Perforin-like holsgf2, its preparation method and use

Abstract

The application discloses a holin HolSGF2 and a preparation method and application thereof, and belongs to the technical field of genetic engineering. The application discloses a holin HolSGF2 and a preparation method and application thereof, and the preparation method comprises the following steps: obtaining a prokaryotic expression vector plasmid HolSGF2-pET-32a containing a holin HolSGF2 gene through gene synthesis, obtaining a recombinant plasmid through transformation of a DH5 alpha competent cell, and introducing the recombinant plasmid into an escherichia coli BL21 expression system to realize massive expression of protein. Then, the holin HolSGF2 is purified through affinity medium, and efficient expression of the holin HolSGF2 in vitro is realized. The HolSGF2 has good bactericidal activity, has good inhibiting effects on gram-negative bacteria and gram-positive bacteria, and has a good application prospect in the development of a new phage drug.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically to a phage SGF2 perforin HolSGF2, its preparation method, and its application. Background Technology

[0002] Shigella, Staphylococcus aureus, and Escherichia coli (E. coli) are the main pathogens causing most serious infectious foodborne illnesses, with Shigella causing inflammatory diarrhea and Shigella disease. Bacterial infections are a major cause of morbidity in clinical practice. In the past, antibiotics were mainly used to treat infections, but long-term antibiotic use has led to drug resistance in pathogens, resulting in worsening of patient conditions. Antibiotic resistance can be acquired genetically and is persistent; a small percentage of cells exhibit non-genetic phenotypic resistance to antibiotics. Multidrug-resistant Shigella isolates have been reported worldwide, thus requiring more effective antibacterial agents. Expressing bacteriophage functional proteins can enhance the bactericidal effect of antibiotics. However, bacteriophages exhibit specificity in their lytic activity against pathogens, and their antibacterial activity is greatly affected by the environment; changes in temperature and pH alter their antibacterial activity, thus hindering the development of phage drugs. Perforin, as a functional protein of bacteriophages, can effectively inhibit the growth of host bacteria and even other bacteria. It is not easily affected by the environment, so perforin has good development and market application value as an antibacterial agent.

[0003] Therefore, providing a perforin HolSGF2, its preparation method, and its application is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0004] In view of this, the present invention provides a perforin HolSGF2, its preparation method and application.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A perforin, HolSGF2, has the amino acid sequence shown in SEQ ID NO.6.

[0007] Furthermore, the nucleotide sequence encoding the perforin HolSGF2 is shown in SEQ ID NO. 5.

[0008] Furthermore, a recombinant expression vector is provided, wherein the recombinant expression vector is obtained by recombination of the nucleotide sequence and the expression vector.

[0009] Furthermore, the expression vector is pET-32a.

[0010] Furthermore, a method for preparing perforin HolSGF2 includes the following steps:

[0011] (1) The nucleotide sequence was constructed into the expression vector pET-32a to obtain the recombinant plasmid HolSGF2-pET-32a;

[0012] (2) The recombinant plasmid HolSGF2-pET-32a was transformed into the Escherichia coli BL21 expression strain, liquid culture was performed, expression was induced, the bacterial culture was collected, and the perforin HolSGF2 was extracted and purified.

[0013] Furthermore, the application of HolSGF2, a perforin, in inhibiting bacterial activity.

[0014] Furthermore, the bacteria are Shigella dysenteriae 1.1869, Shigella flexneri 1.1868, Shigella flexneri 1.10599, Staphylococcus aureus 1.8721, and Escherichiacoli Sakai.

[0015] As can be seen from the above technical solution, compared with the prior art, this invention discloses a perforin HolSGF2, its preparation method, and its application. To achieve soluble protein expression, the expression gene sequence of perforin was analyzed. This perforin possesses a signal peptide and a transmembrane region, exhibiting strong hydrophobicity. The signal peptide and transmembrane region sequences were then deleted. A prokaryotic expression vector plasmid HolSGF2-pET-32a containing the gene encoding perforin HolSGF2 was synthesized. This plasmid was then transformed into DH5α competent cells to obtain a recombinant plasmid, which was introduced into the *E. coli* BL21 expression system for high-level protein expression. Subsequently, purification using affinity media was performed to achieve efficient in vitro expression of perforin HolSGF2. HolSGF2 exhibits good bactericidal activity, showing good inhibitory effects against both Gram-negative and Gram-positive bacteria, and has promising applications in the development of phage drugs. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0017] Figure 1 The attached figure is an SDS-PAGE diagram of the recombinant plasmid HolSGF2(1)-pGEX-6p-1 protein expression of the present invention;

[0018] Wherein, M: Marker; Lane 1: pGEX-6p-1 empty vector cell lysis buffer; Lane 2: pGEX-6p-1 empty vector cell lysis supernatant; Lane 3: pGEX-6p-1 empty vector cell lysis precipitate; Lane 4: HolSGF2(1)-pGEX-6p-1 cell lysis buffer with IPTG; Lane 5: HolSGF2(1)-pGEX-6p-1 cell lysis supernatant with IPTG; Lane 6: HolSGF2(1)-pGEX-6p-1 cell lysis precipitate with IPTG;

[0019] Figure 2 The attached figure is an SDS-PAGE diagram of the recombinant plasmid HolSGF2(1)-pGEX-6p-1 protein expression after changing the induction conditions according to the present invention.

[0020] Wherein, M: Marker; Lane 1: pGEX-6p-1 cell lysis buffer with 1mM IPTG; Lane 2: pGEX-6p-1 cell lysis supernatant with 1mM IPTG; Lane 3: HolSGF2(1)-pGEX-6p-1 cell lysis buffer with 1mM IPTG; Lane 4: HolSGF2(1)-pGEX-6p-1 cell lysis supernatant with 1mM IPTG; Lane 5: HolSGF2(1)-pGEX-6p-1 cell lysis buffer with 0.5mM IPTG; Lane 6: HolSGF2(1)-pGEX-6p-1 cell lysis supernatant with 0.5mM IPTG; Lane 7: 0.05mM IPTG... HolSGF2(1)-pGEX-6p-1 cell lysis buffer with IPTG; Lane 8: HolSGF2(1)-pGEX-6p-1 cell lysis supernatant with 0.05 mM IPTG;

[0021] Figure 3 The attached figure is an SDS-PAGE diagram of the recombinant plasmid HolSGF2-pET-32a protein expression of the present invention;

[0022] Wherein, M: Marker; Lane 1: HolSGF2 cell lysis buffer with IPTG; Lane 2: HolSGF2 cell lysis supernatant with IPTG; Lane 3: HolSGF2 cell lysis precipitate with IPTG;

[0023] Figure 4 The attached figure is an SDS-PAGE image of the recombinant plasmid HolSGF2-pET-32a protein expression and purification of the present invention;

[0024] Wherein, M: Marker; Lane 1: Purified HolSGF2;

[0025] Figure 5The attached figure shows the inhibitory effect of the purified perforin HolSGF2 of this invention on the host bacterium Shigella flexneri 1.1868 (L-2);

[0026] Among them, E2, E3, and E4 are all HolSGF2 protein samples; PBS: negative control;

[0027] Figure 6 The attached figure illustrates the effect of different pH values ​​on the relative cleavage activity of perforin HolSGF2 according to the present invention.

[0028] Figure 7 The attached figure shows the effect of different temperatures on the relative cleavage activity of HolSGF2 according to the present invention. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1 Preparation of perforin HolSGF2(1)

[0031] Source of phage SGF2: Genomic characterization of a novel virulent phageinfecting Shigella fiexneri and isolated from sewage[J]. Virus Research: AnInternational Journal of Molecular and Cellular Virology, 2020, 283. DOI: 10.1016 / j.virusres.2020.197983.

[0032] 1) The original sequence of the perforin expression gene of Shigella flexneri phage SGF2 is SEQ ID NO.1, denoted as HolSGF2(1).

[0033] ATGAAAATGATGAAACAAGCTGCATATGGTTTAATCTGTTTAGGCAGGTGTTGTGGTATCAACCGCAGTTCCTGTAAGCGCAGTTCATCCATTAGTAACTGTCCCTGCTCCAGTAGAGAAGCTTAGCTCTCAATGCAAGCAGAGAACAATTCAAAATAACCTAGTTGTTATTTGTCCTGATGCTGAATCTAACGCTCTGAGAAAGATACTACCTTAA; SEQ ID NO.1.

[0034] The genome of phage SGF2 was extracted using a phage gene extraction kit, and the target fragment was amplified by PCR to obtain the PCR product.

[0035] The primer sequences used for PCR amplification are as follows:

[0036] F: 5'-CGCGGATCCATGAAAATGATGAAACAA-3'; SEQ ID NO.2;

[0037] R: 5'-CCGCTCGAGTTAAGGTAGTATCTTTCTCAG-3'; SEQ ID NO. 3.

[0038] The PCR reaction system (total 50 μl) is as follows: 10× buffer 5 μl, dNTPS 4 μl, primer F 1 μl, primer R 1 μl, Taq enzyme 0.5 μl, DNA template 1 μl, ddH2O 37.5 μl.

[0039] The PCR reaction program is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 1 min, 57℃ annealing for 1 min, 72℃ extension for 1 min, for a total of 35 cycles; 72℃ for 10 min, 4℃ ∞.

[0040] 2) Using pGEX-6p-1 as a vector, the recombinant plasmid HolSGF2(1)-pGEX-6p-1 was constructed: The PCR product and plasmid pGEX-6p-1 were double-digested with BamHI and XhoI. The purified digested products were ligated with T4 ligase to form the recombinant plasmid HolSGF2(1)-pGEX-6p-1. pGEX-6p-1 and the recombinant plasmid were transformed into Escherichia coli DH5α and stored at -20℃ / -80℃.

[0041] 3) The recombinant plasmid HolSGF2(1)-pGEX-6p-1 was extracted from Escherichia coli DH5α using a plasmid extraction kit and transformed into Escherichia coli BL21. It was stored at -20℃ / -80℃.

[0042] 4) The preserved Escherichia coli BL21 containing recombinant plasmid HolSGF2(1)-pGEX-6p-1 was inoculated at a rate of 1% into 20 ml LB liquid medium with 50 μg / ml ampicillin resistance and cultured overnight in a shaker at 37°C.

[0043] 5) Transfer 100 μl of the above seed culture onto an LB solid plate medium and incubate overnight at 37°C.

[0044] 6) Pick a single colony of Escherichia coli BL21 containing the recombinant plasmid from the plate medium, inoculate it into 20 ml of LB liquid medium with 50 μg / ml ampicillin resistance, and culture overnight in a shaker at 37°C to prepare seed culture.

[0045] 7) Transfer the seed culture at an inoculum rate of 1% to 100 ml of LB liquid medium with 50 μg / ml ampicillin resistance, and incubate with shaking in a 37°C incubator. When the OD of the bacterial culture reaches a certain level... 600 When the value reached 0.6-0.8, bacterial culture containing plasmid pGEX-6p-1 was used as a control. 1 mM IPTG was added and the culture was placed in a shaker at 37°C for 4 hours to induce protein expression.

[0046] 8) Collect bacterial cells by centrifugation, wash and resuspend the cells with PBS multiple times, and break the cells using a cell sonicator. Take 1 ml of cell disruption solution as a sample, centrifuge to obtain cell disruption supernatant and cell disruption precipitate, and perform SDS-PAGE detection.

[0047] 9) Add an appropriate amount of protein loading buffer to the protein sample, boil at 100℃ for 5 minutes, cool to room temperature on ice, load the sample, and perform SDS-PAGE detection. Figure 1 As shown, the protein is expressed as an inclusion body (protein size approximately 34 kDa).

[0048] After changing the protein induction conditions (temperature 25℃, IPTG concentrations of 1, 0.5, and 0.05 mM, cultured for 12 hours), bacterial cells were collected and analyzed by SDS-PAGE. Figure 2 As shown.

[0049] from Figure 2 It can be seen that the perforin HolSGF2 obtained by induced expression is still in the form of inclusion bodies, and changing the induced expression conditions still failed to yield a soluble protein.

[0050] Example 2 Preparation of perforin HolSGF2

[0051] 1) The perforin HolSGF2(1) expression gene sequence was analyzed, and its signal peptide and transmembrane region were deleted. The pre-phage SGF2 perforin HolSGF2(1) expression gene sequence is SEQ ID NO.1, and the amino acid sequence is SEQ ID NO.4. The phage SGF2 perforin HolSGF2 expression gene sequence after deleting its signal peptide and transmembrane region is SEQ ID NO.5. The perforin HolSGF2 expression gene sequence SEQ ID NO.5 was obtained through gene synthesis; the amino acid sequence is shown in SEQ ID NO.6.

[0052] HolSGF2(1) original amino acid sequence:

[0053] M KMMKQAAYGLICLGAGVVVSTAVPVSAV HPLVTVPAPVEKLSSQCKQRTIQNNLVVICPDAESNALR KILP ;SEQ ID NO.4.

[0054] ATGCATCCATTAGTAACTGTCCCTGCTCCAGTAGAGAAGCTTAGCTCTCAATGCAAGCAGAGAACAATTCAAAATAACCTAGTTGTTATTTGTCCTGATGCTGAATCTAACGCTCTGAGAAAGATACTACCTTAA; SEQ ID NO.5.

[0055] The final amino acid sequence of the phage SGF2 perforin HolSGF2 to be expressed is shown in SEQ ID NO.6.

[0056] MHPLVTVPAPVEKLSSQCKQRTIQNNLVVICPDAESNALRKILP; SEQ ID NO.6.

[0057] 2) Using pET-32a as a vector, the recombinant plasmid HolSGF2-pET-32a was constructed by double digestion with NcoI and XhoI. The recombinant plasmid was then transformed into Escherichia coli DH5α and stored at -20℃ / -80℃.

[0058] 3) The recombinant plasmid HolSGF2-pET-32a was extracted from Escherichia coli DH5α using a plasmid extraction kit and transformed into Escherichia coli BL21. It was then stored at -20℃ / -80℃.

[0059] 4) The preserved Escherichia coli BL21 containing recombinant plasmid HolSGF2-pET-32a was inoculated at a rate of 1% into 20 ml of LB liquid medium with 50 μg / ml ampicillin resistance, and cultured overnight in a shaker at 37°C.

[0060] 5) Transfer 100 μl of the above seed culture onto an LB solid plate medium and incubate overnight at 37°C.

[0061] 6) Pick a single colony of Escherichia coli BL21 containing the recombinant plasmid from the plate medium, inoculate it into 20 ml of LB liquid medium with 50 μg / ml ampicillin resistance, and culture it overnight in a shaker at 37°C to prepare seed culture;

[0062] 7) Transfer the above BL21 bacterial suspension (seed culture) at an inoculation rate of 1% to 100 ml LB liquid medium containing 50 μg / ml ampicillin resistance, and incubate with shaking in a shaker at 37°C for 3 h. When the OD of the bacterial suspension... 600 When the value reaches 0.4-0.6, add 0.5mM IPTG to induce protein expression, and culture in a shaker at 16℃ for 16h.

[0063] 8) Collect bacterial cells by centrifugation, wash and resuspend the cells with PBS multiple times, break the cells using a cell sonicator, take 1 ml of cell disruption solution as a sample, centrifuge to obtain the supernatant, and perform SDS-PAGE detection.

[0064] 9) Add an appropriate amount of protein loading buffer to the protein sample, boil at 100℃ for 5 minutes, cool to room temperature on ice, load the sample, and perform SDS-PAGE detection. Figure 3 As shown, soluble expression of perforin HolSGF2 was achieved, and the size of perforin HolSGF2 expressed by the recombinant plasmid HolSGF2-pET-32a was approximately 22 kDa.

[0065] from Figure 3 The SDS-PAGE results showed that the target protein was present in the supernatant, achieving soluble expression of perforin HolSGF2 in vitro.

[0066] Example 3 Purification of perforin HolSGF2

[0067] 1) The preserved BL21 strain containing recombinant plasmid HolSGF2-pET-32a was inoculated at a rate of 1% into 20 ml of LB liquid medium with 50 μg / ml ampicillin resistance, and cultured overnight in a shaker at 37°C to prepare seed culture.

[0068] 2) Transfer the seed culture at an inoculum rate of 1% to 1000 ml LB liquid medium with 50 μg / ml ampicillin resistance, and incubate with shaking in a shaker at 37°C. When the OD of the bacterial culture... 600 When the value reaches 0.4-0.6, add 0.5mM IPTG and incubate in a shaker at 16℃ for 16 hours to induce protein expression.

[0069] 3) Collect bacterial cells by centrifugation, wash and resuspend the cells with PBS multiple times, disrupt the cells using a cell sonicator, and collect the supernatant by centrifugation at 13000 rpm / min for 10 min.

[0070] 4) Filter the supernatant obtained in the above steps through a 0.22 μm filter membrane, and then purify the protein using the Ni-NTA protein purification kit (BBI, purchased from Sangon Biotech (Shanghai) Co., Ltd.).

[0071] 5) The storage buffer in the Ni-NTA pre-packed column is drained by gravity.

[0072] 6) Balance the column with twice the column volume of Wash Buffer, allowing the Buffer to slowly flow out of the resin.

[0073] 7) Mix the supernatant obtained after filtration in step 4) with Wash Buffer at a 1:1 ratio to prepare the sample solution, so that the total volume of the sample solution is twice the column volume.

[0074] 8) Add the sample solution to the column and collect the flow-through liquid into a centrifuge tube.

[0075] 9) Load the flow-through liquid obtained in step 8) onto the column again to improve the bonding force between the sample and the packing material.

[0076] 10) Wash the column with twice the column volume of Wash Buffer and collect the flowthrough. Repeat this step using a new collection tube until the flowthrough absorbs light at 280 nm close to the baseline.

[0077] 11) Elute the histidine-tagged protein on the column with twice the column volume of Elution Buffer. Repeat this step twice, storing the eluent separately each time, until the absorbance of the flow-through at 280 nm is close to the baseline.

[0078] 12) Elute the column with 5 column volumes of Elution Buffer, then elute with 5 column volumes of Wash Buffer, and finally wash the column with 5 column volumes of ddH2O. Add 20% ethanol protective solution and store at 4°C.

[0079] 13) The concentration of the eluted protein (approximately 0.9 mg / ml) was determined using a BCA protein concentration assay kit, and SDS-PAGE analysis was performed. Figure 4 As shown, this indicates that HolSGF2 with high purity was obtained.

[0080] 14) The inhibitory activity of purified perforin HolSGF2 against the host bacterium Shigella flexneri 1.1868 (L-2) was detected by inhibition zone assay. The specific steps are as follows: 100 μl of host bacterium L-2 (OD2) cultured overnight was added... 600 =1.092) was spread onto LB solid medium. After complete absorption of the bacterial culture, wells were punched in the plate. 100 μl of approximately 150 μg / ml HolSGF2 protein samples E2, E3, and E4 were added to each well, along with 100 μl of PBS as a negative control. The plates were incubated overnight at 37°C, and the inhibition zones were observed. Results are as follows: Figure 5 As shown, the purified perforin HolSGF2 protein solution can effectively inhibit the growth of host bacteria.

[0081] Example 4: Study on the lytic activity of perforin HolSGF2 against free bacterial cells at different pH and temperature

[0082] 1) Inoculate the host bacteriophage SGF2, Shigella flexneri 1.1868 (L-2), from a glycerol tube into 100 ml of LB liquid medium at a 1% inoculation rate and incubate overnight in a shaker at 37°C.

[0083] 2) OD of the bacterial solution obtained above 600 The bacterial cells of *Shigella flexneri* were collected by centrifugation at 4000 rpm for 20 min when the pH value was between 0.8 and 1.0. The cells were washed with PBS and resuspended. Approximately 10... 11 CFU / ml.

[0084] 3) Transfer 150 μl of the bacterial cells to a 96-well plate. Add 50 μl of 150 μg / ml HolSGF2 perforin to the experimental group, and 50 μl of PBS to the negative control. Perform three replicates for each treatment. Set the pH values ​​to: 3.1, 4.2, 5.1, 6.3, 7.4, 8.2, 9.0, and 10.3. Measure the OD of the bacterial suspension after 2 hours. 600 value.

[0085] 4) Transfer 100 μl of the bacterial cells to a 96-well plate. Add 100 μl of 150 μg / ml HolSGF2 perforin to the experimental group, and 100 μl of PBS to the negative control. Perform three replicates for each treatment. Set the temperature to 4℃, 16℃, 20℃, 25℃, 30℃, 37℃, 45℃, and 55℃. Measure the OD of the bacterial suspension after 5 hours. 600 value.

[0086] 5) Calculate the relative cleavage activity: = {ΔOD600(HolSGF2) - ΔOD600(PBS)} / initial value of OD600, the result is as follows Figure 6 , 7 As shown.

[0087] Figure 6 The effect of different pH values ​​on the relative cleavage activity of perforin HolSGF2 was investigated. Results showed that after 2 hours, different pH values ​​affected the antibacterial activity of HolSGF2, exhibiting varying antibacterial activities against free bacterial cells. Perforin showed high activity within a pH range of 3.1–10.3, with the highest antibacterial activity (31%) at pH 7.4. Therefore, this perforin exhibits high antibacterial activity, is less affected by pH, and shows promising application potential.

[0088] Figure 7 The effect of different temperatures on the relative lytic activity of HolSGF2 was investigated. Results showed that after 5 hours, the activity of perforin against free bacterial cells exhibited a trend of first increasing, then decreasing, and then increasing again when the temperature ranged from 4℃ to 55℃. The antibacterial activity was most pronounced at 55℃, reaching approximately 77%, while the lowest inhibitory activity was approximately 20% at 4℃. These results indicate that the activity of this perforin is not significantly affected by temperature, exhibiting high antibacterial activity over a wide temperature range and demonstrating good environmental adaptability. It shows promising application potential in the preparation of antibacterial agents.

[0089] Example 5: Study on the antibacterial spectrum of HolSGF2 perforin

[0090] 1) Take out the preserved strains Shigella dysenteriae 1.1869, Shigella flexneri 1.1868, Shigella flexneri 1.10599, Staphylococcus aureus 1.8721, Staphylococcus aureus 1.2465, Salmonella typhimurium 1.1174, Salmonellaenteritidis 1.10754, and Escherichia coli Sakai (strain sources are shown in Table 1), and inoculate them into 20 ml of LB liquid medium at a 1% inoculum. Incubate overnight in a shaker at 37°C.

[0091] Table 1. Source of strains

[0092] Shigella flexneri 1.1868 Shigella flexneri 1.1868 CGMCC Shigella dysenteriae 1.1869 Shigella dysenteriae 1.1869 CGMCC Shigella flexneri 1.10599 Shigella flexneri 1.10599 CGMCC Staphylococcus aureus 1.8721 Staphylococcus aureus 1.8721 CGMCC Staphylococcus aureus 1.2465 Staphylococcus aureus 1.2465 CGMCC Escherichia coli Saka Escherichia coli Sakai China CDC Salmonella enteritidis 1.10754 Salmonella enteritidis 1.10754 CGMCC Salmonella typhimurium 1.1174 Salmonella typhimurium 1.1174 CGMCC

[0093] 2) Transfer 100 μl of the above bacterial suspension to each strain (according to the strain order shown in Table 1, OD...). 600 The concentrations of the bacterial culture were 0.8, 0.704, 0.645, 0.674, 0.507, 0.729, 1.161, and 0.637, respectively, and spread onto solid agar plates. After the bacterial culture was evenly spread and completely absorbed, holes were punched in the plates.

[0094] 3) Transfer 100 μl of purified perforin HolSGF2 (or phage SGF2) at a concentration of 150 μg / ml into the wells of a solid plate culture medium, using PBS as a control.

[0095] 4) Place the solid plate culture medium in an incubator at 37°C and incubate overnight. Observe whether an inhibition zone is formed. The experimental results are shown in Table 2.

[0096] Table 2 Antibacterial spectrum of HolSGF2 perforin

[0097] Shigella dysenteriae 1.1869 + - <![CDATA[G - ]]> Shigella flexneri 1.1868 + + <![CDATA[G - ]]> Shigella flexneri 1.10599 + - <![CDATA[G - ]]> Staphylococcus aureus 1.8721 + - <![CDATA[G + ]]> Staphylococcus aureus 1.2465 - - <![CDATA[G + ]]> Salmonella typhimurium 1.1174 - - <![CDATA[G - ]]> Salmonella enteritidis 1.10754 - - <![CDATA[G - ]]> Escherichia coli Sakai + - <![CDATA[G - ]]>

[0098] Note: "+" indicates an inhibition zone, "-" indicates no inhibition zone.

[0099] As shown in Table 2, the perforin has an inhibitory effect on both Gram-positive and Gram-negative bacteria. Compared with bacteriophages, the perforin has a broader antibacterial spectrum, which overcomes the limitation of bacteriophages in specifically lysing the host, and therefore has good application prospects.

[0100] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of perforin HolSGF2 in the preparation of antibacterial drugs, characterized in that, The amino acid sequence of the perforin HolSGF2 is shown in SEQ ID NO. 6; the bacteria are Shigella dysenteriae 1.1869 Shigella flexneri 1.1868 Shigella flexneri 1.10599 Staphylococcus aureus 1.8721 and Escherichia coli Sakai.

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