Perforin derived from staphylococcus warneri mild bacteriophage and application

Abstract

The invention belongs to the technical field of biology, and particularly relates to perforin derived from staphylococcus warneri mild bacteriophage and application. Perforin has a nucleotide sequence as shown in SEQ ID NO: 1. The perforin is a key gene for bacteriophage splitting growth and plays an important role in the splitting process of host bacteria. Therefore, the research aims to verify whether the perforin of which the coding site is positioned on a staphylococcus warneri mild bacteriophage genome can be used as a cell lysis gene and can be used as a potential antibacterial substance or not, and a theoretical basis is provided for prevention and treatment of gram-negative pathogenic bacteria.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a perforin derived from a temperate phage of Staphylococcus worderi and an application thereof. Background Art

[0002] Staphylococci can be identified by observing the irregular grape-like cell clusters formed by them under a microscope. They are Gram-positive, facultative anaerobic, catalase-positive, and non-spore-forming bacteria. They have extremely high tolerance to salt and are part of the normal microbial community on the skin and mucosal surfaces of humans and animals. They are widely present in various ecological niches, including soil, water, air, and various foods. Coagulase is considered to be the main virulence factor of Staphylococci. According to whether they have the ability to coagulate rabbit blood, they can be divided into coagulase-positive bacteria and coagulase-negative bacteria. Among them, coagulase-negative bacteria (CNS) are usually non-pathogenic and are classified as secondary pathogens. They are widely present in the natural environment and show richer genetic diversity. CNS are indispensable fermentation microorganisms in the production of traditional fermented meat products. They have significant enzymatic properties, such as proteases and lipases. Staphylococcus warneri is a more common type of CNS and is widely used as a starter for meat products due to its efficient enzymatic properties. In industrial applications, Staphylococcus walterii not only shows the potential to produce heat-resistant and organic solvent-resistant lipases, but also plays an important role in the field of metal nanoparticles, which has attracted much attention in recent years.

[0003] Phage is a general term for viruses that specifically infect bacteria. According to their different growth cycles when interacting with host bacteria, they can be divided into two categories: virulent phage and temperate phage. Virulent phages will immediately enter the lytic cycle after infecting host bacteria. During this cycle, the phage genome is replicated in large quantities and packaged into progeny phage particles, which are released after programmed lysis of host cells; while temperate phages have a lysogenic cycle and a lytic cycle. During the lysogenic cycle, the phage can integrate its genome into the host chromosome. At this time, the temperate phage is called a prophage, which replicates with the bacterial host chromosome and maintains the lysogenic state by inhibiting the phage's lytic genes. Under the stimulation of inducing bacterial SOS response (such as antibiotic treatment, oxidative stress or DNA damage), the prophage will activate the lytic cycle, lyse bacterial cells like virulent phages and release phage progeny. Most phages in nature are double-stranded DNA (dsDNA) phages. For most dsDNA phages, host cell lysis is due to the synergistic action of two proteins. In the late stage of the phage lytic cycle, endolysin is first produced and accumulated in the cytoplasm, but because it cannot pass through the cytoplasmic membrane to act on the peptidoglycan layer, it requires the help of perforin. When perforin aggregates, it promotes inner membrane depolarization. Perforin dimers are the basic form of its functional assembly. It will then aggregate into oligomers and form holes in the cytoplasmic membrane, allowing endolysin to be released into the periplasm.

[0004] At present, the research on staphylococcal phages is mainly focused on treating infections caused by resistant bacteria such as methicillin-resistant Staphylococcus aureus, providing an alternative treatment method to antibiotics; conducting in-depth analysis of phage genomes to provide a basis for the development of new antibacterial strategies; at the same time, phages have shown great potential in controlling bacterial biofilms and are used to control Staphylococcus aureus contamination in the food industry. Perforin is a key protein in the lytic cycle of virulent phages. Temperate phages usually exist in a lysogenic state, which inhibits the expression of perforin. At present, the research on perforin is mainly divided into four categories: research on perforin as an antibacterial substance; combined use with antibiotics to increase the permeability of antibiotics, thereby significantly improving the bactericidal efficiency; used to design nanoparticles or liposomes for precise targeted drug delivery tools; combining perforin with fluorescent markers for rapid detection of pathogens.

[0005] There are relatively few studies on Staphylococcus worderi temperate phages, and there are no reports on perforins of Staphylococcus worderi temperate phages. Summary of the invention

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

[0007] In order to achieve the purpose of the present invention, the following technical solutions are provided:

[0008] A perforin derived from a temperate bacteriophage of Staphylococcus worderi, wherein the perforin has a nucleotide sequence as 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] An application of the perforin derived from a temperate phage of Staphylococcus vortex, wherein the perforin is used in the lysis of Gram-negative host bacteria.

[0014] An in vivo cell lysis method comprises introducing the perforin into receptor cells, culturing the cells and then achieving receptor cell lysis.

[0015] Specifically, the perforin is amplified, and the amplified product and plasmid are digested and integrated with restriction endonucleases to obtain a recombinant plasmid, and then the recombinant plasmid is transformed into a recipient cell, and the Escherichia coli successfully transformed with the recombinant plasmid is placed in a 50 μg mL -1 Amp was cultured in LB liquid medium until OD 600 The value is about 0.6, and the final concentration is 0.5 mg mL -1 IPTG was used for overnight induction at low temperature to achieve 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 present invention has the following beneficial effects:

[0020] The present invention successfully obtained the perforin gene sequence based on the temperate phage vB_G30_01 of Staphylococcus vortex, and successfully expressed the perforin protein (holin64) in Gram-negative Escherichia coli. At the same time, the perforin protein significantly lyses the host Gram-negative Escherichia coli cells, providing a new theoretical basis for the prevention and treatment of Gram-negative pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a diagram showing the transcription effect of the perforin gene provided in an embodiment of the present invention in the temperate phage vB_G30_01 of Staphylococcus worderi under MMC induction.

[0022] Figure 2 This is a diagram for analyzing the conserved domain of the perforin protein provided in an embodiment of the present invention.

[0023] Figure 3 A predicted map of the transmembrane region of the perforin protein provided in an embodiment of the present invention.

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

[0025] Figure 5 This is the cloning electrophoresis diagram of the perforin gene provided in the embodiment of the present invention, wherein M: 2000Marker; 1-2: holin64.

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

[0027] Figure 7 This is an SDS-PAGE analysis diagram of the expression of the recombinant protein pET32a-holin64 induced by different concentrations of IPTG provided in the embodiment of the present invention, wherein 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 This is a diagram of the bacterial precipitation effect after IPTG induction provided in an embodiment of the present invention, wherein 1: plasmid pET32a(+); 2: recombinant plasmid pET32a-holin64.

[0029] Fig. 9 This is a diagram showing the colony counting effect of diluting and coating plates after the plasmid pET32a(+) and recombinant plasmid pET32a-holin64 provided in the embodiments of the present invention were transformed into Escherichia coli Rosetta-gami2 (DE3).

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

[0031] Fig.11 This is a graph showing the live and dead cell staining results of Escherichia coli Rosetta-gami2 (DE3) transformed with the plasmid pET32a(+) and the recombinant plasmid pET32a-holin64 provided in the embodiments of the present invention before and after IPTG induction.

[0032] Fig.12 A diagram showing the leakage of lactate dehydrogenase (LDH) in Escherichia coli Rosetta-gami2 (DE3) transformed with different plasmids before and after IPTG induction provided in an embodiment of the present invention.

[0033] Fig.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] Fig.14 This is a diagram showing the transcription of the perforin gene in Escherichia coli Rosetta-gami2 (DE3) expressing the recombinant protein pET32a-holin 64 after IPTG induction provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0035] The present invention is further explained by the following examples, but the examples do not constitute any form of limitation to the present invention.

[0036] This perforin is a key gene for phage lytic growth and plays an important role in the lysis process of host bacteria. Therefore, this study aims to verify whether the perforin encoding site located on the genome of Staphylococcus vortex temperate phage, as a cell lysis gene, can serve as a potential antibacterial substance and provide a theoretical basis for the prevention and treatment of Gram-negative pathogens.

[0037] In this experiment, the perforin candidate gene sequence was obtained 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.), its protein structure was predicted, its function was successfully cloned and identified, and the application of the protein encoded by it was explored.

[0038] The experimental methods involved in the following examples, unless otherwise specified, are all conventional experimental methods available in the prior art.

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

[0040] The genome data of bacteriophage vB_G30_01 has been submitted to the NCBI database with the 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 vortex 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] Amino acid sequence of perforin protein (holin64) of Staphylococcus vortex temperate phage vB_G30_01MQFQKNNTLTKLTFKVGFLFYSKGDNQMTSDKLKQYIGLFGGMLGALYLALKASGIEVPFLMPDKLDAWQNFATSIVPFVIAIYGVYKNTYIIHSHSKAQEEYLKENNLK

[0052] 1. Total RNA Extraction

[0053] Staphylococcus waldenii G30 was cultured in beef extract peptone liquid medium until OD 600 About 0.4, and then added to the system to a final concentration of 1 μg mL -1 The cells were induced by mitomycin C (MMC), and an equal amount of fresh beef extract peptone liquid culture medium was added to the control. The bacterial cell pellets were taken at 0h, 2h, 4h, 6h, 8h, and 10h of culture time, respectively, and RNA was extracted using the SteadyPure universal RNA extraction kit.

[0054] 2. Synthesis of Reverse Transcribed cDNA

[0055] Evo M-MLV RT Mix Kit with gDNA Clean for qPCR (containing gDNA removal reagent for qPCR) was used to prepare RNA template solution for reverse transcription reaction.

[0056] 3. Real-time fluorescence quantitative PCR system

[0057] Primers

[0058] Table 1 Primers used in qPCR

[0059]

[0060] Using Premix Pro Taq HS qPCR Kit (ROX Plus) Green Pro Taq HS premixed qPCR kit (including ROX), 16S rDNA was selected as the internal reference gene.

[0061] The experimental results show that ( Figure 1 ), under MMC treatment conditions, the perforin gene of Staphylococcus vortex temperate phage vB_G30_01 began to be transcribed in large quantities, and its relative expression gradually increased over time, indicating that the gene was involved in the lysis and growth process of the phage. At 8 hours after the addition of MMC, the relative expression of the perforin gene reached a peak, indicating that it was a late transcription gene. Example 2 Bioinformatics Analysis of Perforin Protein

[0062] 1. Analysis of the physicochemical properties of perforin protein

[0063] Protparam (https: / / web.expasy.org / protparam / ) software was used to analyze the physicochemical properties of the protein, including the amino acid composition, relative molecular mass, isoelectric point, and hydrophilicity of the protein.

[0064] The physicochemical properties of the protein were analyzed using ProtParam software. The results showed that the perforin protein was composed of 110 amino acids, with a relative molecular mass of 12538.61, an isoelectric point of 9.27, alkaline, 8 negatively charged amino acids, and 12 positively charged amino acids; the molecular formula was C 583 H 896 N 140 O 159 S 4 The total number of atoms is 1782, the instability index is 17.10, the fat index of the protein is 90.45, and the average hydrophilicity is -0.084, so the protein is a stable hydrophobic protein.

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

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

[0067] The perforin protein was compared with the NCBI conserved domain database. The results showed that ( Figure 2 ), perforin has a domain homologous to proteins in the Holin-SPP1 superfamily, which consists of perforins derived from long-tailed bacteriophages with double-stranded DNA and two transmembrane segments.

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

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

[0070] TMHMM-2.0) software was used to predict the transmembrane regions of perforin and endolysin proteins.

[0071] The TMHMM software was used to predict the transmembrane region of perforin protein. The results showed that perforin protein has two transmembrane regions ( Figure 3 ). According to the number of transmembrane regions, perforins can be divided into three types: Type I perforins generally contain three transmembrane regions, Type II perforins generally contain two transmembrane regions, and perforins containing other numbers of transmembrane regions are classified as Type III perforins. Based on this, it was determined that the perforin protein in the mild phage vB_G30_01 of Staphylococcus vorticella belongs to Type II perforin.

[0072] 4. Prediction of the advanced structure of perforin protein

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

[0074] The secondary structure content of perforin protein was analyzed using SOPMA. The results showed that the proportion of α-helix, extended chain, β-fold, and random coil was 40%, 25.45%, 10%, and 24.55%, respectively. The results showed that the secondary structure of perforin protein was mainly α-helix, followed by random coil and extended chain structure, the proportions of the two were similar, and β-fold accounted for the least.

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

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

[0077] 1. Primer Design and Synthesis

[0078] Specific primers designed by Primer Premier 5.0 software (synthesized by Shanghai Shenggong Biotechnology Co., Ltd.) are shown in the table:

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

[0080]

[0081] Note: The underline indicates the restriction site

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

[0083] Staphylococcus waldenii G30 was cultured in 800 mL beef extract peptone liquid medium at 37°C and 180 rpm until OD 600 About 0.4, and then added to the system to a final concentration of 1 μg mL -1 The culture medium was induced with mitomycin C (MMC) at 37℃ and 180rpm for 12h. The culture medium was centrifuged at 12000rpm for 20min, and the supernatant was filtered with 0.45μm and 0.22μm microporous membranes in turn, and extracted with chloroform to obtain crude phage particle extracts. CsCl gradient solutions were prepared and added to centrifuge tubes in order from high density to low density. The crude phage extract was added to the top layer, and placed in a 4℃ ultracentrifuge at 100000g for 3h. The light blue phage concentrate was slowly drawn with a syringe, and finally a 100kDa ultrafiltration tube was used to ultrafilter and remove CsCl to obtain the phage particle concentrate. The genomic DNA of Staphylococcus vortex 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 the genomic DNA of Staphylococcus vortex temperate phage vB_G30_01 as template and holin64 F and holin64 R as primers, PCR amplification was performed respectively ( Figure 5 ).

[0086] PCR reaction system:

[0087] Table 3 PCR reaction system

[0088]

[0089] PCR reaction procedure:

[0090] Table 4 PCR reaction program

[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 refer to the detailed steps of the kit, and the recovered target band was sequenced for verification.

[0093] 4. Construction of prokaryotic expression vector

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

[0095] 5. Growth curve 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 a wavelength of 600 nm. The experimental results showed that ( Figure 6 ), compared with the control, the growth rate of Escherichia coli Rosetta-gami2(DE3) transformed with the recombinant plasmid pET32a-holin64 was significantly slowed down.

[0097] 6. Induce the expression of fusion protein pET32a-holin64 and analyze the expression by protein electrophoresis

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

[0099] The results of the study showed that ( Figure 7 ), the recombinant protein pET32a-holin64 showed specific bands without the addition of IPTG, indicating that its background expression level was relatively high.-1 The specific bands were still visible under the induction of IPTG concentration, so the subsequent experiments decided to use this concentration as the IPTG concentration for inducing protein expression. -1 After IPTG induction at low temperature overnight, the precipitation of Escherichia coli expressing recombinant protein perforin was significantly reduced ( Figure 8 ).

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

[0101] 1. Dilution and coating of E. coli expressing fusion protein

[0102] Escherichia coli Rosetta-gami2 (DE3) that successfully transformed with plasmid pET32a(+) and recombinant plasmid pET32a-holin64 were respectively cultured in 50 μg mL -1 Amp was cultured in LB liquid medium until OD 600 The bacterial solution with a value of 0.6 was diluted and applied to a solution containing 50 μg mL -1 Amp on LB solid medium, invert and culture at 37℃ for 12-16h, observe, photograph and count.

[0103] The experimental results show that ( Fig. 9 ), compared with the control transformed with the empty plasmid, the colony number of Escherichia coli Rosetta-gami2 (DE3) transformed with the recombinant plasmid pET32a-holin64 was significantly reduced.

[0104] 2. IPTG-induced growth curve

[0105] Escherichia coli Rosetta-gami2 (DE3) that successfully transformed with plasmid pET32a(+) and recombinant plasmid pET32a-holin64 were respectively cultured in 50 μg mL -1 Amp was cultured in LB liquid medium until OD 600 The value is 0.6, and the final concentration is 0.5 mg mL -1 The cells were induced with IPTG at 18°C ​​overnight, and the induced growth curve of Escherichia coli Rosetta-gami2 (DE3) was measured at a wavelength of 600 nm every 1 h.

[0106] The results of the study showed that ( Fig.10 ), and the final concentration was 0.5 mg mL -1When induced by IPTG, the light absorption value of Escherichia coli Rosetta-gami2 (DE3) carrying the recombinant plasmid pET32a-holin64 at a wavelength of 600nm decreased rapidly. This phenomenon may be attributed to the large-scale expression of perforin, which will lead to the lysis and death of Escherichia coli. After three hours of induction, the light absorption curve began to rise, which may be due to the increase and sedimentation of lysed bacterial fragments, resulting in an increase in light absorption value.

[0107] 3. Live and dead cell double staining

[0108] The E. coli Rosetta-gami2 (DE3) cells successfully transformed with plasmid pET32a(+) and recombinant plasmid pET32a-holin64 were stained with 50 μg mL -1 Amp was cultured in LB liquid medium until OD 600 The value was 0.6, and the uninduced bacteria were obtained. Then, a final concentration of 0.5 mg mL was added to the system. -1 IPTG was added and induced overnight at 18°C ​​to obtain induced bacteria. The induced bacteria and uninduced bacteria were stained separately. Under a fluorescence microscope, the living cells were yellow-green and the dead cells were red when excited at a wavelength of 490±10nm. Red dead cells could be seen when excited at a wavelength of 528nm.

[0109] The experimental results show that ( Fig.11 ), some dead cells were found in Escherichia coli Rosetta-gami2(DE3) expressing the recombinant protein pET32a-holin64 without induction, but more dead cells were found after induction with IPTG, while no dead cells were found in Escherichia coli Rosetta-gami2(DE3) expressing the empty pET32a(+) after induction with IPTG at low temperature overnight.

[0110] 4. Lactate dehydrogenase detection

[0111] When the cell membrane is damaged, substances inside the cell will leak out and lactate dehydrogenase (LDH) may be released into the culture medium. Therefore, the degree of damage to the Escherichia coli cell membrane caused by perforin and endolysin is evaluated by detecting the content of LDH in the extracellular environment.

[0112] The induced and uninduced E. coli successfully transformed with plasmid pET32a(+) and recombinant plasmid pET32a-holin64 in Experiment 3 of Example 4 were centrifuged to obtain the supernatant, which was placed on ice for testing. For subsequent experimental methods, refer to the instructions for use of the lactate dehydrogenase activity detection kit.

[0113] The experimental results show that ( Fig.12), Escherichia coli Rosetta-gami2 (DE3) expressing the recombinant protein pET32a-holin64 produced LDH leakage before induction, and after IPTG induction, LDH leakage increased significantly.

[0114] 5. Scanning electron microscopy

[0115] The induced and uninduced E. coli successfully transformed with plasmid pET32a(+) and recombinant plasmid pET32a-holin64 in Experiment 3 of Example 4 were centrifuged to obtain bacterial pellets, which were fully dispersed and suspended in glutaraldehyde fixative. After fixation at room temperature in the dark for 30 minutes, the pellets were observed under a microscope.

[0116] The experimental results show that ( Fig.13 ), compared with the pET32a(+) control, the Escherichia coli Rosetta-gami2(DE3) expressing the recombinant protein pET32a-holin64 showed an aggregated state when not induced; after induction with IPTG, the Escherichia coli Rosetta-gami2(DE3) expressing the recombinant protein pET32a-holin64 showed leakage of intracellular substances, and multiple cells aggregated together.

[0117] 6. Detection of transcriptional levels of induced expression of perforin and endolysin genes

[0118] Escherichia coli Rosetta-gami2 (DE3) successfully transformed with the recombinant plasmid pET32a-holin64 was incubated with 50 μg mL -1 Amp was cultured in LB liquid medium until OD 600 The value is 0.6, and the final concentration is 0.5 mg mL -1 The cells were induced at low temperature of 18°C ​​with IPTG, while the control cells were not induced with IPTG. The bacterial cell pellets were taken at 0 h, 3 h, and 6 h of culture time, and RNA was extracted using the SteadyPure universal RNA extraction kit. The subsequent reverse transcription experiments and fluorescence quantitative PCR were the same as in Example 1.

[0119] The experimental results show that ( Fig.14 ), after IPTG induction, the perforin gene of Escherichia coli Rosetta-gami2 (DE3) expressing the recombinant protein pET32a-holin64 was rapidly upregulated, but the upregulation was not obvious after 3 hours, and was significantly upregulated after 6 hours.

[0120] The above research results show 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 treatment of Gram-negative pathogens.

Claims

1. A perforin derived from a temperate phage of Staphylococcus waltneri, characterized in that: The perforin has a nucleotide sequence as shown in SEQ ID NO:

1.

2. The perforin derived from the temperate phage of Staphylococcus vortexis according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the perforin is shown in SEQ ID NO:

2.

3. The perforin derived from the temperate phage of Staphylococcus vortexis according to claim 1, characterized in that: The primer pair for obtaining perforin is: holin64-FGACGCATGGCAAAACTTCGCTAC; holin64-R TTCTTGCGCTTTTGAATGCGAGTG.

4. A use of the perforin derived from the temperate phage of Staphylococcus vortexis according to claim 1, characterized in that: The perforin is used for lysing Gram-negative host bacteria.

5. A method for lysing cells in vivo, characterized in that: The perforin according to claim 1 is introduced into the recipient cells, and the cells are cultured to achieve lysis of the recipient cells.

6. The method for in vivo cell lysis according to claim 5, characterized in that: The perforin of claim 1 is obtained by amplification, and the amplified product and the plasmid are digested and integrated with restriction endonucleases to obtain a recombinant plasmid, and then the recombinant plasmid is transformed into a recipient cell, and the Escherichia coli successfully transformed with the recombinant plasmid is cultured in a medium containing 50 μg mL -1 Amp was cultured in LB liquid medium until OD 600 The value is about 0.6, and the final concentration is 0.5 mg mL -1 IPTG was used for overnight induction at low temperature to achieve host cell lysis.

7. The method for in vivo cell lysis according to claim 5, characterized in that: The primers for amplifying the perforin according to claim 1 are: holin64-FGACGCATGGCAAAACTTCGCTAC; holin64-R TTCTTGCGCTTTTGAATGCGAGTG.

Images