Engineered PVC protein compound for targeted removal of intracellular staphylococcus aureus as well as preparation method and application of engineered PVC protein compound

Abstract

The invention discloses an engineered PVC protein compound for targeted removal of intracellular staphylococcus aureus as well as a preparation method and application of the engineered PVC protein compound, and belongs to the fields of genetic engineering, microbiology and drug delivery. Genetic engineering modification is carried out on PVC, a natural recognition domain of tail fiber protein of the PVC is replaced by a staphylococcus aureus receptor binding structural domain (SRapid BR), and meanwhile, natural toxin protein loaded in an inner cavity of the PVC is replaced by human beta-defensin-3 (HBD-3) with efficient bactericidal activity. The engineered compound can directly deliver HBD-3 into infected cells, effectively remove intracellular staphylococcus aureus which is difficult to kill by conventional antibiotics, and relieve inflammatory response caused by infection. The invention provides a brand-new treatment strategy for resisting intracellular bacterial infection with high targeting, and shows a wide application prospect as a programmable protein delivery platform.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering, microbiology and drug delivery, and specifically relates to an engineered PVC protein complex for targeted elimination of intracellular Staphylococcus aureus, its preparation method and application. Background Technology

[0002] Staphylococcus aureus is a common pathogen that can cause serious illnesses ranging from skin and soft tissue infections to sepsis and endocarditis. Traditional antibiotic therapy is usually effective against free-floating bacteria, but some Staphylococcus aureus can invade and colonize host cells such as epithelial cells and macrophages, forming a difficult-to-eliminate intracellular bacterial reservoir. These intracellular bacteria are protected by the host cell membrane, allowing them to evade recognition by the body's immune system and attack by most antibiotics. Because conventional antibiotics struggle to effectively penetrate cell membranes or reach effective bactericidal concentrations intracellularly, infections become persistent, recurrent, and a source of drug-resistant bacterial transmission and recurrence. Therefore, developing novel treatment strategies that can efficiently target and eliminate intracellular Staphylococcus aureus is a pressing technical challenge in the field of anti-infective therapy.

[0003] To combat intracellular infections, researchers have explored various delivery strategies to enhance intracellular drug concentrations, such as liposomes and polymer nanoparticles. However, these carriers generally suffer from problems such as insufficient targeting, low delivery efficiency, limited endosome escape capabilities, and premature intracellular degradation of the payload, making it difficult to achieve precise and efficient intracellular delivery of therapeutic molecules.

[0004] PVC is a nanoscale complex capable of recognizing specific eukaryotic cells and directly injecting pre-loaded protein effectors into the cytoplasm via tail sheath contraction. In 2023, Zhang Feng's team (published in *Nature*) systematically resolved the structure and function of PVC and demonstrated that by engineering PVC tail fibrous proteins (such as Pvc13), their targeting specificity can be reprogrammed to recognize mammalian cells; simultaneously, the naturally occurring toxin proteins loaded in its lumen can be replaced with functional proteins (such as Cas9, Cre recombinase, etc.). This work develops PVC into a potential programmable protein delivery platform. However, existing publicly available technologies mainly focus on using this platform for gene editing or basic biological research, with payloads primarily consisting of nucleases or reporter proteins. No research has yet applied it to the field of antibacterial infection treatment, let alone addressed how to utilize this platform to solve the challenge of intracellular targeted delivery of antibacterial drugs.

[0005] Human beta-defensin-3 (HBD-3) is an endogenous antimicrobial peptide with broad-spectrum direct killing activity against a variety of Gram-positive and Gram-negative bacteria, including Staphylococcus aureus, and it is not prone to inducing bacterial resistance, making it a promising new antimicrobial agent. However, as a polypeptide molecule, HBD-3 exhibits poor stability in plasma and is easily degraded by proteases; more importantly, it lacks cell-targeting ability, cannot autonomously penetrate cell membranes, and is difficult to effectively reach and eliminate pathogens located within cells. Therefore, although HBD-3 possesses excellent in vitro antimicrobial activity, its inherent pharmaceutical limitations severely restrict its clinical translational application in the treatment of intracellular infections. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to provide an engineered PVC protein complex for targeted elimination of intracellular Staphylococcus aureus, its preparation method, and its application. This invention overcomes the delivery barriers faced by HBD-3 in the treatment of intracellular infections, providing an engineered PVC system that can specifically target infected cells and efficiently deliver HBD-3 into the cell interior.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an engineered PVC protein complex, said engineered PVC protein complex being a recombinant protein assembly obtained through genetic engineering, comprising: (a) Targeting component: PVC tail fibroin with its natural host recognition domain removed is linked to the Staphylococcus aureus receptor binding domain SrapBR; (b) Effector assembly: The PVC cavity, from which the natural toxin proteins have been removed, is connected to human β-defensin-3; The amino acid sequence of the PVC tail fibrous protein with the natural host recognition domain removed is shown in SEQ ID NO.1, and is as follows: MNETRYNATVQEQQTLSNPKAVGPDIDKLKDKFKEGSIPLQTDFNELIDIADIGRKACGQAPQQNGPGEGLKLADDGTLNLKIGTFSNKDFSPLILKDDVLSVDLGSGLTNETNGICVGQGDGITVNTSNVAVKQGNGISVTSSGGVAVKVSANKGLSVDSSGVAVKVNTDKGISVDGNGVAVKVNTSKGISVDNTGVAVIANASKGISVDGSGVAVIANTSKGISVDGSGVAVIANTSKGISVDNTGVAVIANASKGISVDGSGVAVIANTSKGISVDGSGVAVIANTSKGISVDSSGVAVKVKANGGIKVDANGVAIDPNNVLPKGVIVMFSGSTAPTGWALCDGNNGTPNLIDRFILGGKGTDINGVSTNTASGTKNSKLFDFSSDEATLTIDGKTLGRHDHDIKITGTGKHSHKNKVTVPYYILAFIIKL。

[0008] The amino acid sequence of the Staphylococcus aureus receptor-binding domain SrapBR is shown in SEQ ID NO.2, as follows: FASAATTTAVTANTITVNKDNLKQYMTTSGNATYDQSTGIVTLTQDAYSQKGAITLGTRIDSNKSFHFSGKVNLGNKYEGHGNGGDGIGFAFSPGVLGETGLNGAAVGIGGLSNAFGFKLDTYHNTS KPNSAAKANADPSNVAGGGAFGAFVTTDSYGVATTYTSSSTADNAAKLNVQPTNNTFQDFDINYNGDTKVMTVKYAGQTWTRNISDWIAKSGTTNFSLSMTASTGGATNLQQVQFGTFEYTESAVTQ VRYVDVTTGKDIIPPKTYSGNVDQVVTIDNQQSALTAKGYNYTSVDSSYASTYNDTNKTVKMTNAGQSVTYYFTDVKAPTVTVGNQTIEVGKTMNPIVLTTTDNGTGTVTNTVTGLPSGLSYDSATN SIIGTPTKIGQSTVTVVSTDQANNKSTTTFTINVVDTTAPTVTPIGDQSSEVYSPISPIKIATQDNSGNAVTNTVTGLPSGLTFDSTNNTISGTPTNIGTSTISIVSTDASGNKTTTTFKYEVTRN.

[0009] The PVC cavity, from which natural toxin proteins have been removed, is shown in SEQ ID NO.3, as detailed below: MPRYANYQINPKQNIKNSHGKSSSSDFSSGYLSFSNNSLDDPFIRQQVKREFIWEGHMKEIEEASRL.

[0010] The amino acid sequence of the human β-defensin-3 is shown in SEQ ID NO.4, and is as follows: MRIHYLLFALLFLFLVPVPGHGGIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK.

[0011] In the targeting component, the Staphylococcus aureus receptor-binding domain SrapBR is connected to the PVC tail fibrous protein with the natural host recognition domain removed via a flexible linker peptide; in the effector component, human β-defensin-3 is also connected to the PVC lumen with the natural toxin protein removed via a flexible linker peptide.

[0012] Preferably, the amino acid sequence of the flexible linker peptide is as shown in SEQ ID NO.5, specifically as follows: GGSGGGGSGG.

[0013] Preferably, the amino acid sequence of the engineered PVC protein complex is shown in SEQ ID NO.6, specifically as follows: .

[0014] In a second aspect, the present invention provides the use of the above-described engineered PVC protein complex in the preparation of a medicament for eliminating intracellular pathogens, wherein the pathogens are intracellular parasites.

[0015] The intracellular parasite is Staphylococcus aureus.

[0016] The drug for eliminating pathogens within host cells contains an engineered PVC protein complex at a dose of 1.0-4.0 mg / mL, preferably at a dose of 2.5 mg / mL.

[0017] In a third aspect, the present invention provides a method for preparing the above-mentioned engineered PVC protein complex, comprising the following steps: (1) The contents of homologous arms and linker peptides SrapBR The nucleotide sequence was ligated with the linear plasmid pAWP78-PVCpnf1-16 (with the natural host recognition domain removed) to construct the first expression vector pAWP78-Pvc13-Srap; the β-defensin-3 nucleotide sequence containing homologous arms and linker peptides was ligated with the linear plasmid pBR322-Pvc17-22 (with the natural toxin protein removed) to construct the second expression vector pBR322-Pdp1-HBD-3; (2) The first expression vector pAWP78-Pvc13-Srap and the second expression vector pBR322-Pdp1-HBD-3 were introduced into the competent cells of the host bacteria to obtain transformed bacteria, and the expression of engineered PVC protein complex in the transformed bacteria was induced. (3) Collect the cells of the transformed bacteria, wash them with buffer, pre-treat them by cryopreservation at ultra-low temperature, resuspend them in buffer, remove cell debris by centrifugation, and obtain the supernatant containing the engineered PVC protein complex. (4) The supernatant is centrifuged multiple times. After each centrifugation, the precipitate is collected and resuspended in buffer solution. After the last centrifugation, the supernatant is collected to obtain the purified engineered PVC protein complex.

[0018] Preferably, the host bacterium is Escherichia coli EPI300; the co-introduction method is electroporation.

[0019] The [comprising] homologous arms and linker peptides SrapBR The nucleotide sequence is shown in SEQ ID NO.7, and is as follows:

[0020] The β-defensin-3 nucleotide sequence containing the homologous arm and linker peptide is shown in SEQ ID NO.8, and is as follows: ttgaagaagcttcaagattaGGCGGTTCTGGCGGCGGTGGTTCTGGCGGTatgaggatccattatcttctgtttgctttgctcttcctgtttttggtgcctgttccaggtcatggaggaatcataaacacattacag aaatattattgcagagtcagaggcggccggtgtgctgtgctcagctgccttccaaaggaggaacagatcggcaagtgctcgacgcgtggccgaaaatgctgccgaaagaaataatatggattttcatgtcatca.

[0021] After purification as described in step (4), the process also includes a step of removing endotoxins.

[0022] The beneficial effects of this invention are: 1. This invention engineered PVC tail fibrous protein, replacing its natural recognition domain with a SrapBR domain that specifically binds to Staphylococcus aureus infection cell surface markers. This allows the modified PVC system to accurately identify and anchor to infected host cells, thus achieving precise drug delivery. Simultaneously, utilizing PVC's unique syringe-like delivery mechanism, the highly active antimicrobial peptide HBD-3 can be directly injected into infected cells, effectively overcoming the bottleneck of traditional antibiotics' inability to penetrate cell membranes and reach effective intracellular concentrations, achieving highly efficient physical clearance of stubborn intracellular bacterial flora.

[0023] 2. This invention provides a novel, highly targeted therapeutic strategy for intracellular bacterial infections and demonstrates broad application prospects as a programmable protein delivery platform. The engineered PVC complex can directly deliver HBD-3 into infected cells, effectively eliminating intracellular Staphylococcus aureus that is difficult to kill with conventional antibiotics, and reducing the inflammatory response caused by the infection. Experiments have verified that it has no significant toxicity to host cells at effective therapeutic concentrations.

[0024] 3. The engineered PVC system prepared by this invention can not only directly kill intracellular pathogens, but also significantly downregulate the overexpression of key pro-inflammatory factors such as interleukin-6 (IL-6) and CXCL8 in infected cells by eradicating the source of infection, thereby reducing the excessive inflammatory response and related tissue damage caused by infection. Attached Figure Description

[0025] Figure 1 The image shows the gel electrophoresis results of the pAWP78-Pvc13 linear vector (linearized pAWP78-PVCpnf1-16 with the natural recognition domain removed), where channels 1-3 are three replicates of the pAWP78-Pvc13 linear vector.

[0026] Figure 2 Staphylococcus aureus SrapBR Gene gel electrophoresis results, where channels 1-6 are... SrapBR Six-fold repeat sampling of the gene.

[0027] Figure 3 Gel electrophoresis results for the Pdp1 linear vector (linearized pBR322-Pvc17-22 with the natural toxin protein deleted).

[0028] Figure 4 For β-defensin-3 ( HBD-3 Gene gel electrophoresis results.

[0029] Figure 5 The results are from a transmission electron microscope of wild-type PVC, with a magnification of 40,000x (40.0k).

[0030] Figure 6 To target Staphylococcus aureus receptor cells and loaded with HBD-3 Transmission electron microscopy results of PVC (Pvc13-Srap-Pdp1-HBD-3) at a magnification of 60,000 (60.0k).

[0031] Figure 7 These are representative colony growth images of cells from an intracellular bacterial infection model, after treatment with an engineered PVC protein complex for different durations, with the cell lysate spread onto LB agar plates.

[0032] Figure 8 A quantitative statistical bar chart of intracellular bacterial clearance rate after 48 hours of treatment with engineered PVC protein complex.

[0033] Figure 9 This is a quantitative statistical bar chart showing the effect of different concentrations of engineered PVC protein complex on the relative viability of MAC-T cells after treatment at specific time points.

[0034] Figure 10 A graph showing the change in relative cell viability over time after treating MAC-T cells with different concentrations of engineered PVC protein complexes.

[0035] Figure 11A qPCR quantitative statistical graph showing the effect of engineered PVC protein complex treatment on the expression levels of intracellular inflammatory factors IL-6 and CXCL8 mRNA in infected cells. Detailed Implementation

[0036] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0037] The specific embodiments of the present invention will be described in further detail below with reference to examples. The following detailed descriptions are illustrative and intended to provide further explanation of this application, rather than limiting the scope of the invention.

[0038] Example 1: Construction of the first expression vector pAWP78-Pvc13-Srap 1. Preparation of linearized vector backbones with natural recognition domains removed by reverse PCR To replace the natural recognition domain of PVC tail fibrous protein (Pvc13) with the SrapBR domain, a vector backbone with the natural recognition domain coding sequence removed was first constructed. For this purpose, reverse PCR was used, employing the commercial plasmid pAWP78-PVCpnf1-16 (a wild-type PVC tail fibrous protein containing the natural recognition domain; Catalog: 198271, purchased from Addgene) as a template, and a pair of reverse primers (ZT1-F and ZT1-R) were designed. The primers were designed with their 3' ends complementary to the sequences flanking the natural recognition domain coding region, and their orientation was outward. PCR amplified a linearized DNA fragment that fully preserved the PVC tail fibrous protein backbone but precisely deleted the natural recognition domain coding sequence, thus preparing for subsequent homologous recombination insertion of the SrapBR gene fragment.

[0039] Agarose gel electrophoresis confirmed that the size of the obtained linearized vector fragment was in line with expectations. Figure 1 The reverse primer sequence used is as follows: ZT1-F:CACGATCATGATATTAAGAT (SEQ ID NO.9); ZT1-R:TCTCCCCAGTGTTTTACCAT (SEQ ID NO. 10).

[0040] The specific PCR reaction system is shown in Table 1.

[0041] Table 1: Reaction System 2. Preparation SrapBR gene fragments The DNA sequence encoding the Staphylococcus aureus receptor-binding domain SrapBR (NCBI Reference Sequence: YP_501439.1) was synthesized by Sangon Biotech Co., Ltd. During synthesis, linker peptides (amino acid sequence GGSGGGGGSGG) were added to the 5' and 3' ends of the gene, respectively, to connect the two protein domains. Primers containing homologous arms for homologous recombination were used. SrapBR The gene was amplified by PCR. Staphylococcus aureus S rapBR Gene gel electrophoresis results as follows Figure 2 As shown. The primer sequences used are as follows: TYB1-F:atggtaaaacactggggagaGGCGGTTCTGGCGGCGGT (SEQ ID NO.11) TYB1-R:atcttaatatcatgatcgtgACCACCTGAACCACCACCACC (SEQ ID NO.12) 3. Homologous recombination linkage Includes homologous arms and linker peptides SrapBR The nucleotide sequence (shown in SEQ ID NO.7) was ligated into the linearized vector backbone pAWP78-PVCpnf1-16 via homologous recombination. The vector was then transformed into competent DH5α cells by heat shock. After single-clone colonies were picked and sequenced for verification, the plasmid was extracted using the TIANGEN endotoxin-free plasmid mini-prep kit, yielding the first expression vector pAWP78-Pvc13-Srap.

[0042] The homologous recombination reaction system is shown in Table 2.

[0043] Table 2. Reaction System The recombination reaction temperature and time were: 50℃ for 15 minutes; then cooled to 4℃ or immediately placed on ice for cooling.

[0044] The specific steps of the thermal shock conversion method are as follows: (1) Thawing competent cells: Take out DH5α competent cells from the -80℃ freezer, thaw them on ice for about 5-10 minutes, and then immediately transform the DNA sample.

[0045] (2) Add 10 μL of the recombinant product to be transformed into 100 μL of competent cells, then gently tap the bottom of the tube 5 times, and immediately place it on ice for 30 minutes.

[0046] (3) Heat shock treatment: After the ice bath, the centrifuge tube containing competent cells and recombinant products was quickly placed in a 42°C water bath for 45 seconds. Then it was immediately placed on ice and left to stand for 3 minutes.

[0047] (4) Resuscitation culture: Add 900 μL of LB medium preheated at 37℃, mix by inverting, and then place in a constant temperature shaker at 37℃ and 220 rpm for 45 minutes for resuscitation culture.

[0048] (5) Place the revived bacterial culture in a benchtop centrifuge, centrifuge at 5000g at 4℃ for 5 minutes, discard the supernatant, and resuspend the precipitate in about 100μL of supernatant. Spread the precipitate on an LB plate containing 50μg / mL kanamycin.

[0049] (6) Invert the plate and incubate it overnight in a 37°C incubator.

[0050] Example 2: Construction of the second expression vector pBR322-Pdp1-HBD-3 1. Preparation of a linearized vector backbone with natural toxin proteins removed by reverse PCR To replace the naturally occurring toxin proteins loaded in the PVC cavity with HBD-3 First, a vector backbone with the natural toxin protein coding sequence removed needs to be constructed. For this purpose, inverse PCR was employed. Using the commercial plasmid pBR322-Pvc17-22 (a wild-type PVC lumen protein containing the natural toxin protein; Catalog: 198272, purchased from Addgene) as a template, a pair of reverse primers (ZT2-F and ZT2-R) were designed. The 3' ends of these primers are complementary to the sequences flanking the natural toxin protein coding region, and the primers are oriented outwards. This PCR reaction amplifies a linearized DNA fragment that fully preserves the PVC lumen plasmid backbone but precisely deletes the natural toxin protein coding sequence, thus facilitating subsequent homologous recombination insertion. HBD-3 The gene fragments are ready.

[0051] Agarose gel electrophoresis confirmed that the size of the obtained linearized vector fragment was in line with expectations. Figure 3 The reverse primer sequence used is as follows: ZT2-F:TATGGATTTTCATGTCATCAGGAGA (SEQ ID NO. 13); ZT2-R:TAATTCTGAAGCTTCTTCAATCTCC (SEQ ID NO. 14).

[0052] The specific PCR reaction system is shown in Table 1 of Example 1.

[0053] 2. HBD-3 gene fragments The DNA sequence encoding mature human β-defensin-3 (HBD-3) was synthesized by Sangon Biotech Co., Ltd. (NCBI Reference Sequence: NC_000008.11). During synthesis, a linker peptide (amino acid sequence: GGSGGGGGSGG) was added to the 5' end of the gene to connect two protein domains. The β-defensin-3 (HBD-3) gene was amplified by PCR using primers containing homologous arms for homologous recombination. The gel electrophoresis results of the β-defensin-3 (HBD-3) gene are shown below. Figure 4 As shown. The primer sequences used are as follows: TYB2-F:ttgaagaagcttcaagattaGGTGGCTCTGGTGGTGGC (SEQ ID NO.15) TYB2-R:tgatgacatgaaaatccataTTATTTCTTTCTTTCGGCAGCATT (SEQ ID NO.16) 3. Homologous recombination linkage The β-defensin-3 nucleotide sequence (SEQ ID NO. 8) containing a homologous arm and a linker peptide was ligated into the linearized vector backbone pBR322-Pvc17-22 via homologous recombination. After transformation into competent DH5α cells, single colonies were picked and sequenced for verification. Plasmids were then extracted using the TIANGEN endotoxin-free plasmid mini-prep kit to obtain the second expression vector pBR322-Pdp1-HBD-3.

[0054] The homologous recombination reaction system is shown in Table 2 of Example 1.

[0055] Example 3: Preparation and purification of engineered PVC protein complex 1. The first expression vector pAWP78-Pvc13-Srap prepared in Example 1 and the second expression vector pBR322-Pdp1-HBD-3 prepared in Example 2 were electroporated into competent EPI300 cells. After successful transformation, single colonies on LB agar medium were picked and added to LB liquid medium containing 100 μg / mL ampicillin and 50 μg / mL kanamycin. The mixture was incubated overnight at 37°C with constant shaking at 220 rpm.

[0056] 2. Inoculate the overnight shaken bacterial culture into 600ml LB liquid medium at a ratio of 1:100, and incubate at 220 rpm and 30℃ for 24 hours.

[0057] 3. Collect the bacterial precipitate after centrifuging at 4°C and 8000 rpm for 10 minutes using a high-speed centrifuge.

[0058] 4. Discard the supernatant, add 30 ml of 1×PBS buffer to resuspend the bacterial pellet, and centrifuge at 4°C and 4000 rpm for 30 minutes.

[0059] 5. Discard the supernatant and freeze the bacterial precipitate in an ultra-low temperature freezer at -80°C for at least 12 hours.

[0060] 6. Prepare P Buffer: 0.5×Cellytic B lysis buffer, 25 mmol / L -1 Tris (pH=7.4), 200 μg / mL -1 Lysozyme, 50 μg / mL -1 Dnase I, 0.5% Triton X-100, 5mmol L -1 MgCl2, 1× protease inhibitor, mix thoroughly.

[0061] 7. Add 30 ml of P Buffer to the frozen bacteria, resuspend by pipetting, and incubate in a water bath at 37°C for 30 minutes.

[0062] 8. Place in a high-speed refrigerated centrifuge at 4°C and centrifuge at 10,000 rpm for 15 minutes. Collect the supernatant in an ultracentrifuge tube and balance it with P Buffer to ensure balance. Centrifuge at 4°C and 150,000 g for 1.5 hours.

[0063] 9. Discard the supernatant, invert the ultracentrifuge tube to allow it to dry, resuspend the precipitate in 1 ml of 4°C pre-cooled sterile 1×PBS buffer, transfer it to a 1.5 ml EP centrifuge tube, centrifuge at 4°C and 4000 rpm for 10 minutes in a benchtop refrigerated centrifuge, and collect the supernatant.

[0064] 10. Place the supernatant back into the ultracentrifuge tube, add sterile 1×PBS buffer pre-cooled at 4°C to balance the mixture, and centrifuge at 150,000 g for 1.5 hours at 4°C.

[0065] 11. Discard the supernatant, invert the ultracentrifuge tube to allow it to dry, resuspend the precipitate in 200 μl of sterile 1×PBS buffer pre-cooled at 4°C, transfer it to a 1.5 ml Eppendorf tube, and centrifuge at 4°C and 4000 rpm for 10 minutes using a benchtop refrigerated centrifuge.

[0066] 12. Collect the supernatant, measure the PVC concentration at a wavelength of 280 nm using a micro-nucleic acid protein analyzer, and store it in a 4°C refrigerator for later use (stored in a 4°C refrigerator for a maximum of 7 days).

[0067] Endotoxins (chemically lipopolysaccharides) are major components of the cell walls of Gram-negative bacteria. PVC protein complexes extracted from E. coli cells often contain high levels of endotoxin contamination and are highly toxic to cells. Therefore, they should be removed before conducting cell biology experiments. The following steps were taken to remove endotoxins using a liquid-phase endotoxin remover: 1) Add the pre-cooled liquid endotoxin remover (Beyotime, C0268S) at a ratio of 1:10 to the extracted PVC solution, mix well by pipetting, and place on ice for 10 minutes.

[0068] 2) Place in a constant temperature water bath and heat at 37°C for 25 minutes, until the solution becomes turbid or separates into layers.

[0069] 3) Centrifuge at 13,000 rpm for 10 minutes at room temperature, and collect the supernatant, i.e., the PVC solution. Be careful not to collect the precipitate or the bottom liquid.

[0070] The above steps can be repeated multiple times to achieve complete removal of endotoxins.

[0071] Transmission electron microscopy revealed that the purified wild-type PVC (wild-type PVC is the original commercially available pAWP78-PVCpnf1-16 and pBR322-PVCpnf17-22 that were not modified and were directly purified using the same purification steps as above) was observed. Figure 5 ) and the engineered PVC protein complex of the present invention ( Figure 6 All exhibited typical and structurally intact 'syringe'-shaped nanoparticle morphology, with uniform particle size and distribution. This indicates that the targeted modification of PVC did not affect the correct assembly of its protein subunits or the integrity of the overall complex structure.

[0072] Example 4: Evaluation of the in vitro clearance effect of engineered PVC protein complex on intracellular Staphylococcus aureus 1. Construction of an intracellular infection model of Staphylococcus aureus: (1) After digesting and counting bovine mammary epithelial MAC-T cells in good growth condition, the cells were divided into groups of 3.5 × 10⁶ cells per well. 5 Cells were seeded at a density of 1,000 cells per well in 6-well cell culture plates and cultured at 37°C in a 5% CO2 incubator until the cells reached 80% confluence.

[0073] (2) Take 1 mL of overnight cultured Staphylococcus aureus culture into a 1.5 mL EP centrifuge tube, centrifuge at 4℃ and 3000×g for 10 minutes to collect the cells, and discard the supernatant.

[0074] (3) Gently resuspend the bacterial pellet in sterile 1×PBS buffer, centrifuge and wash again, and repeat this step a total of 3 times.

[0075] (4) The washed bacteria were resuspended in 1 mL of DMEM-F12 basal medium without FBS and antibiotics, and the bacteria were counted using a hemocytometer.

[0076] (5) Discard the cell culture supernatant from the 6-well plate and gently wash the cells twice with pre-warmed PBS. Based on the cell count, add an appropriate amount of bacterial suspension to each well (to a final volume of 2 mL / well with basal medium) at a ratio of 30:1 (30 Staphylococcus aureus per MAC-T cell). Incubate the culture plate in an incubator for 2 hours to allow the bacteria to fully invade the host cells.

[0077] (6) After incubation, the bacterial culture medium was aspirated, and the cells were washed three times with PBS to remove all uninvaded adhering bacteria. Then, 2 mL of complete culture medium (DMEM-F12 basal medium + 10% fetal bovine serum) containing 100 μg / mL gentamicin was added to each well, and the cells were cultured for another hour. Gentamicin effectively killed all remaining extracellular bacteria, thus establishing a pure intracellular Staphylococcus aureus infection model.

[0078] 2. Determination of intracellular bacterial survival rate The cells that had been used to establish the infection model were randomly divided into the following three groups: (1) Blank control group: not infected with Staphylococcus aureus, without PVC treatment, containing only Mac-T cells and complete culture medium.

[0079] (2) Infection model group (positive control group): infected with Staphylococcus aureus, without PVC treatment, and with an equal amount of 1×PBS.

[0080] (3) PVC treatment group: infected with Staphylococcus aureus, and added to a complete culture medium containing 2.5 mg / mL PVC protein complex.

[0081] When the Staphylococcus aureus infection model was completed, i.e. at "0 hours", the blank control group and the infection model group were replaced with complete culture medium, and the PVC treatment group was replaced with complete culture medium containing 2.5 mg / ml PVC. The cells were then returned to the CO2 incubator for further culture.

[0082] The intracellular bacterial survival rate was determined after 0, 12, 24, and 48 hours of incubation. The method is as follows: (1) At the above time points, discard the culture medium, wash with 1×PBS, add 0.1% Triton X-100 solution, and mix by pipetting to lyse the cells and release intracellular bacteria.

[0083] (2) Perform serial dilutions of the cell lysate (10 ppm). -1 10 -2 10 -3 Take 5 μl and spread it on LB agar medium.

[0084] (3) After incubation at 37℃ for 24 hours, count the number of colonies (CFU) and calculate the number of CFUs at each time point. Compared with the "0-hour" infection model group, calculate the change in intracellular bacterial survival rate after PVC treatment. Intracellular bacterial survival rate = (PVC treatment group / 0-hour infection model group) × 100%.

[0085] The test results show: Figure 7 Representative photographs of colony growth on LB agar plates after treatment with engineered PVC protein complex in an intracellular bacterial infection model for different time periods are presented. It can be clearly seen that the number of colonies in the infection model group was relatively high and did not decrease significantly within 0–48 h on all plates; while in the PVC treatment group, the number of colonies decreased significantly after 12 h of treatment; after 24 h of treatment, the number of colonies was significantly lower than that in the model group; and after 48 h of treatment, the colonies were extremely sparse, approaching the detection limit. These results clearly demonstrate that engineered PVC can effectively eliminate intracellular Staphylococcus aureus.

[0086] Figure 8 The intracellular bacterial survival rate statistical curve showed that the intracellular bacterial survival rate in the PVC treatment group decreased significantly over time, with a survival rate of 45% at 12h, 18% at 24h, and 8% at 48h. In contrast, the survival rate in the infection model group remained above 90%, further verifying the intracellular bactericidal effect of engineered PVC and the time-dependent bactericidal effect.

[0087] Example 5: Cytotoxicity assessment of engineered PVC protein complexes To quantitatively assess the potential toxicity of different concentrations of engineered PVC protein complexes to host cells (MAC-T), determine their safe concentration range, and provide a safety basis for dose selection in efficacy trials, the following cell types and reagents were selected for cytotoxicity assessment experiments: Cells: Bovine mammary epithelial cells (MAC-T). Complete culture medium: DMEM / F12 supplemented with 10% fetal bovine serum. Detection reagent: CCK-8 cell counting kit. Sample to be tested: PVC protein complex prepared in Example 3, diluted to the required concentration with complete culture medium.

[0088] MAC-T cells were seeded at a density of 6000 cells per well in 96-well plates and cultured overnight for adhesion. The following experimental groups were set up: Blank control group: Only fresh complete culture medium was used.

[0089] PVC treatment group: The culture medium was replaced with complete culture medium containing 1.0 mg / mL, 2.5 mg / mL, and 4.0 mg / mL PVC protein complex, respectively.

[0090] Each group has 6 replicates. The treated cells are then returned to the incubator for further culture.

[0091] Tests were performed at 12, 24, 48, and 72 hours after treatment: (1) Add 10 μL of CCK-8 solution directly to each well, mix gently, and continue incubation for 1.5 hours.

[0092] (2) The absorbance of each well was measured at 450 nm using an ELISA reader. Cell viability = (OD value of PVC treatment group - OD value of blank control group) / (OD value of cell control group - OD value of blank control group) × 100%.

[0093] Figure 9 The experimental results showed that at concentrations of 1.0 mg / mL and 2.5 mg / mL, the PVC protein complex had no significant effect on the viability of MAC-T cells at each time point (12-72 hours), and the relative viability of cells remained above 95%, with no statistical difference compared with the blank control group (p>0.05).

[0094] Figure 10 Experimental results showed that when the concentration was increased to 4.0 mg / mL, a statistically significant decrease in cell viability was observed after 72 hours of treatment (p<0.05), with a relative viability of approximately 85%. Figure 10 This indicates that mild cytotoxicity may occur under this high concentration and long duration of exposure.

[0095] Overall trends indicate that PVC protein complexes exhibit excellent biocompatibility at effective concentrations (≤2.5 mg / mL).

[0096] The above test results show that the engineered PVC protein complex of the present invention is non-toxic to host cells in the concentration range of ≤2.5 mg / mL, proving that it has good biosafety at the effective dose for antibacterial effect.

[0097] Example 6: Evaluation of the regulatory effect of engineered PVC protein complex on the inflammatory response of infected cells To investigate the regulatory role of the PVC protein complex in clearing intracellular bacteria on infection-related inflammatory responses and to evaluate its additional benefits in alleviating infectious tissue damage, a stable intracellular Staphylococcus aureus infection model of MAC-T cells was constructed according to the description in Example 4.

[0098] Two processing groups were designed: Control group: Not infected, no PVC treatment added.

[0099] PVC treatment group: For Staphylococcus aureus infection, add PVC to a final concentration of 2.5 mg / mL.

[0100] Each group has 3 replicate wells, and the experiment is repeated 3 times.

[0101] Cells were collected at 0, 12, 24, and 48 hours after treatment (or medium change).

[0102] Total RNA was extracted from cells using the TIANGEN TRNzol Universal kit, and its concentration and purity were determined (A260 / A280 ratio between 1.8 and 2.0). gRNA was removed and cDNA was synthesized via reverse transcription using the EasyScript One-Step kit.

[0103] Targeting inflammatory factor genes ( IL-6, CX-8 ) and internal reference gene ( GAPDH Specific primers were designed for real-time quantitative PCR. The primer sequences used are as follows: IL-6-F:GTGAGAAGAATGATGAGTGTGAAAGC (SEQ ID NO. 17); IL-6-R: ATCGCCTGATTGAACCCAGATTG (SEQ ID NO. 18).

[0104] CX-8-F:ATGACTTCCAAGCTGGCTGTTG (SEQ ID NO. 19); CX-8-R: TGGCATCGAAGTTCTGTACTCATTC (SEQ ID NO. 20).

[0105] GAPDH-F: CGGCACAGTCAAGGCAGAGAAC (SEQ ID NO. 21); GAPDH-R: CCACATACTCAGCACCAGCATCAC (SEQ ID NO. 22).

[0106] Using cDNA as a template, amplification reactions were performed on an Applied Biosystems 7500 real-time quantitative PCR instrument using ChamQ SYBR qPCR Master Mix. The reaction program was: 95°C pre-denaturation for 30 seconds; 95°C denaturation for 5 seconds; 60°C annealing / extension for 30 seconds, for a total of 40 cycles. Two... –ΔΔCtThe method calculates the relative expression level of the target gene.

[0107] Experimental results are as follows Figure 11 As shown in the figure, qPCR results showed that engineered PVC treatment significantly inhibited the expression of both inflammatory cytokine genes in a time-dependent manner. Compared with the 0-hour infection model group, the IL-6 mRNA expression level in the PVC-treated group began to decrease significantly at 12 hours and remained at extremely low levels at 24 and 48 hours. This indicates that engineered PVC can rapidly and potently inhibit IL-6 gene transcription triggered by intracellular bacterial infection; the expression trend of CXCL8 was similar to that of IL-6. After PVC treatment, the mRNA expression level of CXCL8 also showed a significant decrease, with a substantial reduction in expression levels at 24 and 48 hours after treatment compared to the 0-hour model group.

[0108] The above test results demonstrate that the engineered PVC protein complex of this invention can not only directly eliminate intracellular Staphylococcus aureus, but also effectively inhibit the excessive secretion of pro-inflammatory factors IL-6 and CXCL8 by host cells by eradicating the source of infection, thereby exerting a dual therapeutic effect in reducing infection-related inflammatory damage. This further highlights its comprehensive advantages as a therapeutic strategy targeting intracellular infections.

[0109] The above description is merely a preferred embodiment of this application and is not intended to limit the application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications made within the spirit and principles of this application are not permitted. Equivalent substitutions and improvements should all be included within the scope of protection of this application.

Claims

1. An engineered PVC protein complex for targeted elimination of intracellular Staphylococcus aureus, characterized in that, The engineered PVC protein complex is a recombinant protein assembly obtained through genetic engineering, comprising: (a) Targeting component: PVC tail fibroin with its natural host recognition domain removed is linked to the Staphylococcus aureus receptor binding domain SrapBR; (b) Effector assembly: The PVC cavity, from which the natural toxin proteins have been removed, is connected to human β-defensin-3; The amino acid sequence of the PVC tail fibroin with the natural host recognition domain removed is shown in SEQ ID NO.1, and the amino acid sequence of the Staphylococcus aureus receptor binding domain SrapBR is shown in SEQ ID NO.

2. The amino acid sequence of the PVC lumen after natural toxin proteins have been removed is shown in SEQ ID NO.3, and the amino acid sequence of the human β-defensin-3 is shown in SEQ ID NO.

4.

2. The engineered PVC protein composite according to claim 1, characterized in that, In the targeting component, the Staphylococcus aureus receptor-binding domain SrapBR is connected to the PVC tail fibrous protein with the natural host recognition domain removed via a flexible linker peptide; in the effector component, human β-defensin-3 is also connected to the PVC lumen with the natural toxin protein removed via a flexible linker peptide.

3. The engineered PVC protein composite according to claim 2, characterized in that, The amino acid sequence of the flexible linker peptide is GGSGGGGGSGG.

4. The engineered PVC protein complex according to any one of claims 1-3, characterized in that, The amino acid sequence of the engineered PVC protein complex is shown in SEQ ID NO.

6.

5. The use of the engineered PVC protein complex of claim 1 in the preparation of a drug for eliminating pathogens within host cells, characterized in that, The pathogen is an intracellular parasite.

6. The application according to claim 5, characterized in that, The intracellular parasite is Staphylococcus aureus.

7. The application according to claim 5, characterized in that, The drug for eliminating pathogens within host cells contains an engineered PVC protein complex at a dose of 1.0-4.0 mg / mL.

8. A method for preparing the engineered PVC protein complex according to claim 1, characterized in that, Includes the following steps: (1) The contents of homologous arms and linker peptides SrapBR The nucleotide sequence was ligated with the linear plasmid pAWP78-PVCpnf1-16 (with the natural host recognition domain removed) to construct the first expression vector pAWP78-Pvc13-Srap; the β-defensin-3 nucleotide sequence containing homologous arms and linker peptides was ligated with the linear plasmid pBR322-Pvc17-22 (with the natural toxin protein removed) to construct the second expression vector pBR322-Pdp1-HBD-3; (2) The first expression vector pAWP78-Pvc13-Srap and the second expression vector pBR322-Pdp1-HBD-3 were introduced into the competent cells of the host bacteria to obtain transformed bacteria, and the expression of engineered PVC protein complex in the transformed bacteria was induced. (3) Collect the cells of the transformed bacteria, wash them with buffer, pre-treat them by cryopreservation at ultra-low temperature, resuspend them in buffer, remove cell debris by centrifugation, and obtain the supernatant containing the engineered PVC protein complex. (4) The supernatant is centrifuged multiple times. After each centrifugation, the precipitate is collected and resuspended in buffer solution. After the last centrifugation, the supernatant is collected to obtain the purified engineered PVC protein complex.

9. The preparation method according to claim 8, characterized in that, The host bacterium is Escherichia coli EPI300; the co-introduction method is electroporation.

10. The preparation method according to claim 8, characterized in that, The [comprising] homologous arms and linker peptides SrapBR The nucleotide sequence is shown in SEQ ID NO.7, and the β-defensin-3 nucleotide sequence containing the homologous arm and linker peptide is shown in SEQ ID NO.8.

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