Cat allergen protein self-assembled nanoparticles as well as preparation method and application thereof

Abstract

The invention provides cat allergen protein self-assembled nanoparticles as well as a preparation method and application thereof, and belongs to the technical field of biology. The nanoparticles are formed by displaying cat allergen protein on the surface of a T7 bacteriophage through Spy Tag and Spy Catcher. The invention also provides a preparation method of the nanoparticle. The preparation method comprises the following steps: preparing the recombinant T7 bacteriophage nanoparticle with Spy Tag polypeptide displayed on the surface; preparing a fusion protein formed by the Spy Catcher protein and the cat allergen protein; and mixing the recombinant T7 bacteriophage nanoparticles with the fusion protein to obtain the nanoparticles. According to the invention, the Spy Tag is displayed on the surface of the T7 phage for the first time, and a covalent bond is spontaneously formed by the Spy Tag and the Spy Catcher-Fel d1 fusion protein, so that the efficient assembly of the allergen protein on the T7 phage is realized, the immunogenicity of Fel d1 is remarkably improved, and the development and application prospects are broad.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a self-assembled nanoparticle of feline allergen protein, its preparation method, and its application. Background Technology

[0002] Cats are common companion animals to humans, but also a significant source of allergic reactions. A cat's sebaceous glands, salivary glands, and anal glands secrete various allergenic proteins. These proteins adhere to the surface of the fur when the cat grooms itself and are dispersed into the environment with the fur and dander, thus triggering allergic reactions in those exposed. To date, eight feline allergens (Fel d1-Fel d8) have been identified, with Fel d1 being the primary sensitizing component. Neutralizing allergens with specific antibodies is an effective anti-allergy treatment strategy. However, obtaining natural allergens for immunization using extraction methods is costly and complex, while genetically engineered allergen proteins often result in low antibody titers due to insufficient immunogenicity. Therefore, there is an urgent need to establish a simple, low-cost strategy for preparing allergen proteins that can induce a strong immune response to meet the needs of specific antibody preparation. Summary of the Invention

[0003] The purpose of this invention is to provide a self-assembled nanoparticle of feline allergen protein. By displaying a Spy Tag on the surface of T7 phage and then forming a covalent link with the Spy Catcher-Fel d1 fusion protein, a directional, efficient, and stable nanoparticle-like antigen is achieved, thereby enhancing the immunogenicity of feline allergen protein and significantly improving the immune effect. The preparation method is simple and low in cost.

[0004] The objective of this invention is achieved through the following technical solution: A self-assembled nanoparticle of feline allergen protein was formed by displaying feline allergen protein on the surface of T7 phage using Spy Tag and Spy Catcher.

[0005] In this invention, the cat allergen protein is Fel d1.

[0006] This invention also provides a method for preparing the self-assembled nanoparticles of the feline allergen protein, comprising the following steps: (1) Preparation of recombinant T7 phage nanoparticles displaying Spy Tag peptides on their surface; (2) Preparation of a fusion protein formed by Spy Catcher protein and feline allergen protein; (3) The recombinant T7 phage nanoparticles described in step (1) are mixed with the fusion protein described in step (2), and the cat allergen protein self-assembled nanoparticles are obtained by specific covalent linkage between Spy Tag peptide and Spy Catcher protein.

[0007] In this invention, the recombinant T7 phage displaying the Spy Tag polypeptide is obtained by inserting the coding gene of the Spy Tag polypeptide into the T7-Select 415-1b phage vector and rescuing it through reverse genetics.

[0008] In this invention, the gene sequence encoding the Spy Tag polypeptide is shown in SEQ ID NO:1, and the amino acid sequence of the SpyCatcher polypeptide is shown in SEQ ID NO:8.

[0009] In this invention, the cat allergen protein comprises at least one of Fel d1, Fel d3, Fel d4, Fel d7 or Fel d8.

[0010] In this invention, the amino acid sequence of the feline allergen protein Fel d1 is as shown in SEQ ID NO:3, or a variant that has at least 80% sequence identity with SEQ ID NO:3 and retains the immunogenicity of feline allergen.

[0011] In this invention, the fusion protein formed by the SpyCatcher protein and the feline allergen protein is formed by linking SpyCatcher and Fel d1 together via a linker, the amino acid sequence of which is shown in SEQ ID NO:6.

[0012] In this invention, the fusion protein formed by the Spy Catcher protein and the cat allergen protein is obtained by expression through a prokaryotic expression system or a eukaryotic expression system; preferably, the expression system is an Escherichia coli expression system, a yeast expression system, an insect cell expression system, or a mammalian cell expression system.

[0013] The present invention also provides the application of the self-assembled nanoparticles of the feline allergen protein in the preparation of anti-feline allergen egg yolk antibodies.

[0014] T7 phage display technology can express exogenous peptides or proteins fused onto the capsid protein p10B. This invention is the first to propose displaying a Spy Tag on the surface of T7 phage, which spontaneously forms a covalent bond with the pre-expressed Spy Catcher-Fel d1 fusion protein, achieving efficient assembly of allergen proteins onto T7 phage nanoparticles (see...). Figure 1Compared with existing technologies, this invention not only overcomes the limitation of T7 phage in displaying large protein molecules at high copy numbers, but also significantly enhances the immunogenicity of the allergen protein Fel d1 with its nanoparticle-like structure. Furthermore, through a novel linker design, it was unexpectedly discovered that linkers composed of different amino acids significantly affect the covalent linkage of Spy Tag / Spy Catcher on the T7 phage surface and the immunogenicity of the nanoparticle-like antigen. This strategy overcomes the limitations of T7 phage surface display capabilities and enhances the immunogenicity of allergen proteins with a nanoparticle-like structure. Simultaneously, the in vitro assembly method offers good modularity, making it suitable for other feline allergen proteins or protective antigens of pathogenic microorganisms, and possesses broad development and application prospects. Attached Figure Description

[0015] Figure 1 A roadmap for the preparation of subunit antigen-autogenous nanoparticles.

[0016] Figure 2 This image shows the rescue and identification of T7-Spy Tag phage. Image A is an electrophoresis image of T7-Spy Tag phage PCR identification; lane 1 represents the PCR amplification product of T7 phage without the Spy Tag gene inserted, and lanes 2-4 represent the PCR amplification products of phage plaques. Image B is an SDS-PAGE electrophoresis image of T7-Spy Tag phage culture medium; lane 1 is a 10-fold concentrated T7-Spy Tag phage culture medium (concentration is 1×10⁻⁶). 11 Lane 1 contains T7-Spy Tag phage culture medium (concentration 1×10⁻⁶ pfu / mL), and Lane 2 contains T7-Spy Tag phage culture medium (concentration 1×10⁻⁶ pfu / mL). 10 pfu / mL); C is a Western blot image of T7-Spy Tag phage culture medium, and lane 1 is a 10-fold concentrated T7-Spy Tag phage culture medium (concentration is 1×10⁻⁶ pfu / mL). 11 The p10B fusion protein formed by Spy Tag in the pfu / mL concentration, lane 2 is T7-Spy Tag phage culture medium (concentration 1×10⁻⁶ pfu / mL), lane 2 is T7-Spy Tag phage culture medium (concentration 1×10⁻⁶ pfu / mL). 10 The fusion protein formed by p10B and Spy Tag in (pfu / mL); D is the electron microscopy observation result of T7-SpyTag phage.

[0017] Figure 3Expression and identification of the Spy Catcher-Fel d1 fusion protein. A shows the double enzyme digestion electrophoresis images of each recombinant plasmid vector; lanes 1-3 show the double enzyme digestion products of the recombinant plasmid vectors expressing the fusion proteins Spy Catcher-Fel d1-a, Spy Catcher-Fel d1-b, and Spy Catcher-Fel d1-c, respectively. B shows the PCR identification electrophoresis image of the recombinant rod-like particle; lane 1 is the negative control (the template is DH10Bac without the recombinant vector inserted). E. coli Lanes 2-4 show the templates for the PCR products as recombinant rod cells expressing SpyCatcher-Fel d1-a, SpyCatcher-Fel d1-b, and SpyCatcher-Fel d1-c, respectively. Lane C is a Western blot diagram identifying the SpyCatcher-Fel d1-c fusion protein expression. Lanes 1-3 show the culture supernatant of control cells (SF9 cells not transfected with rod cells), the lysate supernatant of control cells (SF9 cells not transfected with rod cells), and the lysate pellet of control cells (SF9 cells not transfected with rod cells), respectively. Lanes 4-6 show the culture supernatant of cells infected with the recombinant virus expressing SpyCatcher-Fel d1-c, the lysate supernatant of cells infected with the recombinant virus expressing SpyCatcher-Fel d1-c, and the lysate pellet of cells infected with the recombinant virus expressing SpyCatcher-Fel d1-c, respectively. Lane D shows the lysate pellet of each fusion protein after Elution Buffer treatment. 3. Western blot diagram of the eluent obtained by elution. Lanes 1-3 are the purified Spy Catcher-Fel d1-a, Spy Catcher-Fel d1-b, and Spy Catcher-Fel d1-c fusion proteins, respectively.

[0018] Figure 4Preparation and identification of nanoparticle-like allergen proteins. A shows the identification of the binding ability of each fusion protein to T7-Spy Tag phage. Spotting positions 2 and 4 are PBS and T7 phage, respectively, and spotting positions 1 and 3 are Spy Tag protein and T7-Spy Tag phage, respectively. (a): PVDF membranes with samples added at positions 1-4 are immersed in Spy Catcher-Fel d1-a solution. (b): PVDF membranes with samples added at positions 1-4 are immersed in Spy Catcher-Fel d1-b solution. (c): PVDF membranes with samples added at positions 1-4 are immersed in Spy Catcher-Fel d1-c solution. B shows the Western blot identification of the assembled nanoparticle-like antigens. Lane 1 is the BL21 host bacteria, lane 2 is the T7-Spy Tag phage, and lanes 3-5 are Spy Catcher-Feld1-a, Spy Catcher-Fel d1-b, and Spy Catcher-Fel d1-c with T7-Spy Tag phage, respectively. Tag forms self-assembled nanoparticles.

[0019] Figure 5 This study describes the detection of anti-Fel d1 specific antibody titers in laying hen serum and egg yolk. A shows the antibody titer detection in serum; B shows the antibody titer detection in egg yolk. Detailed Implementation

[0020] The present invention will be further illustrated below with reference to specific embodiments, and the technical features and advantages of the present invention will become clearer as the description unfolds. However, the embodiments are merely exemplary and do not constitute any limitation on the scope of protection of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but these modifications are still within the scope of protection of the present invention.

[0021] Example 1: Construction and identification of recombinant T7 phages displaying Spy Tag on their surface 1. Construction of recombinant T7 phage displaying Spy Tag on surface To display the Spy Tag polypeptide in the T7 phage capsid protein, a DNA sequence encoding the Spy Tag (SEQ ID NO.1, amino acid sequence as shown in SEQ ID NO.8) was first synthesized by a commercial company and cloned into the multiple cloning site of the pUC19 vector to obtain the pUC19-Spy Tag plasmid. This sequence contains EcoRI and HindIII restriction endonuclease sites at its 5' and 3' ends, respectively. The pUC19-Spy Tag plasmid and the T7-Select 415-1b phage vector (Novagen) were double-digested with EcoRI and HindIII, and the corresponding fragments were detected and recovered by agarose gel electrophoresis. The recovered Spy Tag fragment was ligated with the T7-Select 415-1b vector using T4 DNA ligase. The ligation product was reacted with the T7 Packaging Extracts (Novagen) system at 22 °C for 2 h, and then the reaction was terminated by adding LB medium. The resulting packaging product was mixed with BL21 host bacteria and plated for amplification. The mixture was then cultured at 37 °C until plaques were formed, thus completing the reverse genetic rescue of the T7-Spy Tag phage.

[0022] 2. Identification of T7-Spy Tag recombinant phage Single-clonal phage plaques were selected and screened by PCR using T7 Select universal primers (including T7 Select up primer: 5'-GGAGCTGTCGTATTCCAGTC-3' and T7 Select down primer: 5'-AACCCCTCAAGACCCGTTTA-3') to identify positive recombinant phages. The total reaction volume was 30 μL, including 0.5 μL of T7 Select up primer, 0.5 μL of T7 Select down primer, 0.5 μL of exudate from a single phage plaque, 15 μL of ExTaq Mix (Takara), and 13.5 μL of ddH2O. The PCR amplification program was as follows: 94 ℃ pre-denaturation for 3 min; 30 cycles of 94 ℃ for 30 s, 54 ℃ for 30 s, and 72 ℃ for 30 s; extension at 72 ℃ for 10 min. The exudate from a single phage plaque was obtained as follows: a 10 μL pipette tip was used to gently touch the phage plaque, and the exudate was collected by siphoning. The amplification products were analyzed by 1.5% agarose gel electrophoresis. Figure 2(A) The results showed that the negative control band (PCR amplification product of T7 phage without the Spy Tag gene inserted) was approximately 200 bp, while the positive recombinant phage band was approximately 300 bp, consistent with the expected fragment size of the Spy Tag sequence insertion. The positive recombinant phage was named T7-Spy Tag phage.

[0023] Glycerin cryopreservation E. coli BL21 strain was streaked onto a flat surface of LB agar medium and incubated overnight at 37°C; [The text abruptly ends here, likely due to an incomplete sentence or missing information.] E . coli A single colony of BL21 was inoculated into 5 mL of LB liquid medium and incubated overnight at 37 °C with shaking at 200 rpm. 3 mL of the overnight culture was then inoculated into 300 mL of LB liquid medium and incubated until OD reached [growth rate]. 600 The concentration of T7-Spy Tag phage was approximately 0.8. The culture was inoculated at 37 °C with shaking at 100 rpm for 2-3 hours, until the bacterial suspension changed from turbid to clear. The phage was recovered using PEG-NaCl precipitation, and the titer was determined using the double-layer agar sandwich method. The phage concentration was adjusted to 1 × 10⁸ with PBS buffer. 10 pfu / mL. SDS-PAGE was used to detect the recovered T7-Spy Tag phage; Western blot was used to detect the fusion protein formed by the phage capsid protein p10B and the Syp Tag using anti-T7 tag-HRP secondary antibody (Novagen). Results are as follows. Figure 2 As shown in Figures B and C, a single target band is displayed at 43 kD, with the size consistent with expectations, indicating that the Spy Tag protein is correctly displayed on the surface of T7 phage.

[0024] Take 20 μL of the recovered T7-Spy Tag phage, drop it onto a 200-mesh carbon-coated copper grid, and after adsorption and fixation, perform negative staining with 3% phosphotungstic acid. Observe the morphology of the recombinant phage under a scanning electron microscope. The results are as follows: Figure 2 As shown in Figure D, the results indicate that the SpyTag protein on the surface does not affect the assembly of T7 phage and still maintains the nanoparticle structure.

[0025] Example 2: Expression and Identification of Spy Catcher-Fel d1 Fusion Protein

[0026] 1. Construction of recombinant rod granules expressing fusion proteins To achieve high-copy binding of the Fel d1 protein on the surface of T7-Spy Tag phage and significantly enhance the immunogenicity of Fel d1, multiple linkers were designed for the connection between Spy Catcher and Fel d1, forming the fusion protein Spy Catcher-Fel d1, which consists of Spy Catcher, Linkers, and Fel d1 linked sequentially from the 5' to the 3' end. Experiments revealed that the amino acid structure of the Linkers significantly affects the covalent binding efficiency of the Spy Catcher-Fel d1 fusion protein to T7-Spy Tag phage, ultimately influencing the immunogenicity of Fel d1. Linker 3 forms a flexible, foldless structure, providing the fusion protein with free spatial conformation while introducing moderate electrostatic repulsion to prevent domain interference. Therefore, only the Spy Catcher-Fel d1-c fusion protein can efficiently bind to T7-Spy Tag phage and induce a high level of immune response.

[0027] The amino acid composition of Spy Catcher-Fel d1-c is shown in Table 1, and the encoding gene sequence is shown in SEQ ID NO.7. Table 1 also lists two other fusion proteins: Spy Catcher-Fel d1-a and Spy Catcher-Fel d1-b.

[0028]

[0029] BamHI restriction sites were added to the 5' end and HindIII restriction sites to the 3' end of the coding genes of the three fusion proteins mentioned above. The coding genes of the fusion proteins with restriction sites at both ends were synthesized by a commercial company. These genes were then inserted into the pFastBac1 vector (Invitrogen) via the BamHI restriction site at the 5' end and the HindIII restriction site at the 3' end. Recombinant plasmid vectors expressing each fusion protein were constructed, with the recombinant vector expressing Spy Catcher-Fel d1-c named pFastbac1-Spy Catcher-Fel d1-c. Double enzyme digestion was used to identify each positive recombinant plasmid vector, as shown below. Figure 3 As shown in Figure A, after electrophoresis of the double enzyme digestion products of the three recombinant plasmid vectors, a target band of approximately 1200 bp was observed, which is in line with expectations.

[0030] 200 ng of pFastbac1-Spy Catcher-Fel d1-c vector was slowly added to DH10Bac E. coliIn Beyotime competent cells, gently mix. Incubate on ice for 30 min, then heat shock at 42°C for 90 s. Immediately incubate on ice for 5 min, then add 800 μL of SOC medium (Solarbio) and incubate at 37°C with shaking at 225 r / min for 4 h. Spread 100 μL of the bacterial culture onto LB agar plates containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, 10 μg / mL tetracycline, 100 mg / mL IPTG, and 20 mg / mL X-gal, and incubate upside down at 37°C for 48 h. Using the same method, introduce Spy Catcher-Feld1-a and Spy Catcher-Feld1-b into DH10Bac, respectively. E. coli (Beyotime) competent cells and screening.

[0031] White monoclonal colonies were picked and identified by PCR using primers M13F and M13R (M13F: 5'-GTTTTCCCAGTCACGAC-3'; M13R: 5'-CAGGAAACAGCTATGAC-3'). The results are as follows. Figure 3 As shown in Figure B, the negative control (DH10Bac without the recombinant vector inserted) E. coli No target band was observed. Positive colonies expressing Spy Catcher-Fel d1-a, SpyCatcher-Fel d1-b, and Spy Catcher-Fel d1-c amplified the target band. PCR-positive clones were inoculated into LB medium containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, and 10 μg / mL tetracycline. After overnight incubation, 1.8 mL of bacterial culture was centrifuged at 12000 r / min, 4℃ for 2 min, and the supernatant was discarded. Recombinant rod-like particles carried by the bacteria were extracted using the BAC / PAC DNA mini-extraction kit (Omega bio-teck). DNA was finally eluted with 50 μL of elution buffer and stored at -20℃.

[0032] 2. Rescue of recombinant bacillus viruses expressing various fusion proteins SF9 cells were cultured to the logarithmic growth phase using SF 900 II medium (Gibco) for transfection with recombinant bacteria. Specifically, 5 × 10⁶ cells were seeded into 6-well plates. 5SF9 cells / 2 mL / well, cultured at 27°C for 1 h to allow cell adhesion. During this period, rod-particle complexes expressing each fusion protein were prepared with transfection reagent: 12 μg recombinant rod-particles were diluted with 200 μL SF 900 II medium (without antibiotics or FBS). Before use, the transfection reagent Lipofectamine™ 3000 (Invitrogen) was warmed to room temperature for 30 min. 5 μL of the transfection reagent was mixed with the diluted recombinant rod-particles and incubated at room temperature for 45 min. The incubated mixture was added to one well of a 6-well plate seeded with SF9 cells and incubated at 27°C for 4–6 days. The supernatant was aspirated to obtain P1 generation viruses expressing the fusion proteins Spy Catcher-Fel d1-a, Spy Catcher-Fel d1-b, and Spy Catcher-Fel d1-c, respectively.

[0033] SF9 cells were cultured to the logarithmic growth phase using 20 mL of SF 900 II medium (Gibco). 400 μL of P1 generation virus was added, and after 4-6 days of culture, the cells were centrifuged at 1500 r / min for 5 min. The supernatant was collected to harvest the P2 generation virus. The obtained virus solution was stored at 4℃ in the dark. Separate samples of the supernatant and cells were prepared and Western blotted using His Tag-HRP secondary antibody (Abcam). The Western blot results for the recombinant virus expressing Spy Catcher-Fel d1-c are as follows: Figure 3 In lane C, no target band was detected in the culture supernatant of control cells (SF9 cells not transfected with rod granules), the lysate supernatant of control cells (SF9 cells not transfected with rod granules), or the lysate pellet of control cells (SF9 cells not transfected with rod granules). However, the target band was visible at 41 kD in the culture supernatant, lysate supernatant, and lysate pellet of cells infected with the recombinant virus expressing Spy Catcher-Fel d1-c, with the highest concentration in lane 4, indicating secretory expression. Similarly, the fusion proteins Spy Catcher-Fel d1-a and Spy Catcher-Fel d1-b were also secreted in the recombinant virus.

[0034] 3. Expression and purification of Spy Catcher-Fel d1 fusion protein SF9 cells were cultured to the logarithmic growth phase in 200 mL of SF 900Ⅱ medium (Gibco). One mL of P2 generation virus expressing Spy Catcher-Fel d1-a, Spy Catcher-Fel d1-b, and Spy Catcher-Fel d1-c fusion proteins was added to each cell. After 4–6 days of culture, the cells were centrifuged at 5000 r / min for 5 min, and the supernatant was collected to obtain the supernatant expressing each fusion protein. SDS-PAGE and Western blot analysis confirmed that each fusion protein was correctly expressed in the supernatant. The supernatant expressing each fusion protein was dialyzed overnight at a 1:20 volume ratio (using an aqueous solution containing 50 mM Tris and 300 mM NaCl, pH 7.5). Using a low-pressure chromatography system, the mixture of the dialyzed culture supernatant and the incubated Ni column was slowly added to a purification column. Equilibrate the Ni column with Balance Buffer (an aqueous solution containing 50 mM Tris and 300 mM NaCl, pH 8.5) at a flow rate of 0.5 mL / min until the effluent OD... 280 The value reached baseline. Wash with Washing Buffer (an aqueous solution containing 20 mM imidazole, 50 mM Tris, and 300 mM NaCl, pH 8.5) at a flow rate of 1 mL / min until the effluent OD... 280 Once the baseline value is reached, elute the target protein with Elution Buffer 1 (an aqueous solution containing 50 mM imidazole, 50 mM Tris, and 300 mM NaCl, pH 8.5) at a flow rate of 1 mL / min and collect the eluent. Elute the target protein with Elution Buffer 2 (an aqueous solution containing 100 mM imidazole, 50 mM Tris, and 300 mM NaCl, pH 8.5) at a flow rate of 1 mL / min and collect the eluent. Elute the target protein with Elution Buffer 3 (an aqueous solution containing 250 mM imidazole, 50 mM Tris, and 300 mM NaCl, pH 8.5) at a flow rate of 1 mL / min and collect the eluent. Elute the target protein with Elution Buffer 4 (an aqueous solution containing 500 mM imidazole, 50 mM Tris, and 300 mM NaCl, pH 8.5) at a flow rate of 1 mL / min and collect the eluent. The purity of each fusion protein under four elution conditions was determined by Western blot. Figure 3As shown in Figure D, the target protein bands obtained by eluting each fusion protein with Elution Buffer 3 were relatively simple. Therefore, the eluent obtained by eluting each fusion protein with Elution Buffer 3 was collected as the purified protein. The concentrations of the three fusion proteins were determined using a Nanodrop spectrophotometer and adjusted to 1 mg / mL, then frozen at -20 ℃ for later use.

[0035] Example 3: Preparation and Identification of Self-Assembled Nanoparticles of Feline Allergen Protein

[0036] 1. Identification of the binding ability of Spy Catcher-Fel d1 fusion protein to T7-Spy Tag phage particles The binding ability of the fusion proteins Spy Catcher-Fel d1-a, Spy Catcher-Fel d1-b, and Spy Catcher-Fel d1-c to T7-Spy Tag phage particles was identified.

[0037] Immerse the PVDF membrane in methanol for 10-15 seconds, wash three times with PBS at pH 7.4, and air dry until no droplets remain on the surface. Add 10 μL of Spy Tag protein (synthesized by Jier Biochemical (Shanghai) Co., Ltd., amino acid sequence as shown in SEQ ID NO. 8), PBS buffer at pH 7.4, T7-Spy Tag phage (prepared in Example 1), and T7 phage to the predetermined positions on the membrane, allowing it to air dry on the membrane surface. Repeat this process for five membranes. Then immerse the membrane in 1% BSA blocking solution overnight at 4°C. The next day, wash the membrane three times with PBST buffer and incubate it in 100 ng / mL Spy Catcher-Fel d1-a, Spy Catcher-Fel d1-b, and Spy Catcher-Fel d1-c solutions at 37°C for 1 hour. Wash three times with PBST buffer, then place the membrane in a 1:5000 diluted solution of anti-His Tag-HRP secondary antibody (Abcam) and incubate at 37°C for 1 h. Wash three times with PBST buffer, then place the membrane in DAB chromogenic solution and react at room temperature for 5-10 min. Transfer the membrane to PBS buffer to terminate the reaction.

[0038] The results are as follows Figure 4Spots A, 2, and 4 contain PBS and T7 phage, respectively, and do not contain Spy Catcher-Fel d1 proteins, therefore no color development was observed. Spots 1 and 3, however, contain Spy Tag protein and T7-Spy Tag phage, respectively, and show clear color development. This indicates that the fusion proteins Spy Catcher-Fel d1-a, Spy Catcher-Fel d1-b, and Spy Catcher-Fel d1-c can adsorb onto the surface of T7-Spy Tag phage particles. Under the same reaction conditions, Spy Catcher-Fel d1-c exhibits strong binding to T7-Spy Tag, resulting in a larger and deeper color development area. Conversely, Spy Catcher-Fel d1-a and Spy Catcher-Fel d1-b show weak binding to T7-Spy Tag, resulting in a small and pale color development area. The above results indicate that Linker 3 is beneficial for optimizing the spatial conformation of the Spy Cathe-Fel d1 fusion protein, enabling it to bind more efficiently to T7-SpyTag phage.

[0039] 2. Preparation and Identification of Self-Assembled Nanoparticles of Feline Allergen Protein The fusion proteins Spy Catcher-Fel d1-a, Spy Catcher-Fel d1-b, and Spy Catcher-Fel d1-c were respectively bound to T7-Spy Tag phage particles to prepare corresponding nanoparticle-like Fel d1 allergen proteins.

[0040] Because the icosahedral head of T7 phage is composed of 415 copies of capsid protein, each T7-Spy Tag phage displays 415 copies of the Spy Tag on its surface. Take 10 mL of a 5×10⁻⁶ titer. 11 Based on the assumption that all 415 copies of the Spy Tag on the surface of each T7-Spy Tag phage are bound to the Spy Catcher-Fel d1 fusion protein, 2.075 × 10⁻⁶ pfu / mL of T7-Spy Tag phage would require 2.075 × 10⁻⁶ pfu / mL of T7-Spy Tag phage. 15 The number of fusion protein molecules (N) corresponds to an amount of substance (M) of 3.32 × 10⁻⁶. -9 Based on the formula: mass (m) = amount of substance (M) × molecular weight of fusion protein (41kD), the calculated amount of each SpyCatcher-Fel d1 fusion protein added was 136 μg. At a titer of 5 × 10⁻⁶ mol / L, the total amount of SpyCatcher-Fel d1 fusion protein added was 136 μg. 11Add 200 μL of Spy Catcher-Fel d1-c fusion protein (1 mg / mL) dropwise to T7-Spy Tag phage at pfu / mL and mix gently. Place the mixture in a 4 °C refrigerator, invert and mix thoroughly, and allow to react for 24 hours to obtain self-assembled nanoparticles of feline allergen protein. At this point, the concentration of the fusion protein in the system is 20 μg / mL. Using the same method, prepare self-assembled nanoparticles with Spy Catcher-Fel d1-a and Spy Catcher-Feld1-b respectively with T7-Spy Tag phage, the only difference being that the same mass of SpyCatcher-Fel d1-a and Spy Catcher-Fel d1-b is used instead of Spy Catcher-Fel d1-c.

[0041] BL21 host bacteria, T7-Spy Tag phage, and three self-assembled nanoparticles were subjected to SDS-PAGE electrophoresis. After transfer, the T7-Spy Tag phage and the new protein formed after covalent binding with Spy Catcher-Fel d1 were detected by Western blot using HRP-labeled T7 Tag secondary antibody (Novagen). The results are as follows. Figure 4 As shown in Figure B, lane 1 contains the BL21 host bacteria, which shows no banding due to the absence of T7 phage capsid protein; lane 2 contains the T7-Spy Tag phage, where a band of the p10B-Spy Tag fusion protein, approximately 43 kD in size, is visible; lanes 3-5 contain self-assembled nanoparticles formed by three fusion proteins and T7-SpyTag, respectively. These lanes show both individual p10B-Spy Tag bands and an additional band of approximately 80 kD, the size of which is the sum of the p10B-Spy Tag and Spy Catcher-Fel d1 bands. The results indicate that the Spy Catcher-Feld1-a, Spy Catcher-Fel d1-b, and Spy Catcher-Fel d1-c fusion proteins can form stable and irreversible covalent bonds on the surface of the T7-Spy Tag, but they fail to bind to all p10B-Spy Tags. Among them, Spy Catcher-Fel d1-c has a significantly stronger binding ability to the T7-Spy Tag, and the proportion of nanoparticles formed after covalent binding is significantly higher than that formed by the other two fusion proteins. Therefore, the binding ability of Spy Catcher-Fel d1-a and Spy Catcher-Fel d1-b is relatively weak.

[0042] Example 4: Preparation of egg yolk antibodies using self-assembled nanoparticles of feline allergen protein 1. Vaccine preparation and group immunization The extracted natural Fel d1 allergen protein (purchased from INDOOR biotechnongies) was adjusted to a concentration of 1 mg / mL and 0.2 mg / mL, and emulsified with ISA 206 adjuvant at a volume ratio of 1:1 to prepare vaccines with two antigen concentrations, Fel d1-H and Fel d1-L, with the natural Fel d1 allergen protein content being 500 μg / mL and 100 μg / mL, respectively.

[0043] Following the method described in Example 3, Spy Catcher-Fel d1-c fusion protein was reacted with T7-Spy Tag phage to prepare self-assembled nanoparticles of feline allergen protein, denoted as the self-assembled nanoparticles of this invention. The self-assembled nanoparticles of this invention were mixed with ISA206 adjuvant at a volume ratio of 1:1 and emulsified to obtain the T7-Spy Tag / Spy Catcher-Fel d1-c vaccine, wherein the concentration of the Spy Catcher-Fel d1-c fusion protein was 10 μg / mL.

[0044] Forty newly laying hens were selected and divided into four groups, each immunized with one of the three vaccines mentioned above. A non-immunized group was also included as a negative control. Each hen was immunized with 0.2 mL of vaccine: the high-dose natural allergen group received 100 μg of natural Fel d1 allergen protein per hen; the low-dose natural allergen group received 20 μg of natural Fel d1 allergen protein per hen; and the self-assembled nanoparticle group of this invention received 2 μg of Spy Catcher-Fel d1-c allergen protein per hen. Two weeks after the first immunization, a second immunization was administered at the same dose; then, two weeks later, a third and fourth immunization were administered at double the initial dose. Blood samples and eggs were collected two weeks after the first immunization.

[0045] 2. Serum and egg yolk antibody titer determination Using artificially extracted natural Fel d1 protein (INDOOR biotechnongies) as the coating antigen, an ELISA detection method was established to evaluate the level of anti-Fel d1 specific antibodies in serum and egg yolk. The specific operating procedure is as follows: Dilute the natural Fel d1 protein to a concentration of 10 μg / mL with coating buffer (0.05M carbonate buffer, pH 9.6), add 100 μL / well of coating antigen, and incubate overnight at 4°C; the next day, wash the plate 3 times with PBST buffer (PBS buffer containing 0.5% Tween-20, pH 7.4), add 300 μL / well of PBST buffer containing 0.5% BSA (bovine serum albumin), and block at 4°C for 2 hours; wash the plate 3 times with PBST buffer, add 100 μL / well of the test sample diluted 1:1000 (serum and egg yolk are both diluted 1000 times with PBS buffer at pH 7.4), and incubate at 37°C for 1 hour; wash the plate 5 times with PBST buffer, add 100 μL / well of goat anti-chicken IgY-HRP secondary antibody (Abcam, code ab6877) diluted 1:8000, and incubate at 37°C for 1 hour; wash the plate 5 times with PBST buffer, add 100 μL / well of goat anti-chicken IgY-HRP secondary antibody diluted 1:8000, and incubate at 37°C for 1 hour; wash the plate 5 times with PBST buffer, add 100 μL / well of goat anti-chicken IgY-HRP secondary antibody (Abcam, code ab6877), ... Add μL / well of TMB chromogenic solution, incubate at 37°C in the dark for 10 min, then add 100 μL / well of stop solution and read the value at 450 nm using a microplate reader. From serum antibody levels ( Figure 5 (A) and egg yolk antibody levels ( Figure 5 As shown in Figure B), the serum and egg yolk of laying hens immunized with the self-assembled nanoparticle vaccine of this invention showed significantly higher anti-Fel d1 antibody titers than those immunized with the natural allergen vaccine. Only 2 μg of Spy Catcher-Feld1-c fusion efficiently binds to T7-Spy Tag phage to form a nanoparticle-like structure, resulting in antibody levels higher than those immunized with 20 μg or 100 μg of natural Fel d1 allergen protein alone.

Claims

1. A self-assembled nanoparticle of feline allergen protein, characterized in that... This is achieved by displaying feline allergens on the surface of T7 phages using Spy Tag and Spy Catcher.

2. The self-assembled nanoparticles of feline allergen protein according to claim 1, characterized in that... The cat allergen protein in question is Fel d1.

3. The method for preparing the self-assembled nanoparticles of feline allergen protein according to claim 1, characterized in that, Includes the following steps: (1) Preparation of recombinant T7 phage nanoparticles displaying Spy Tag peptides on their surface; (2) Preparation of a fusion protein formed by Spy Catcher protein and feline allergen protein; (3) The recombinant T7 phage nanoparticles described in step (1) are mixed with the fusion protein described in step (2), and the cat allergen protein self-assembled nanoparticles are obtained by specific covalent linkage between SpyTag peptide and Spy Catcher protein.

4. The preparation method according to claim 3, characterized in that, The recombinant T7 phage displaying the Spy Tag polypeptide on the surface was obtained by reverse genetic rescue after inserting the coding gene of the Spy Tag polypeptide into the T7-Select 415-1b phage vector.

5. The preparation method according to claim 4, characterized in that, The gene sequence encoding the Spy Tag polypeptide is shown in SEQ ID NO:1, and the amino acid sequence of the Spy Catcher polypeptide is shown in SEQ ID NO:

8.

6. The preparation method according to claim 5, characterized in that, The cat allergen contains at least one of Fel d1, Feld3, Fel d4, Fel d7, or Fel d8.

7. The preparation method according to claim 6, characterized in that, The amino acid sequence of the feline allergen protein Fel d1 is shown in SEQ ID NO:3, or a variant that has at least 80% sequence identity with SEQ ID NO:3 and retains the immunogenicity of the feline allergen.

8. The preparation method according to claim 7, characterized in that, The fusion protein formed by Spy Catcher protein and feline allergen protein is composed of Spy Catcher and Fel d1 linked together by a linker, the amino acid sequence of which is shown in SEQ ID NO:

6.

9. The preparation method according to claim 8, characterized in that, The fusion protein formed by the Spy Catcher protein and the feline allergen protein is obtained by expression through a prokaryotic expression system or a eukaryotic expression system; preferably, the expression system is an Escherichia coli expression system, a yeast expression system, an insect cell expression system, or a mammalian cell expression system.

10. The use of the self-assembled cat allergen protein nanoparticles of claim 1 in the preparation of anti-cat allergen egg yolk antibodies.

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