A perforin derived from Staphylococcus warwick temperate phage and its application

Abstract

This invention belongs to the field of biotechnology, specifically relating to a perforin derived from *Staphylococcus warwickiana* temperate bacteriophage and its applications. The perforin has the nucleotide sequence shown in SEQ ID NO:1. This perforin is a key gene for phage lysis and growth, playing a crucial role in the lysis of host bacteria. Therefore, this study aims to verify whether the perforin, whose coding site is located on the genome of *Staphylococcus warwickiana* temperate bacteriophage, as a cell lysis gene, can serve as a potential antibacterial substance, providing a theoretical basis for the prevention and control of Gram-negative pathogens.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a perforin derived from Staphylococcus warwick temperate phage and its applications. Background Technology

[0002] Staphylococci can be identified by observing their irregular, grape-like cell clusters under a microscope. They are Gram-positive, facultative anaerobic, catalase-positive, and non-spore-forming bacteria. They have extremely high salt tolerance and are part of the normal microbiota on the skin and mucous membranes of humans and animals, widely distributed in various ecological niches, including soil, water, air, and various foods. Coagulase is considered the main virulence factor of Staphylococci. Based on their ability to coagulate rabbit blood, they can be divided into coagulase-positive and coagulase-negative bacteria. Coagulase-negative bacteria (CNS) are generally non-pathogenic and are classified as minor pathogens. They are widely distributed in the natural environment and exhibit richer genetic diversity. CNS are indispensable fermentation microorganisms in the traditional production of fermented meat products, possessing significant enzymatic properties such as proteases and lipases. Staphylococcus warneri is a common CNS, widely used as a fermentation agent for meat products due to its highly efficient enzymatic properties. In industrial applications, Staphylococcus warwick not only demonstrates the potential to produce heat- and organic solvent-resistant lipases, but also plays an important role in the field of metal nanoparticles, which has received much attention in recent years.

[0003] Bacteriophages are a general term for viruses that specifically infect bacteria. Based on their different growth cycles during their interaction with host bacteria, they can be divided into two categories: virulent phages and temperate phages. Virulent phages immediately enter the lysis cycle after infecting host bacteria. During this cycle, the phage genome is replicated extensively and packaged into progeny phage particles, which are released after programmed lysis of the host cell. Temperate phages, on the other hand, have both a lysogenic and a lysis cycle. During the lysogenic cycle, the phage can integrate its genome into the host chromosome; at this stage, the temperate phage is called a prophage. It replicates along with the host chromosome and maintains its lysogenic state by inhibiting the phage's lysis genes. Under stimulation that induces a bacterial SOS response (such as antibiotic treatment, oxidative stress, or DNA damage), the prophage activates the lysis cycle, lysing the bacterial cell and releasing progeny phages, just like virulent phages. Most bacteriophages in nature are double-stranded DNA (dsDNA) phages. For most dsDNA phages, host cell lysis is caused by the synergistic action of two proteins. Late in the phage lysis cycle, endolysins are first produced and accumulate in the cytoplasm, but because they cannot cross the cytoplasmic membrane to function in the peptidoglycan layer, they require the assistance of perforin. When perforin aggregates, it promotes inner membrane depolarization. Perforin dimers are the basic form of its functional assembly, subsequently polymerizing into oligomers and forming pores in the cytoplasmic membrane, thus allowing endolysins to be released into the periplasm.

[0004] Current research on staphylococcal bacteriophages mainly focuses on treating infections caused by methicillin-resistant Staphylococcus aureus (MRSA) and other drug-resistant bacteria, providing an alternative treatment method to antibiotics; in-depth analysis of the bacteriophage genome provides a basis for developing new antibacterial strategies; simultaneously, bacteriophages have shown great potential in controlling bacterial biofilms and have been used to control Staphylococcus aureus contamination in the food industry. Perforin is a key protein in the lysis cycle of virulent bacteriophages, while temperate bacteriophages are usually in a lysogenic state, which inhibits perforin expression. Current research on perforin is mainly divided into four categories: research on perforin as an antibacterial substance; combined use with antibiotics to increase antibiotic permeability, thereby significantly improving bactericidal efficiency; use in the design of nanoparticles or liposomes for precise targeted drug delivery tools; and combining perforin with fluorescent labels for rapid pathogen detection.

[0005] Research on mild phages of Staphylococcus wartii is relatively limited, and there are no reports on perforin of mild phages of Staphylococcus wartii. Summary of the Invention

[0006] The purpose of this invention is to provide a perforin derived from Staphylococcus warwick temperate phage and its application.

[0007] To achieve the objectives described in this invention, the following technical solutions are provided:

[0008] A perforin derived from the temperate bacteriophage of Staphylococcus wartii, the perforin having the nucleotide sequence shown in SEQ ID NO:1.

[0009] The amino acid sequence of the protein encoded by the perforin is shown in SEQ ID NO:2.

[0010] The primer pair for obtaining perforin is:

[0011] holin64-FGACGCATGGCAAAACTTCGCTAC;

[0012] holin64-R TTCTTGCGCTTTTGAATGCGAGTG.

[0013] The application of the perforin derived from Staphylococcus warwick temperate phage, wherein the perforin is used in the lysis of Gram-negative host bacteria.

[0014] A method for lysing cells in vivo involves introducing the perforin into recipient cells, culturing them, and then lysing the recipient cells.

[0015] Specifically, the process involves: amplifying the perforin, digesting the amplification product and plasmid with restriction endonucleases, integrating them to obtain a recombinant plasmid, and then transforming the recombinant plasmid into recipient cells. Successfully transformed *E. coli* cells are then incubated with a solution containing 50 μg / mL... -1 Amp cultured in LB liquid medium until OD 600 The value was approximately 0.6, and the final concentration added was 0.5 mg / mL. -1 IPTG-induced overnight low-temperature induction leads to host cell lysis.

[0016] The primers for amplifying the perforin are:

[0017] holin64-FGACGCATGGCAAAACTTCGCTAC;

[0018] holin64-R TTCTTGCGCTTTTGAATGCGAGTG.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] This invention successfully obtained the perforin gene sequence based on the mild bacteriophage vB_G30_01 of Staphylococcus warwick and successfully expressed the perforin protein (holin64) in Gram-negative Escherichia coli. Simultaneously, this perforin protein significantly lyses host Gram-negative Escherichia coli cells, providing a new theoretical basis for the prevention and control of Gram-negative pathogens. Attached Figure Description

[0021] Figure 1 The diagram shows the transcription effect of the perforin gene in Staphylococcus warwick and temperate phage vB_G30_01 under MMC induction, as provided in the embodiments of the present invention.

[0022] Figure 2 This is an analysis diagram of the conserved domains of the perforin protein provided in an embodiment of the present invention.

[0023] Figure 3 This is a prediction map of the transmembrane region of perforin protein provided in an embodiment of the present invention.

[0024] Figure 4 This is a predicted tertiary structure diagram of perforin protein provided in an embodiment of the present invention.

[0025] Figure 5 The image shows a cloned electrophoresis diagram of the perforin gene provided in an embodiment of the present invention, where M: 2000 Marker; 1-2: holin64.

[0026] Figure 6 The growth curves of Escherichia coli Rosetta-gami2(DE3) with transformation plasmid pET32a(+) and recombinant plasmid pET32a-holin64 provided in the embodiments of the present invention.

[0027] Figure 7 The following is an SDS-PAGE analysis of recombinant protein pET32a-holin64 expression induced by different concentrations of IPTG provided in this embodiment of the invention, where M: Marker; 1-5: recombinant protein pET32a-holin64; 1: 1 mg·mL -1 IPTG; 2: 0.75 mg / mL -1 IPTG; 3: 0.5 mg / mL -1 IPTG; 4: 0.25 mg / mL -1 IPTG; 5:0 mg·mL -1 IPTG.

[0028] Figure 8 The image shows the bacterial cell precipitation effect after IPTG induction provided in the embodiment of the present invention, where 1: plasmid pET32a(+); 2: recombinant plasmid pET32a-holin64.

[0029] Figure 9 The image shows the colony count results after dilution and plating of Escherichia coli Rosetta-gami2(DE3) with plasmid pET32a(+) and recombinant plasmid pET32a-holin64 provided in the embodiments of the present invention.

[0030] Figure 10 The growth curves of Escherichia coli Rosetta-gami2(DE3) transformed with IPTG-induced plasmid pET32a(+) and recombinant plasmid pET32a-holin64 provided in the embodiments of the present invention.

[0031] Figure 11 The images show the staining results of live and dead cells of Escherichia coli Rosetta-gami2(DE3) before and after IPTG induction with the transformation plasmid pET32a(+) and recombinant plasmid pET32a-holin64 provided in the embodiments of the present invention.

[0032] Figure 12 The diagram shows the leakage of lactate dehydrogenase (LDH) in Escherichia coli Rosetta-gami2(DE3) transformed with different plasmids before and after IPTG induction, as provided in the embodiments of the present invention.

[0033] Figure 13 Scanning electron microscope (SEM) images of Escherichia coli Rosetta-gami2(DE3) transformed with different plasmids before and after IPTG induction, provided in the embodiments of the present invention.

[0034] Figure 14 This diagram illustrates the transcription of the perforin gene in Escherichia coli Rosetta-gami2(DE3) expressing the recombinant protein pET32a-holin 64 after IPTG induction, as provided in this embodiment of the invention. Detailed Implementation

[0035] The following examples further illustrate this study, but the examples do not constitute any limitation on the present invention.

[0036] Perforin is a key gene for phage lysis and growth, playing a crucial role in the lysis of host bacteria. Therefore, this study aims to verify whether perforin, whose coding site is located on the genome of *Staphylococcus warwickii* temperate phage, as a cell lysis gene, can serve as a potential antibacterial substance, providing a theoretical basis for the prevention and control of Gram-negative pathogens.

[0037] This experiment obtained the perforin candidate gene sequence from Staphylococcus warneri temperate phage vB_G30_01 (Pu,F.;Zhang,N.;Pang,J.;Zeng,N.;Baloch,FB;Li,Z.;Li,B.Deciphering the Genetic Architecture of Staphylococcus warneri Prophage vB_G30_01:A Comprehensive Molecular Analysis.Viruses 2024,16,1631.), predicted its protein structure, successfully cloned and identified its function, and explored the application of the protein it encodes.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional experimental methods already existing in the prior art.

[0039] Example 1: Detection of perforin gene transcription level in Staphylococcus warwick temperate phage vB_G30_01 after mitomycin C (MMC) treatment

[0040] The genome data of bacteriophage vB_G30_01 has been submitted to the NCBI database with accession number PP213047. The perforin gene sequence was obtained from the whole genome sequencing of bacteriophage vB_G30_01 and named holin64.

[0041] SEQ ID NO:1

[0042] Nucleotide sequence (5'-3') of the perforin gene (holin64) of Staphylococcus warwick temperate phage vB_G30_01.

[0043] ATGCAATTCCAGAAAAACAATACACTCACAAAGCTAACCTTTAAGGTT

[0044] GGTTTTTTATTTTACTCAAAAGGAGATAATCAAATGACTTCAGATAAAT

[0045] TAAAACAATATATTGGCTTATTTGGTGGTATGTTAGGGGCTTTATACCTT

[0046] GCATTAAAAGCAAGTGGAATCGAAGTTCCTTTTTTAATGCCCGATAAA

[0047] TTAGACGCATGGCAAAACTTCGCTACGTCAATAGTACCTTTTGTAATTG

[0048] CGATATATGGCGTCTATAAAAACACATATATTATTCACTCGCATTCAAAA

[0049] GCGCAAGAAGAATACTTAAAAGAAAATAATTTAAAATAG

[0050] SEQ ID NO:2

[0051] The amino acid sequence of the perforin protein (holin64) of *Staphylococcus warwick* temperate phage vB_G30_01 is as follows: MQFQKNNTLTKLTFKVGFLFYSKGDNQMTSDKLKQYIGLFGGMLGALYLALKASGIEVPFLMPDKLDAWQNFATSIVPFVIAIYGVYKNTYIIHSHSKAQEEYLKENNLK

[0052] 1. Total RNA extraction

[0053] Staphylococcus warwick G30 was cultured in beef extract peptone liquid medium until OD. 600 The concentration was approximately 0.4, and then a final concentration of 1 μg·mL⁻¹ was added to the system. -1 Mitomycin C (MMC) induction was performed, and an equal volume of fresh beef extract peptone liquid medium was added to the control. Bacterial cell pellets were collected at 0h, 2h, 4h, 6h, 8h, and 10h of culture time, and RNA was extracted using the SteadyPure Universal RNA Extraction Kit.

[0054] 2. Synthesis of reverse-transcribed cDNA

[0055] RNA template solutions were prepared using the Evo M-MLV RT Mix Kit with gDNA Clean for qPCR (Evo M-MLV Reverse Transcription Premix Kit, which includes gDNA removal reagent for qPCR), and the reverse transcription reaction was performed.

[0056] 3. Real-time quantitative PCR system

[0057] Primers

[0058] Table 1 Primers used for qPCR

[0059]

[0060] Using the Premix Pro Taq HS qPCR Kit (ROX Plus) The Green Pro Taq HS premixed qPCR kit (containing ROX) uses 16S rDNA as the internal reference gene.

[0061] Experimental results show that ( Figure 1 Under MMC treatment conditions, the perforin gene of *Staphylococcus wartii* temperate phage vB_G30_01 began to be transcribed in large quantities. As time progressed, its relative expression level gradually increased, indicating that this gene participated in the phage lysis and growth process. The relative expression level of the perforin gene reached its peak 8 hours after the addition of MMC, indicating that it was a late-transcription gene. Example 2: Bioinformatics Analysis of Perforin Protein

[0062] 1. Physicochemical property analysis of perforin protein

[0063] The physicochemical properties of proteins were analyzed using the Protparam software (https: / / web.expasy.org / protparam / ), including the protein's amino acid composition, relative molecular mass, isoelectric point, and hydrophilicity / hydrophobicity.

[0064] The physicochemical properties of the protein were analyzed using ProtParam software. The results showed that perforin protein consists of 110 amino acids, with a relative molecular mass of 12538.61, an isoelectric point of 9.27, and is slightly alkaline. It contains 8 negatively charged amino acids and 12 positively charged amino acids. Its molecular formula is C1. 583 H 896 N 140 O 159 S4 has a total of 1782 atoms, an instability index of 17.10, a lipid index of 90.45, and an average hydrophilicity of -0.084. Therefore, this protein is a stable hydrophobic protein.

[0065] 2. Prediction of conserved domains of perforin proteins

[0066] The domains of perforin were analyzed using the NCBI (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) online software for conserved structural domains.

[0067] According to the comparison of perforin proteins based on the NCBI conserved domain database, the results showed that ( Figure 2 Perforin possesses a homologous domain to the Holin-SPP1 superfamily of proteins, which consists of perforin derived from long-tailed phages with two transmembrane segments of double-stranded DNA.

[0068] 3. Prediction of the transmembrane region of perforin

[0069] Using TMHMM (https: / / services.healthtech.dtu.dk / service.php?)

[0070] The TMHMM-2.0 software predicts the transmembrane regions of perforin and endosomal proteins.

[0071] The transmembrane region of perforin was predicted using TMHMM software, and the results showed that perforin has two transmembrane regions ( Figure 3 Based on the number of transmembrane regions, perforin can be classified into three types: Type I perforin generally contains three transmembrane regions, Type II perforin generally contains two transmembrane regions, and perforin containing other numbers of transmembrane regions is classified as Type III perforin. Based on this, the perforin protein in *Staphylococcus warwickii* temperate phage vB_G30_01 is determined to be a Type II perforin.

[0072] 4. Prediction of the higher-order structure of perforin proteins

[0073] The secondary structure of perforin was analyzed using the SOPMA tool (https: / / npsaprabi.ibcp.fr / cgibin / npsa_automat.pl?page=npsa_sopma.htmL). The tertiary structure of perforin was predicted using AlphaFold (https: / / colab.research.google.com / github / sokrypton / Colab Fold / blob / main / AlphaFold2.ipynb), and a three-dimensional structure diagram of the protein was drawn.

[0074] SOPMA analysis of the secondary structure content of perforin revealed that it comprises 40% α-helices, 25.45% extended chains, 10% β-sheets, and 24.55% random coils. This indicates that the secondary structure of perforin is primarily α-helices, followed by random coils and extended chains in similar proportions, with β-sheets being the least abundant.

[0075] The tertiary structure of perforin was predicted using AlphaFold2 software, and the prediction results were modeled to generate 3D visualizations. The output results show that ( Figure 4 The three-dimensional model of perforin protein shows that its tertiary structure is mainly α-helix with fewer β-turn structures, which is basically consistent with the results of secondary structure prediction.

[0076] Example 3: Cloning of the perforin gene and construction of a prokaryotic expression system

[0077] 1. Primer design and synthesis

[0078] The specific primers designed using Primer Premier 5.0 software (synthesized by Shanghai Sangon Biotech Co., Ltd.) are shown in the table below:

[0079] Table 2. PCR cloning primer sequences and restriction enzyme sites

[0080]

[0081] Note: Underlined areas indicate enzyme cleavage sites.

[0082] 2. Extraction of genomic DNA from Staphylococcus warwick temperate phage vB_G30_01

[0083] Staphylococcus warwick G30 was cultured in 800 mL of beef extract peptone liquid medium at 37°C and 180 rpm until OD reached. 600 The concentration was approximately 0.4, and then a final concentration of 1 μg·mL⁻¹ was added to the system. -1 Mitomycin C (MMC) was induced at 37℃ and 180 rpm for 12 h. After centrifugation at 12000 rpm for 20 min, the supernatant was filtered sequentially through 0.45 μm and 0.22 μm microporous membranes, and extracted with chloroform to obtain crude phage particle extract. A CsCl gradient solution was prepared and added to centrifuge tubes sequentially from high to low density. The crude phage extract was added on top, and the tubes were centrifuged at 100000g for 3 h at 4℃. The pale blue phage concentrate was slowly aspirated using a syringe. Finally, the phage particle concentrate obtained from CsCl was removed by ultrafiltration using a 100 kDa ultrafiltration tube. Genomic DNA of Staphylococcus warwick temperate phage vB_G30_01 was extracted using the phenol-chloroform method. The extracted DNA was dissolved in sterile water and stored at -20℃.

[0084] 3. Gene cloning

[0085] Using Staphylococcus warwick temperate phage vB_G30_01 genomic DNA as a template, and holin64 F and holin64 R as primers, PCR amplification was performed. Figure 5 ).

[0086] PCR reaction system:

[0087] Table 3 PCR reaction system

[0088]

[0089] PCR reaction procedure:

[0090] Table 4 PCR reaction procedures

[0091]

[0092] The PCR reaction solution was subjected to agarose gel electrophoresis, and the target band was recovered using the SanPrep column DNA gel recovery kit. The operation steps were followed according to the detailed steps of the kit. The recovered target band was then sequenced for verification.

[0093] 4. Construction of prokaryotic expression vectors

[0094] The known plasmid pET32a(+) and the target gene without mutations after sequencing were double-digested using restriction endonucleases Sac I and BamHI to construct the recombinant plasmid pET32a-holin64. The recombinant plasmid pET32a-holin64 was transformed into Escherichia coli Rosetta-gami2(DE3).

[0095] 5. Growth curve determination of E. coli expressing fusion protein

[0096] The growth curves of *E. coli* Rosetta-gami2(DE3) successfully transformed with empty plasmid pET32a(+) and recombinant plasmid pET32a-holin64 were measured at 600 nm. The experimental results showed that ( Figure 6 Compared with the control, the growth rate of Escherichia coli Rosetta-gami2(DE3) transformed with recombinant plasmid pET32a-holin64 was significantly slowed down.

[0097] 6. Induction of expression of the fusion protein pET32a-holin64 and analysis of its expression by protein electrophoresis.

[0098] The expression strain Rosetta-gami2(DE3), which was successfully transformed with plasmid pET32a-holin64, was subjected to a solution containing 50 μg·mL⁻¹. - 1 Amp cultured in LB liquid medium until OD 600 The value was 0.6, and the final concentration was 0 mg·mL⁻¹. -1 0.25 mg·mL -1 0.5 mg·mL -1 0.75 mg·mL -1 1 mg·mL -1 IPTG was added at a certain ratio, and the mixture was induced and cultured overnight at 16°C. Then, SDS-PAGE protein electrophoresis was performed to analyze protein expression.

[0099] The research results indicate that ( Figure 7 The recombinant protein pET32a-holin64 showed a specific band in the absence of IPTG, indicating a relatively high basal expression level. Furthermore, at 0.5 mg / mL... -1Even with IPTG induction, the specific band remained visible; therefore, this concentration was chosen as the IPTG concentration for inducing protein expression in subsequent experiments. The final concentration was 0.5 mg / mL. -1 After overnight induction with IPTG at low temperature, the cell precipitation of E. coli expressing recombinant protein perforin was significantly reduced. Figure 8 ).

[0100] Example 4: Effect of pET32a-holin64 fusion protein expression on expression strains

[0101] 1. Plate dilution and plating of E. coli expressing the fusion protein

[0102] Rosetta-gami2(DE3) E. coli successfully transformed with plasmid pET32a(+) and recombinant plasmid pET32a-holin64 were respectively subjected to 50 μg·mL⁻¹ of irradiated medium. -1 Amp cultured in LB liquid medium until OD 600 Dilute the bacterial suspension with a pH of 0.6 and spread it onto a plate containing 50 μg / mL. -1 Incubate Amp on LB solid medium at 37°C inverted for 12-16 hours, observe, photograph, and count.

[0103] Experimental results show that ( Figure 9 Compared with the control group transformed with the empty plasmid, the number of Escherichia coli Rosetta-gami2(DE3) transformed with the recombinant plasmid pET32a-holin64 was significantly reduced.

[0104] 2. IPTG-induced growth curve

[0105] Rosetta-gami2(DE3) E. coli successfully transformed with plasmid pET32a(+) and recombinant plasmid pET32a-holin64 were respectively subjected to 50 μg·mL⁻¹ of irradiated medium. -1 Amp cultured in LB liquid medium until OD 600 The value was 0.6, and the final concentration added was 0.5 mg·mL. -1 IPTG was used to induce Escherichia coli Rosetta-gami2(DE3) overnight at 18°C, and the induction growth curve was measured at 600 nm every 1 hour.

[0106] The research results indicate that ( Figure 10 ), when the final concentration is 0.5 mg·mL -1During IPTG induction, the absorbance of E. coli Rosetta-gami2(DE3) carrying the recombinant plasmid pET32a-holin64 decreased rapidly at a wavelength of 600 nm. This phenomenon may be attributed to the fact that the large expression of perforin leads to the lysis and death of E. coli. After three hours of induction, the absorbance curve began to rise, which may be due to the increase and sedimentation of lysed bacterial fragments, resulting in an increase in absorbance.

[0107] 3. Double staining of live and dead cells

[0108] The live and dead cell staining kit was used to stain E. coli Rosetta-gami2(DE3) successfully transformed with plasmid pET32a(+) and recombinant plasmid pET32a-holin64 in 50 μg / mL solution. -1 Amp cultured in LB liquid medium until OD 600 The value was 0.6, resulting in uninduced bacterial cells. Then, a final concentration of 0.5 mg / mL was added to the system. -1 Induced bacterial cells were obtained by IPTG induction overnight at 18°C. The induced and uninduced cells were stained separately and examined under a fluorescence microscope at a wavelength of 490±10 nm. Live cells appeared yellow-green, while dead cells appeared red. Excitation at 528 nm revealed red dead cells.

[0109] Experimental results show that ( Figure 11 In E. coli Rosetta-gami2(DE3) expressing the recombinant protein pET32a-holin64, some dead cells were found without induction, but more dead cells were found after IPTG induction. In contrast, E. coli Rosetta-gami2(DE3) expressing the empty vector pET32a(+) showed no dead cells after IPTG induction at low temperature overnight.

[0110] 4. Lactate dehydrogenase detection

[0111] When the cell membrane is damaged, intracellular substances leak out, and lactate dehydrogenase (LDH) may be released into the culture medium. Therefore, the extent of damage to the E. coli cell membrane by perforin and endosomalin is assessed by detecting the LDH content in the extracellular environment.

[0112] Take the E. coli that were successfully transformed with plasmid pET32a(+) and recombinant plasmid pET32a-holin64 in Experiment 3 of Example 4, centrifuge and collect the supernatant, place it on ice for testing, and refer to the instructions for use of the lactate dehydrogenase activity assay kit for subsequent experimental methods.

[0113] Experimental results show that ( Figure 12Escherichia coli Rosetta-gami2(DE3) expressing the recombinant protein pET32a-holin64 already exhibited LDH leakage even without induction, and LDH leakage significantly increased after IPTG induction.

[0114] 5. Scanning electron microscope

[0115] Take the E. coli that were successfully transformed with plasmid pET32a(+) and recombinant plasmid pET32a-holin64 in Experiment 3 of Example 4, centrifuge the bacterial pellet, thoroughly disperse and suspend it in glutaraldehyde fixative, fix it at room temperature in the dark for 30 min, and then observe it under a microscope.

[0116] Experimental results show that ( Figure 13 Compared with the pET32a(+) control, E. coli Rosetta-gami2(DE3) expressing the recombinant protein pET32a-holin64 showed an aggregated state when not induced; after IPTG induction, E. coli Rosetta-gami2(DE3) expressing the recombinant protein pET32a-holin64 showed intracellular leakage and multiple cells aggregated together.

[0117] 6. Detection of transcriptional levels of perforin and endosomalin genes induced to express

[0118] E. coli Rosetta-gami2(DE3) successfully transformed with recombinant plasmid pET32a-holin64 was subjected to a solution containing 50 μg·mL⁻¹ -1 Amp cultured in LB liquid medium until OD 600 The value was 0.6, and the final concentration added was 0.5 mg·mL. -1 IPTG was used to induce the bacteria at a low temperature of 18℃, while the control group was induced without IPTG. Bacterial cell pellets were collected at culture times of 0h, 3h, and 6h, and RNA was extracted using the SteadyPure Universal RNA Extraction Kit. Subsequent reverse transcription experiments and quantitative real-time PCR were performed as in Example 1.

[0119] Experimental results show that ( Figure 14 In E. coli Rosetta-gami2(DE3) expressing the recombinant protein pET32a-holin64, the perforin gene was rapidly upregulated after IPTG induction. The upregulation was not obvious at 3 hours, but it became significantly upregulated again at 6 hours.

[0120] The above results indicate that the expression of recombinant perforin has a significant inhibitory effect on the growth of its expression host, Escherichia coli Rosetta-gami2 (DE3), which provides a theoretical basis for the prevention and control of Gram-negative pathogens.

Claims

1. The application of a perforin derived from Staphylococcus warwick temperate phage, characterized in that: The application of the perforin in the lysis of Gram-negative host bacteria; Perforin has the nucleotide sequence shown in SEQ ID NO:1; The amino acid sequence of the protein encoded by the perforin is shown in SEQ ID NO:2; The primer pair for obtaining perforin is: holin64 -F GACGCATGGCAAAACTTCGCTAC; holin64 -R TTCTTGCGCTTTTGAATGCGAGTG。 2. A method for lysing cells in vivo, characterized in that: The perforin described in claim 1 is introduced into recipient cells and cultured to achieve lysis of the recipient cells.

3. The method for lysing cells in vivo according to claim 2, characterized in that: The perforin of claim 1 was amplified, and the amplification product and plasmid were digested and integrated using restriction endonucleases to obtain a recombinant plasmid. The recombinant plasmid was then transformed into recipient cells, and the successfully transformed *E. coli* were cultured in a solution containing 50 μg / mL... -1 Amp cultured in LB liquid medium until OD 600 The value was 0.6, and the final concentration added was 0.5 mg·mL. -1 IPTG-induced overnight low-temperature induction leads to host cell lysis.

4. The method for lysing cells in vivo according to claim 3, characterized in that: The primers for amplifying the perforin according to claim 1 are: holin64 -F GACGCATGGCAAAACTTCGCTAC; holin64 -R TTCTTGCGCTTTTGAATGCGAGTG。

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