A degradation-targeting chimera, nanomedicine, and composition of an anti-PRRSV biotype protein and its applications

Abstract

This invention discloses a degradation-targeting chimera, nanomedicine, and composition of an anti-PRRSV biotype protein, and their applications, belonging to the field of bioengineering technology. Specifically, this invention discloses a degradation-targeting chimera of an anti-PRRSV biotype protein, wherein the degradation-targeting chimera is obtained by linking a PRRSV N protein-specific nanobody to the BTB domain of the E3 ubiquitin ligase adaptor protein SPOP via Nnb-SPOP. This lays a research foundation for the efficient in vivo delivery of novel biological drugs and their clinical application against PRRSV, and provides a new strategy for the comprehensive prevention and eradication of PRRSV in my country.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a degradation-targeting chimera, nanomedicine, and composition of an anti-PRRSV biotype protein, and their applications. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by Porcine reproductive and respiratory syndrome virus (PRRSV). The main clinical symptoms in affected piglets include severe respiratory disorders, and in pregnant sows, premature birth, fetal death, and mummified fetuses are severe reproductive problems. It is also prone to secondary infections with other viruses and bacteria. Since PRRSV was first isolated in my country in 1996, the virus has spread to major pig-farming areas nationwide, becoming a major pathogen restricting the healthy development of the pig farming industry. From 2006 onwards, a highly pathogenic strain (HP-PRRSV) broke out in southern my country, causing complex clinical symptoms and posing a significant threat to the development of my country's pig farming industry over the following decade. In 2014, the introduced NADC30 strain recombinated with local strains, giving rise to the NADC30 / 34 recombinant variant strain, further exacerbating the difficulty of PRRSV control and posing a new challenge to my country's comprehensive swine disease prevention and control system.

[0003] PRRSV's characteristics, including genetic diversity, high variability, polysaccharide shielding, and antibody-dependent enhancement, which allow it to evade the host's humoral immune response, continuously present new challenges for PRRSV vaccine development. Currently, existing PRRSV vaccines have significant limitations: while live attenuated vaccines offer good protection against homologous strains and can induce cellular immunity, their protective effect against heterologous strains is limited, and they carry the risk of virulence reversion and adverse drug reaction (ADE); inactivated and subunit vaccines, although offering improved safety, have poor ability to stimulate host cellular immunity, low immunogenicity, and insufficient protective efficacy. This suggests that existing commercial vaccines cannot provide complete immune protection for pig herds, necessitating the development of novel anti-PRRSV drugs for emergency prevention and control of PRRS. However, traditional anti-PRRSV drug development faces two major bottlenecks: pathogen-targeted drugs (such as ribavirin and remdesivir) suffer from high cytotoxicity and easy drug resistance; host-targeted drugs (such as morula ketone C and vidofluradim) have unstable efficacy and are prone to causing cytokine storms and other side effects. In contrast, nanobody (Nb) as a biological drug has become a hot topic in antiviral drug development due to its advantages such as easy genetic manipulation, strong permeability, and good safety. Summary of the Invention

[0004] The purpose of this invention is to provide an effective pharmaceutical composition for treating porcine reproductive and respiratory syndrome.

[0005] This invention provides a degradation-targeting chimera of an anti-PRRSV biotype protein, wherein the degradation-targeting chimera is Nnb-SPOP obtained by linking a PRRSV N protein-specific nanobody to the BTB domain of the E3 ubiquitin ligase adaptor protein SPOP; and a flexible polypeptide (GGGGS)2 is used for linking.

[0006] This invention provides a degradation-targeting chimeric composition of a biotype protein against PRRSV, wherein the degradation-targeting chimeric composition comprises Nnb-SPOP and (Nsp9nb)2-SPOP as described in claim 1. ΔNLS Mixing; the (Nsp9nb)2-SPOP ΔNLS It was obtained by deleting the nuclear localization signal sequence in (Nsp9nb)2-SPOP; the amino acid sequence of the nuclear localization signal sequence is shown in SEQ ID NO.6.

[0007] Progress is limited to one location, Nnb-SPOP and (Nsp9nb)2-SPOP ΔNLS The concentration ratio is 1:1.

[0008] Further specifying, the amino acid sequence of Nnb-SPOP is shown in SEQ ID NO.3; (Nsp9nb)2-SPOP ΔNLS The amino acid sequence is shown in SEQ ID NO.4.

[0009] The present invention provides a nucleotide sequence of a degradation-targeting chimera encoding the above-mentioned anti-PRRSV biological protein.

[0010] The present invention provides an expression vector containing the above-described nucleotide sequence.

[0011] The present invention provides a host cell containing the above-described nucleotide sequence.

[0012] The present invention provides the use of the above-mentioned degradation-targeting chimera of the anti-PRRSV biotype protein, the above-mentioned degradation-targeting chimera composition of the above-mentioned anti-PRRSV biotype protein, the above-mentioned nucleotide sequence, the above-mentioned expression vector or the above-mentioned host cell in the preparation of a drug or vaccine for inhibiting porcine reproductive and respiratory syndrome virus.

[0013] The present invention provides nanoparticles for combating porcine reproductive and respiratory syndrome virus (PRRSV), which are formed by assembling the above-mentioned degradation-targeting chimera of anti-PRRSV biotype protein or the above-mentioned composition of degradation-targeting chimera of anti-PRRSV biotype protein with bacteriophage P22 virus-like particles.

[0014] This invention provides the application of the above-mentioned anti-porcine reproductive and respiratory syndrome virus nanoparticles in the preparation of drugs or vaccines that inhibit porcine reproductive and respiratory syndrome virus.

[0015] Beneficial Effects: This invention utilizes a strategy combining PRRSV Nsp9 and N protein-specific nanobodies with E3 ubiquitin ligase ligands (SPOP) to develop bioPROTACs molecules capable of efficiently degrading PRRSV Nsp9 and N proteins. By modifying the SPOP BTB domain to remove the nuclear localization signal, the developed novel bioPROTACs molecules exhibit stronger in vitro anti-PRRSV effects. Furthermore, the P22 VLPs-bioPROTACs nanobiomedicine developed in this invention possesses both precise targeting of lung macrophages and efficient degradation of viral proteins, laying a research foundation for the efficient in vivo delivery of novel biopharmaceuticals and their clinical application against PRRSV, and providing a new strategy for the comprehensive prevention and control of PRRSV in my country. Attached Figure Description

[0016] Figure 1 To verify Nnb-SPOP and (Nsp9nb)2-SPOP ΔNLS The eukaryotic expression and degradation function of Nnb-SPOP were studied; Figure A shows the results of Western blotting (WB) detection of Nnb-SPOP eukaryotic expression (M: Protein Marker; 1: 1 μg pCDNA3.1 empty vector; 2: 1 μg pCDNA3.1-Nnb-SPOP); Figure B shows the results of Western blotting (WB) detection of (Nsp9nb)2-SPOP. ΔNLS Eukaryotic expression results (M: Protein Marker; 1: 1 μg pCDNA3.1 empty vector; 2: 1 μg pCDNA3.1-(Nsp9nb)2-SPOP) ΔNLS Figure C. Western blotting detection of Nnb-SPOP's ability to degrade N protein (Control: pCDNA3.1 empty vector plasmid); Figure D. Western blotting detection of (Nsp9nb)2-SPOP ΔNLS Degradation ability of Nsp9 protein (Control is pCDNA3.1 empty vector plasmid).

[0017] Figure 2 To verify Nnb-SPOP and (Nsp9nb)2-SPOP ΔNLS The in vitro anti-PRRSV function was demonstrated; Figure A shows the viral titer assay to verify the ability of cells overexpressing two bioPROTACs to inhibit viral replication; Figure B shows the indirect immunofluorescence assay to detect the expression level of PRRSV N protein in each cell line.

[0018] Figure 3The expression and purification of multifunctional P22 VLPs-bioPROTACs nanoparticles were carried out. Figure A shows SDS-PAGE verification of prokaryotic expression of P22 VLPs-Nnb-SPOP (M: Protein Marker; 1. Induces; 2. Not induces); Figure B shows SDS-PAGE verification of P22 VLPs-(Nsp9nb)2-SPOP. ΔNLS Prokaryotic expression of P22 VLPs-Nnb-SPOP (M: Protein Marker; 1. Induces); Figure C. Layered image of P22 VLPs-Nnb-SPOP after sucrose density gradient centrifugation; Figure D. Transmission electron microscopy results of P22 VLPs-Nnb-SPOP; Figure E. Immunoelectron microscopy results of P22 VLPs-Nnb-SPOP.

[0019] Figure 4 To verify the macrophage delivery capability of P22 VLPs-Nnb-SPOP;

[0020] Figure 5 To verify the ability of P22 VLPs-bioPROTACs to inhibit PRRSV replication; Figure A shows the expression level of PRRSV N protein in PAMs detected by IFA; Figure B shows the statistical analysis of fluorescence intensity in Figure A using ImageJ software. Detailed Implementation

[0021] (Nsp9nb)2-SPOP is described in the patent document with application number 202310342828.4.

[0022] Nucleotide sequence of the BTB domain in the porcine E3 ubiquitin ligase adaptor protein SPOP: (SEQ ID NO.25) TCCGTCAACATTTCTGGCCAGAATACCATGAATATGGTGAAGGTTCCCGAGTGCCGGTTGGCAGATGAGTTAGGAGGACTGTGGGAGAATTCCCGGTTCACAGACTGTTGTTTGTGCGTTGCTGGCCAGGAATTCCAGGCTCACAAAGCTATCTTAGCAGCTCGTTCTCCAGTTTTTAGTGCCATGTTTGAACATGAAATGGAGGAGAGCAAAAAGAATCGGGTTGAAATCAATGATGTGGAGCCTGAAGTTTTTAAGGAAATGATGTGTTTCATTTACACGGGGAAGGCGCCGAACCTGGACAAGATGGCTGATGATTTGCTGGCAGCTGCTGACAAGTATGCCTTGGAGCGTTTAAAGGTCATGTGTGAGGATGCGCTCTGCAGTAACCTCTCCGTCGAGAACGCCGCCGAAATTCTCATCCTGGCTGACCTCCACAGCGCAGATCAATTGAAAACTCAGGCAGTGGATTTCATCAACTATCATGCTTCGGATGTCCTGGAGACCTCCGGGTGGAAGTCCATGGTGGTGTCACATCCTCACTTGGTGGCGGAGGCGTACCGTTCTCTGGCTTCAGCACAGTGCCCGTTTCTGGGACCCCCACGCAAACGCCTGAAGCAATCC;

[0023] Amino acid sequence of the BTB domain in the porcine E3 ubiquitin ligase adaptor protein SPOP: (SEQ ID NO.26) SVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQS;

[0024] NLS nucleotide sequence: caggcccttttctgggacccccacgcaaacgcctgaagcaatcc (SEQ ID NO. 5);

[0025] NLS sequence amino acid sequence: QCPFLGPPRKRLKQS (SEQ ID NO.6).

[0026] Example 1. Construction of a degradation-targeting chimeric Nnb-SPOP for an anti-PRRSV biotype protein

[0027] Using the Nnb eukaryotic expression plasmid pCDNA3.1-FLAG-Nnb (a vector containing the Nnb sequence) preserved in our laboratory as a template, a linearized plasmid with added homologous arms (3.1-FLAG-Nnb sequence) was obtained by PCR amplification using primers 3.1-FLAG-Nnb F / R. Using porcine alveolar macrophage genomic cDNA as a template, the SPOP sequence with added homologous arms was amplified using primers SPOP F / R. The 3.1-FLAG-Nnb sequence and the SPOP sequence were then ligated using a flexible polypeptide (GGGGS)2 via homologous recombinase to obtain pCDNA3.1-FLAG-Nnb-SPOP. The relevant primer sequences are shown in Table 1 and were synthesized by Ruiboxingke Biotechnology Co., Ltd. The nucleotide sequence of Nnb-SPOP is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.3.

[0028] Example 2. A degradation-targeting chimeric (Nsp9nb)2-SPOP of an anti-PRRSV biotype protein. ΔNLS Construction

[0029] Using the (Nsp9nb)2-SPOP eukaryotic expression plasmid pCDNA3.1-FLAG-(Nsp9nb)2-SPOP (disclosed in patent application number 202310342828.4) previously constructed in our laboratory as a template, primer 3.1-SPOP was used. ΔNLS F / R amplified the mutant plasmid sequence by high-fidelity Taq polymerase PCR, obtaining a SPOP nuclear localization sequence missing with 9 bp homologous arms at both the 5' and 3' ends. After PCR product recovery and purification, the template plasmid was digested with Dpn 1 enzyme. The digested PCR product was then transformed into competent cells, plated, picked, and sequenced to obtain the eukaryotic recombinant plasmid pCDNA3.1-FLAG-(Nsp9nb)2-SPOP. ΔNLS Eukaryotic expression plasmid. The relevant primer sequences are shown in Table 1, and were synthesized by Ruiboxingke Biotechnology Co., Ltd. (Nsp9nb)2-SPOP ΔNLSThe nucleotide sequence is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.4.

[0030] Table 1 Primer Sequences

[0031]

[0032] Example 3. Verification of the target protein degradation function of Nnb-SPOP

[0033] HEK293T cells were seeded into 6-well plates until 70% colony-forming. Using PEI transfection reagent, 1 μg of pCDNA3.1-FLAG-Nnb-SPOP was transfected into each well at a plasmid:PEI ratio of 1:3. After 24 h, the cell supernatant was discarded, and 200 μL of cell lysis buffer (RIPA + 10% PMSF) was added to each well to extract cell proteins. The samples were collected and centrifuged at 10,000 rpm for 10 min, and the supernatant was collected for SDS-PAGE. The protein gel samples were transferred to PVDF membranes using a transfer apparatus and blocked with 5% skim milk at room temperature for 1 h. Western blotting was then performed using a mouse Flag-tagged monoclonal antibody as the primary antibody and DyLight 800-labeled goat anti-mouse antibody as the secondary antibody. The results are as follows: Figure 1 A shows that the Nnb-SPOP (37.6 kDa) size is consistent with the SnapGene prediction, indicating that bioPROTACs are successfully expressed in cells.

[0034] Cells were co-transfected with 1 μg pCAGGS-HA-N and 1, 2, and 3 μg pCDNA3.1-FLAG-Nnb-SPOP. Cell protein samples were collected 24 h post-transfection for Western blot analysis to verify the degradation effect of Nnb-SPOP on N protein. Results are as follows: Figure 1 As shown in C, Nnb-SPOP achieves a 70% efficiency in targeting and degrading N protein.

[0035] Example 4. (Nsp9nb)2-SPOP ΔNLS Validation of target protein degradation function

[0036] First, HEK293T cells were seeded into six-well plates until they reached 70% occupancy. Then, using PEI transfection reagent, 1 μg of pCDNA3.1-FLAG-(Nsp9nb)2-SPOP was transfected at a plasmid:PEI ratio of 1:3. ΔNLSCells were transfected into wells. After 24 h, the cell supernatant was discarded, and 200 μL of cell lysis buffer (RIPA + 10% PMSF) was added to each well to extract cell proteins. The samples were collected, centrifuged at 10,000 rpm for 10 min, and the supernatant was collected for SDS-PAGE. The protein gel samples were transferred to PVDF membranes using a transfer apparatus and blocked with 5% skim milk at room temperature for 1 h. Western blotting was then performed using a mouse Flag-tagged monoclonal antibody as the primary antibody and DyLight 800-labeled goat anti-mouse antibody as the secondary antibody. The results are as follows: Figure 1 B shows (Nsp9nb)2-SPOP, which are consistent with the SnapGene predicted size. ΔNLS (52.6 kDa) indicates that bioPROTACs were successfully expressed in cells.

[0037] pCAGGS-HA-Nsp9 was mixed with 1, 2, and 3 μg of pCDNA 3.1-FLAG-(Nsp9nb)2-SPOP ΔNLS Transfect 293T cells at the same ratio as described above, and collect samples for Western blotting verification 24 hours later. Results Figure 1 D Display (Nsp9nb) 2-SPOP ΔNLS It exhibits dose-dependent degradation of the target protein, and its degradation efficiency at a 1:3 plasmid transfection ratio is higher than that of (Nsp9nb)2-SPOP (disclosed in the patent application with application number 202310342828.4), reaching up to 80%.

[0038] Example 5. Analysis of the antiviral effect of Nnb-SPOP

[0039] 1 μg of plasmid pCDNA3.1-FLAG-Nnb-SPOP was transfected into Marc-145 cells grown to 60%-70% confluence in 12-well plates. 24 h post-transfection, cells were inoculated with 0.01 MOI of HP-PRRSV (HuN4), and 1 h after inoculation, the medium was replaced with maintenance medium containing 2% serum. Cell supernatants were collected at 24 h, 48 h, and 72 h post-inoculation for viral titer determination (TCID). 50 The samples were then observed and photographed using an inverted fluorescence microscope after 72 hours of incubation. Results are as follows: Figure 2 As shown, the viral titer and cytopathic effect of PRRSV-infected cell supernatants transfected with Nnb-SPOP were significantly lower than those of the PRRSV-infected control group. Furthermore, the bioPROTACs transfection group exhibited significantly stronger cytopathic inhibitory activity than (Nsp9nb)2-SPOP, which was previously invented in our laboratory. These results indicate that Nnb-SPOP and (Nsp9nb)2-SPOP... ΔNLSAll small molecules possess the ability to inhibit PRRSV replication, significantly reduce the expression level of N protein in PRRSV-infected cells, and their antiviral effect is superior to (Nsp9nb)2-SPOP.

[0040] Example 6. (Nsp9nb)2-SPOP ΔNLS Antiviral efficacy analysis

[0041] 1 μg of plasmid pCDNA3.1-FLAG-(Nsp9nb)2-SPOP ΔNLS Marc-145 cells grown to 60%-70% confluence in 12-well plates were transfected. 24 h post-transfection, cells were inoculated with 0.01 MOI of HP-PRRSV (HuN4), and 1 h later, the medium was replaced with maintenance medium containing 2% serum. Cell supernatants were collected at 24 h, 48 h, and 72 h post-inoculation for viral titer determination (TCID). 50 The samples were then observed and photographed using an inverted fluorescence microscope after 72 hours of incubation. Results are as follows: Figure 2 As shown, PRRSV infection transfected with (Nsp9nb)2-SPOP ΔNLS The viral titer and cytopathic effect induced by the small molecules in the cell supernatant were significantly lower than those in the PPRSV-infected control group. The cytopathic effect of the bioPROTACs transfection group was significantly stronger than that of (Nsp9nb)2-SPOP previously invented in our laboratory. This preliminarily demonstrates that the above-mentioned bioPROTACs molecules exhibit superior antiviral activity in vitro compared to (Nsp9nb)2-SPOP previously invented in our laboratory.

[0042] Example 7. Development of Multifunctional P22 VLPs-Nnb-SPOP Nanobiomedicine

[0043] 1. Construction and prokaryotic expression of P22 VLPs-Nnb-SPOP plasmid: Using pCDNA3.1-FLAG-Nnb-SPOP as a template, primers Nnb-SPOP F / R were designed for PCR amplification of the Nnb-SPOP gene fragment with added homologous arm sequences; using pET-28a-P22 VLP stored in the laboratory as a vector template (published in the article: Su S, Shen X, Shi X, et al. Cell-penetrating peptides TAT ​​and 8R functionalize P22 virus-like particles to enhance tissue distribution and retention in vivo[J]. Frontiers in Veterinary Science, 2024.DOI:10.3389 / fvets.2024.1460973.), P22 VLPs with added homologous arm sequences were amplified by PCR using primers SP-NF / R. The Nnb-SPOP coding sequence was successfully subcloned into the 5' end of the P22 VLPs SP protein nucleotide sequence by fusing the two fragments through homologous recombination. Further, using the above intermediate plasmid as a template, a linearized P22 VLPs plasmid with a homologous arm sequence added to the 3' end of the CP protein nucleotide sequence was amplified using CP F / R primers. Using a synthesized porcine Fc fragment (NCBI accession number: AK405781) eukaryotic expression vector (synthesized by Ruiboxingke Biotechnology Co., Ltd.) as a template, the IgG Fc fragment was amplified using primers Fc F / R. The two fragments were then fused through homologous recombination, successfully subcloning the IgG Fc sequence into the 3' end of the P22 VLPs CP protein nucleotide sequence, ultimately yielding the multifunctional pET-28a-P22 VLPs-Nnb-SPOP plasmid. The relevant primer sequences are shown in Table 2 and were synthesized by Ruiboxingke Biotechnology Co., Ltd.

[0044] The plasmid was transformed into competent ROSETTA (DE3) cells of prokaryotic protein expression bacteria. Single colonies were picked and cultured in LB medium containing kanamycin resistance. Once the OD value of the bacterial culture reached between 0.4 and 0.8, a final concentration of 0.1 mM IPTG was added, and expression was induced overnight at 180 r / min and 16℃. The bacterial cells were collected, sonicated, and centrifuged at 10,000 rpm for 20 min. The supernatant was collected and subjected to SDS-PAGE. The molecular weight of each component protein in the VLPs was predicted using SnapGene 6.02 software. The results are as follows: Figure 3As shown in Figure A, the 57 kDa Nnb-SPOP-SP and 72.1 kDa CP-Fc proteins were successfully expressed, demonstrating the successful expression of P22 VLPs-Nnb-SPOP nanoparticles.

[0045] Table 2 Primer Sequences

[0046]

[0047] 2. Purification and characterization of P22 VLPs-Nnb-SPOP nanoparticles

[0048] The supernatant after filtration at 0.22 μM was transferred to 40 mL polypropylene centrifuge tubes and ultracentrifuged at 28,000 rpm for 2 h at 4 °C. For further purification, four sucrose concentration gradients (25%–55%) were prepared in 40 mL centrifuge tubes, and the concentrated protein was carefully added to the top of the sucrose. The tubes were then centrifuged at 28,000 rpm for 3 h at 4 °C. The VLP bands were carefully collected from the tubes and dissolved in PBS, then centrifuged under the same conditions to remove residual sucrose. After the purified VLPs were dissolved in a small amount of PBS and further confirmed by SDS-PAGE, a distinct white aggregate layer formed in the ultracentrifuged tubes, as shown in the image. Figure 3 As shown in C. After collection, the samples were centrifuged once with PBS at 28,000 rpm for 3 h at 4 °C to remove sucrose. Finally, the samples were resuspended with a small amount of PBS, and a portion was aliquoted for subsequent evaluation, while the remainder was stored at -20 °C.

[0049] A small amount of sample was placed on a copper grid and incubated at 25 °C for 10 min. The sample was then negatively stained with 2.5% phosphotungstic acid solution for 1 min. Excess stain was blotted with filter paper, and the morphological characteristics of VLPs were examined using transmission electron microscopy. Under the microscope, uniformly sized, clearly structured, and approximately 80 nm in diameter P22 VLPs-Nnb-SPOP nanoparticles were observed. Figure 3 D). For immunoelectron microscopy experiments, a 1:200 dilution of porcine IgG Fc monoclonal antibody was used, and visualization was performed using gold-particle-labeled anti-mouse secondary antibody. The results are as follows. Figure 3 As shown in Figure E, the clearly visible gold nanoparticles on the exogenous VLPs demonstrate the successful display of porcine IgG Fc on their surface. Following the above procedures, a multifunctional P22 VLPs-Nnb-SPOP nanobiomedicine was finally obtained, internally encapsulated with bioPROTACs (Nnb-SPOP) and externally chimeric with porcine Fc.

[0050] 3. Uptake of P22 VLPs-Nnb-SPOP nanobiomedicine by alveolar macrophages

[0051] P22 VLPs-Nnb-SPOP at a final concentration of 50 μg / mL was added to PAMs pre-seeded for 4-6 h and to Marc-145 cells that had grown into a monolayer. After 6 h of incubation, the cells were fixed, permeabilized, and blocked for IFA detection. A His-tagged monoclonal antibody on the VLP surface was used as the primary antibody, and goat anti-mouse IgG was used as the secondary fluorescent antibody to stain P22 VLPs-Nnb-SPOP. Cell nuclei were then counterstained with DAPI. Observation under an inverted fluorescence microscope revealed that PAMs exhibited specific uptake of P22 VLPs-Nnb-SPOP, while Marc-145 cells failed to successfully uptake it. Figure 4 The above results demonstrate that P22 VLPs-Nnb-SPOP possesses good macrophage targeting ability. At a final concentration of 50 μg / mL, P22 VLPs-Nnb-SPOP exhibited good macrophage delivery capability and was not taken up by Marc-145 cells.

[0052] Example 8. Multifunctional P22 VLPs-(Nsp9nb)2-SPOP ΔNLS Development of nanobiomedicine

[0053] 1.P22VLPs-(Nsp9nb)2-SPOP ΔNLS Construction and prokaryotic expression of plasmids: using pCDNA3.1-FLAG-(Nsp9nb)2-SPOP ΔNLS Using Nsp9nb as a template, primers (Nsp9nb)2-SPOP were designed. ΔNLS F / R was used for PCR amplification with (Nsp9nb)2-SPOP containing the homologous arm sequence. ΔNLS Gene fragments; using laboratory-preserved pET-28a-P22 VLPs as a vector template (published in the article: Su S, Shen X, Shi X, et al. Cell-penetrating peptides TAT ​​and 8R functionalize P22 virus-like particles to enhance tissue distribution and retention in vivo[J]. Frontiers in Veterinary Science, 2024.DOI:10.3389 / fvets.2024.1460973.), linearized plasmids of P22 VLPs with added homologous arm sequences were obtained by PCR amplification using primers SP-NF / R. The two fragments were then fused via homologous recombination, successfully generating (Nsp9nb)2-SPOP. ΔNLSThe coding sequence was subcloned to the 5' end of the P22 VLPs SP protein nucleotide sequence. Further, using the aforementioned intermediate plasmid as a template, a linearized P22 VLPs plasmid with a homologous arm sequence added to the 3' end of the CP protein nucleotide sequence was amplified using CP F / R primers. Using a synthesized porcine Fc fragment eukaryotic expression vector (synthesized by Ruiboxing Biotechnology Co., Ltd.) as a template, an IgG Fc fragment was amplified using primers Fc F / R. The two fragments were then fused via homologous recombination, successfully subcloning the IgG Fc sequence to the 3' end of the P22 VLPs CP protein nucleotide sequence, ultimately yielding the multifunctional pET-28a-P22 VLPs-(Nsp9nb)2-SPOP. ΔNLS Plasmids. The relevant primer sequences are shown in Table 2 and were synthesized by Ruiboxing Biotechnology Co., Ltd.

[0054] The plasmid was transformed into competent ROSETTA (DE3) cells of prokaryotic protein expression bacteria. Single colonies were picked and cultured in LB medium containing kanamycin resistance. Once the OD value of the bacterial culture reached between 0.4 and 0.8, a final concentration of 0.1 mM IPTG was added, and expression was induced overnight at 180 r / min and 16℃. The bacterial cells were collected, sonicated, and centrifuged at 10,000 rpm for 20 min. The supernatant was collected and subjected to SDS-PAGE. The molecular weight of each component protein in the VLPs was predicted using SnapGene 6.02 software. The results are as follows: Figure 3 As shown in B, a (Nsp9nb)2-SPOP of size 75 kDa was successfully expressed. ΔNLS -SP and 72 kDa CP-Fc protein, demonstrating P22VLPs-(Nsp9nb)2-SPOP ΔNLS Nanoparticles were successfully expressed.

[0055] 2. Purification of P22 VLPs-(Nsp9nb)2-SPOP nanoparticles

[0056] The supernatant filtered at 0.22 μM was transferred to 40 mL polypropylene centrifuge tubes and ultracentrifuged at 28,000 rpm for 2 h at 4 °C. For further purification, four sucrose concentration gradients (25%–55%) were prepared in 40 mL centrifuge tubes, and the concentrated protein was carefully added on top of the sucrose. The tubes were then centrifuged at 28,000 rpm for 3 h at 4 °C. The distinct white aggregate that formed in the centrifuge tubes after ultracentrifugation was carefully collected, and the samples were washed once with PBS at 28,000 rpm for 3 h at 4 °C to remove sucrose. After confirmation by SDS-PAGE, the samples were stored at -20 °C.

[0057] Example 9. Validation of in vitro anti-PRRSV efficacy

[0058] 1. Obtaining hybrid P22 VLPs-bioPROTACs

[0059] P22 VLPs-Nnb-SPOP and P22 VLPs-(Nsp9nb)2-SPOP ΔNLS Mix them at a concentration ratio of 1:1.

[0060] 2. Validation of the antiviral efficacy of P22 VLPs-Nnb-SPOP nanobiomedicine in PAMs

[0061] Porcine alveolar macrophages (PAMs) were seeded into 24-well plates. After reaching 70% confluence, cells were inoculated with a virus at an MOI of 0.01. After 1 h of incubation, the cells were washed three times with PBS and replaced with maintenance medium containing 2% serum. The cells were divided into four groups: a virus-inoculated control group, P22 VLPs-Nnb-SPOP, and P22 VLPs-(Nsp9nb)2-SPOP. ΔNLS The groups receiving the drug alone and the groups receiving the drug in combination.

[0062] 3. Measure the final concentration of 50 μg / mL P22 VLPs-bioPROTACs (P22 VLPs-Nnb-SPOP, P22 VLPs-(Nsp9nb)2-SPOP). ΔNLS Or P22 VLPs-Nnb-SPOP and P22 VLPs-(Nsp9nb)2-SPOP ΔNLS The mixture was added to the cells and cultured further. Cells were fixed with 3.7% paraformaldehyde at 24 hpi and subjected to IFA staining for PRRSV N protein. Observation and photographic recording were performed under a microscope. Figure 5 Fluorescence results showed that the PRRSV N protein content in cells incubated with P22 VLPs-bioPROTACs after infection was significantly lower than that in the control group, with P22 VLPs-Nnb-SPOP and P22 VLPs-(Nsp9nb)2-SPOP being particularly high. ΔNLS The inhibitory effects of the single-drug groups on cytopathic effects were all above 80%. Furthermore, P22 VLPs-Nnb-SPOP and P22 VLPs-(Nsp9nb)2-SPOP... ΔNLS The combined administration group showed an inhibition rate of up to 90% against PRRSV replication. These results indicate that P22VLPs-bioPROTACs have a significant inhibitory effect on PRRSV replication in PAMs, particularly P22VLPs-Nnb-SPOP and P22VLPs-(Nsp9nb)2-SPOP. ΔNLSThe combined application showed better results than the use of individual drugs. This experiment demonstrated that the P22 VLPs-bioPROTACs nanobiomedicine constructed using P22 VLPs modified with Fc fragments as delivery vectors exhibited good antiviral activity in vitro, and that the combined application of multiple targets resulted in even better antiviral effects.

Claims

1. A degradation-targeted chimera of a biotype protein against PRRSV, characterized in that, The degradation targeting chimera is Nnb-SPOP obtained by connecting a PRRSV N protein specific nanobody to the BTB domain of the E3 ubiquitin ligase linker protein SPOP, the amino acid sequence of which is shown as SEQ ID NO. 26; a flexible polypeptide (GGGGS)2; the amino acid sequence of Nnb-SPOP is shown as SEQ ID NO.

3.

2. A biotype protein of PRRSV-degrading targeted chimera composition, characterized in that, The degradation-targeting chimera composition is (Nsp9nb)2-SPOP ΔNLS mixed with (Nsp9nb)2-SPOP ΔNLS The amino acid sequence of the (Nsp9nb)2-SPOP ΔNLS is obtained by deleting the nuclear localization signal sequence in the (Nsp9nb)2-SPOP; the amino acid sequence of the nuclear localization signal sequence is shown as SEQ ID NO.

6.

3. The degradation-targeting chimeric composition of the anti-PRRSV biotype protein according to claim 2, characterized in that, Nnb-spop and (nsp9nb)2-spop ΔNLS The concentration ratio is 1:

1.

4. A gene encoding the degradation targeting chimera of the anti-PRRSV biotype protein as claimed in claim 1.

5. An expression vector containing the gene as claimed in claim 4.

6. A host cell containing the gene as claimed in claim 4.

7. Use of the degradation targeting chimera of the anti-PRRSV biotype protein as claimed in claim 1, the degradation targeting chimera composition of the PRRSV biotype protein as claimed in any one of claims 2-3, the gene as claimed in claim 4, the expression vector as claimed in claim 5, or the host cell as claimed in claim 6 in the preparation of a medicine for inhibiting porcine reproductive and respiratory syndrome virus.

8. A nanoparticle against porcine reproductive and respiratory syndrome virus, characterized in that, The degradation targeting chimera of the anti-PRRSV biotype protein as claimed in claim 1 or the degradation targeting chimera composition of the anti-PRRSV biotype protein as claimed in any one of claims 2-3 is assembled with bacteriophage P22 virus-like particles.

9. Use of the anti-porcine reproductive and respiratory syndrome virus nanoparticle as claimed in claim 8 in the preparation of a medicine for inhibiting porcine reproductive and respiratory syndrome virus.

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