37°C in the dark for 5 min. X. Termination: The reaction was terminated using 2M HCl, 50 μL / well; XI. Reading: Using a microplate reader, read the OD value. 450 The titers of anti-short peptide nanobodies in serum were measured to assess their potency.

[0054] Immunization was performed on a healthy 3-year-old male Bactrian camel with no underlying diseases, a blank immune background, and moderate body size. The protein titer (OD) of the short peptide immunotransmitter reached its peak after the 7th immunization. 450 ≥1), the result is as follows Figure 7 .

[0055] Table 1. Results of protein titer assay for short peptide immunocarrier proteins and short peptide epitopes.

[0056] During continuous immunization, the serum antibody titers of the tested camels showed a significant dose-dependent increase, reaching peak titers after the 7th immunization, indicating that the immunization protocol successfully induced a high-titer antibody response. No significant signal (OD) was detected in the negative control group (BSA-coated). 450 The result was <0.1, confirming that the nanobodies in serum have high specificity for the target epitope.

[0057] In summary, this embodiment demonstrates through rigorous controlled experiments that the nanobodies obtained in the serum specifically target the epitopes, and that the immunization strategy can stably induce high-titer antibody responses, providing a reliable foundation for the subsequent isolation and application of nanobodies.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A short peptide immune carrier protein, characterized in that, The short peptide immunocarrier protein comprises ferritin and a short peptide antigenic epitope protein, which are linked by a flexible linker chain, and its amino acid sequence is shown in SEQ ID NO.

1.

2. A short peptide epitope titer detection protein, characterized in that, The short peptide epitope potency detection protein comprises a short peptide antigen epitope protein and a carrier protein containing a GST tag, the amino acid sequence of which is shown in SEQ ID NO.

2.

3. A recombinant expression plasmid for inserting a gene encoding the short peptide immune vector protein of claim 1 or the short peptide epitope titer detection protein of claim 2.

4. An engineered bacterium carrying the recombinant expression plasmid as described in claim 3.

5. A method for preparing the short peptide immunocarrier protein of claim 1 or the short peptide epitope titer detection protein of claim 2, characterized in that, The gene encoding the short peptide immunocarrier protein of claim 1 or the short peptide epitope titer detection protein of claim 2 is cloned into prokaryotes such as Escherichia coli, eukaryotes, and yeast systems, and protein expression is induced, followed by isolation and purification to obtain the protein.

6. The preparation method according to claim 5, characterized in that, Preparation of short peptide immunocarrier protein: The gene encoding the short peptide immunocarrier protein of claim 1 was inserted into the pET26b(+) expression vector to obtain a recombinant expression vector, which was transformed into Escherichia coli. Positive clones were obtained by screening with kanamycin. The obtained positive clones were inoculated into a culture medium for culture. Protein expression was induced by IPTG. The bacterial cells were collected, lysed, and purified by nickel column affinity chromatography to obtain the short peptide immunocarrier protein. Preparation of short peptide epitope potency detection protein: The gene encoding the short peptide epitope potency detection protein of claim 2 was inserted into the pET42a(+) expression vector to obtain a recombinant expression vector, which was transformed into Escherichia coli. Positive clones were obtained by screening with kanamycin, and the obtained positive clones were inoculated into a culture medium for culture. Protein expression was induced by IPTG, the bacterial cells were collected, and after disruption, the short peptide epitope potency detection protein was obtained by purification by nickel column affinity chromatography.

7. The application of the short peptide immunocarrier protein according to claim 1 in the preparation of nanobodies.

8. The application according to claim 7, characterized in that, Obtain specific nanobodies.

9. The application of the short peptide epitope titer detection protein according to claim 2 in detecting antibody titers in enzyme-linked immunosorbent assay (ELISA).

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