Recombinant bacteriophage lyase and application thereof

Abstract

The invention discloses recombinant bacteriophage lyase and application thereof. The amino acid sequence of the recombinant bacteriophage lyase E8-3 is as shown in SEQ ID NO: 43, and an in-vitro minimum inhibitory concentration experiment shows that the recombinant bacteriophage lyase E8-3 has a bacteriostatic effect on helicobacter pylori and acinetobacter baumannii, and particularly has a wide application prospect in the development of anti-helicobacter pylori drugs.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a recombinant phage lysin and its applications. Background Technology

[0002] Helicobacter pylori ( Helicobacter pylori Helicobacter pylori (H. pylori) is an obligate microaerophilic, helical or curved rod-shaped Gram-negative bacterium that specifically colonizes the epithelial surface and mucus layer of the human gastric mucosa. Since its first isolation and confirmation of its causal relationship with gastritis and peptic ulcers by Barry Marshall and Robin Warren in 1982, this pathogen has become one of the most important chronic infectious agents worldwide. H. pylori infection has a significant global impact, affecting more than 4.4 billion people worldwide, with infection rates reaching 70-90% in developing countries and approximately 30-50% in developed countries. Infection usually occurs in childhood and can persist throughout life without effective intervention, constituting a dynamic infection-immunity-tissue damage process.

[0003] The clinical consequences of Helicobacter pylori infection are extremely severe and diverse, constituting a spectrum of diseases ranging from benign inflammation to malignant lesions. The vast majority of infected individuals present with asymptomatic chronic active gastritis. However, approximately 10-20% of infected individuals will develop peptic ulcers (gastric or duodenal ulcers), the mechanism of which is closely related to the strong local inflammatory response induced by bacterial virulence factors (such as CagA and VacA), dysregulation of gastric acid secretion, and impaired mucosal defense mechanisms. More seriously, Helicobacter pylori infection is the most significant controllable risk factor for gastric cancer. The International Agency for Research on Cancer (IARC) classified it as a Group 1 carcinogen as early as 1994. It is estimated that approximately 78% of new gastric cancer cases worldwide are attributed to Helicobacter pylori infection. Its carcinogenic mechanism involves the accumulation of gene mutations caused by persistent chronic inflammation, abnormal host cell signaling pathways (such as the Wnt / β-catenin and NF-κB pathways), epigenetic alterations, and the formation of the tumor microenvironment. Furthermore, Helicobacter pylori infection is directly associated with the development and progression of gastric mucosa-associated lymphoid tissue (MALT) lymphoma, and eradication therapy can even reverse some early-stage MALT lymphomas. In recent years, studies have also found that infection may be associated with an increased risk of various extra-gastrointestinal diseases, such as idiopathic thrombocytopenic purpura, iron deficiency anemia, and certain cardiovascular and neurological diseases, further exacerbating its disease burden.

[0004] For decades, the standard therapy for eradication of H. pylori has mainly relied on the combination of proton pump inhibitors (PPI) and antibiotics, such as the classic triple therapy (PPI+ amoxicillin+ clarithromycin / metronidazole) and the bismuth quadruple therapy which was promoted with the increasing resistance rate. However, in the actual application, there are many problems such as poor compliance, adverse drug reactions and increasing proportion of drug-resistant strains, which lead to the increase of eradication failure rate and the increase of recurrence risk. The rapid development of antibiotic resistance has become the most serious challenge for global eradication therapy. Due to the abuse of antibiotics, non-standard treatment and the adaptive evolution of bacteria, the resistance rate of H. pylori to key first-line antibiotics has risen sharply. The World Health Organization has listed clarithromycin-resistant H. pylori as a 'high priority' drug-resistant pathogen. Global monitoring data shows that the primary resistance rate of clarithromycin has exceeded 30% in some areas, the resistance rate of metronidazole is as high as 60-70% in many countries, and the resistance rate of quinolones such as levofloxacin is also showing a rapid upward trend. High resistance rate directly leads to a significant increase in the failure rate of standard regimens (the first eradication failure rate can reach 20-30% in some areas), forcing clinicians to use more complex, longer and more side effects of remedial regimens, not only increasing the economic and physiological burden of patients, but also exacerbating the further spread of drug resistance. In addition, long-term or repeated use of broad-spectrum antibiotics will irreversibly destroy the intestinal flora homeostasis of the host, increasing the risk of Clostridium difficile infection, antibiotic-associated diarrhea and long-term metabolic diseases. Therefore, in addition to traditional antibiotics, there is an urgent need in current clinical practice to find new antibacterial strategies with new mechanisms of action, high specificity, low ecological interference and effective overcoming of drug resistance.

[0005] Bacteriophage is a virus that can specifically infect and lyse bacteria. Endolysin is a kind of hydrolytic enzyme synthesized and secreted by bacteriophage at the end of infection, which can efficiently and accurately degrade the peptidoglycan layer of bacterial cell wall, leading to the lysis and death of bacteria due to osmotic pressure imbalance. Compared with traditional antibiotics, endolysin has multiple advantages: unique target, not easy to produce cross-resistance with existing antibiotics; high specificity to host bacteria, can accurately target pathogenic bacteria without destroying beneficial flora; rapid action, strong bactericidal effect; also shows potential in combating biofilm. Therefore, endolysin is considered as a promising alternative or supplement to antibiotics in the post-antibiotic era.

[0006] Although significant progress has been made in the study of lytic enzymes against gram-positive bacteria, the development and application of lytic enzymes against gram-negative bacteria are relatively lagging due to the protection of outer membrane structure. The study of bacteriophage lytic enzymes against Helicobacter pylori is currently in the early stage, and the number of reported natural or recombinant lytic enzymes is limited. For example, Chinese patent CN118240809A discloses an Acinetobacter baumannii bacteriophage lytic enzyme LysZHSHW, but its activity, stability and expression amount need to be further optimized. Therefore, it has important scientific value and application prospect to explore and modify new efficient anti-Helicobacter pylori lytic enzyme. SUMMARY

[0007] In order to overcome the above-mentioned deficiencies existing in the prior art, the purpose of the present application is to provide a recombinant bacteriophage lytic enzyme and its application.

[0008] The purpose of the present application is at least achieved by one of the following technical solutions.

[0009] The present application provides a recombinant bacteriophage lytic enzyme E8-3, and the amino acid sequence thereof is shown as SEQ ID NO: 43.

[0010] The present application also provides a nucleic acid molecule encoding the recombinant bacteriophage lytic enzyme E8-3, and the nucleotide sequence thereof is shown as SEQ ID NO: 42, or a nucleic acid sequence complementary to SEQ ID NO: 42.

[0011] Further, the nucleic acid molecule further comprises an expression regulatory element for improving translation efficiency, and the expression regulatory element is selected from: a ribosome binding site (RBS) optimization sequence and / or a 5' untranslated region (5'-UTR) optimization sequence.

[0012] The present application also provides a recombinant expression vector, and the recombinant expression vector comprises the nucleic acid molecule encoding the recombinant bacteriophage lytic enzyme E8-3, and is operably linked to a regulatory sequence capable of driving the transcription and / or translation of the nucleic acid molecule encoding the recombinant bacteriophage lytic enzyme E8-3 in a host cell.

[0013] Further, the recombinant expression vector comprises an inducible promoter and / or a constitutive promoter.

[0014] Further, the recombinant expression vector comprises a sequence for promoting expression and / or purification, and the sequence for promoting expression and / or purification is selected from one or more of: an affinity purification tag, a solubility / expression-promoting fusion tag and a secretion signal peptide.

[0015] Further, the affinity purification tag is one or more of: a His tag, a Strep tag and a FLAG tag.

[0016] Further, the solubilization / expression-promoting fusion tag is selected from one or more of SUMO, thioredoxin (Trx), MBP, GST and NusA.

[0017] Further, the expression vector is pET-28a(+).

[0018] The present application also provides a recombinant host cell comprising the recombinant expression vector.

[0019] Further, the host cell is E. coli, Bacillus or yeast cell.

[0020] Further, the host cell is E. coli BL21 (DE3).

[0021] The present application also provides a method for preparing the recombinant bacteriophage lytic enzyme E8-3 protein, which specifically comprises the following steps: (1) culturing the recombinant host cell and expressing the E8-3 protein; (2) collecting the expression product and performing separation and / or purification to obtain the recombinant bacteriophage lytic enzyme E8-3 protein.

[0022] Further, the purification comprises an affinity chromatography step and / or an ion exchange chromatography step and / or a gel filtration chromatography step.

[0023] The present application also provides the use of the recombinant bacteriophage lytic enzyme E8-3 protein in the preparation of an antibacterial preparation.

[0024] Further, the antibacterial preparation is used for inhibiting or killing Helicobacter pylori and / or Acinetobacter baumannii.

[0025] Further, the antibacterial preparation comprises a drug for preventing and / or treating Helicobacter pylori infection and related diseases, a functional food additive with the function of inhibiting the growth of Helicobacter pylori, and a disinfectant for inhibiting or killing Helicobacter pylori.

[0026] Further, the related diseases comprise Helicobacter pylori-associated gastritis and / or peptic ulcer.

[0027] The present application also provides an antibacterial preparation comprising the recombinant bacteriophage lytic enzyme E8-3.

[0028] Compared with the prior art, the present application has the following advantages and beneficial effects: (1) The recombinant bacteriophage lytic enzyme E8-3 of the present application shows good antibacterial effect on Helicobacter pylori in the in vitro minimum inhibitory concentration (MIC) determination, with an MIC of 31 μg / mL, indicating that the recombinant bacteriophage lytic enzyme E8-3 can be used as an antibacterial protein against Helicobacter pylori for the development of antibacterial preparations.

[0029] (2) The MIC of the recombinant bacteriophage lyase E8-3 for Acinetobacter baumannii in the in vitro MIC determination is 125 μg / mL, indicating that the recombinant bacteriophage lyase E8-3, in addition to having an inhibitory effect on Helicobacter pylori, can also be used for inhibiting gram-negative pathogenic bacteria, and has potential value for expanding to the prevention and control of a variety of pathogenic bacteria. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 A construction map of the recombinant expression plasmid of Example 1.

[0031] Figure 2 Hydrolysis circle images containing each recombinant bacteriophage lyase E8-OMPs in Example 3.

[0032] Figure 3 A gel electrophoresis map of the recombinant bacteriophage lyase E8-3 of Example 6. DETAILED DESCRIPTION

[0033] The specific implementation of the present application is further illustrated below in combination with the drawings and examples, but the implementation and protection of the present application are not limited thereto. It should be noted that if the following processes are not specifically and in detail described, they can be implemented or understood by those skilled in the art with reference to the prior art. If the reagents or instruments used are not marked with the manufacturer, they are considered to be conventional products that can be obtained by commercial purchase.

[0034] Example 1 Gene synthesis of bacteriophage lyase E8 and transmembrane peptide According to the preference of E. coli codons, nucleic acid sequences encoding lyase E8 and 40 transmembrane peptide (OMP) nucleotide sequences were synthesized.

[0035] The nucleic acid sequence encoding lyase E8 (SEQ ID NO: 1): ATGAAAACGAGCAACCCGGGCGTGGATCTGATTAAAGGCTTTGAAGGCCTGCGCCTGAAAGCGTATGATGATGGCGTGGGCGTGTGGACCATTGGCTTTGGCACCATTAAATATCCGAACGGCGTGCGCGTGAAAAAAGGCGATACCTGCACCGAAAGCCAAGCGGAAGAATATCTGCGCAACGATCTGGTGGTGTTTGAAAGCGCGATTAACCGCCTGGTGAAAGTGCCGCTGAATCAGAATCAGTTTGATGCGCTGGCGAGCTTTACCTATAACCTGGGCGAAGGCAACCTGAGCATTAGCACCCTGCTGAAAAAACTGAACGCGAAAGACTATAAAGGCGCGGCCGCGGAATTTCCGAAATGGAACAAAGCGGGCGGCCGCGTGCTGGCGGGCCTGGTGAAACGCCGCAAAGCGGAAATGGAACTGTTTCTGAAAAATAGTACCTCTAATAGTAGCACCAATTCAGGCTCTACCGGTAAAGTGAGTCTGCCGAATCGTGTGATTCGCGTGACCAAACCGATTGTTCATGGTAGCGATGTGCTGGCAATTCAGAAAGCACTGTCAAGCCTGTATTTTTATCCGGAAAAAGGTGCCAAAGATAATGGTTCCGATAGCTATTATGGTCCGAAAACCGCCAATGCCGTTAAACGCTTTCAGTCTGTTAATGGCTTAGTTGCAGATGGCGTGTATGGCCCGAAAACCCGCGCAGCCATTCTGAAAAAACTG GCGGGTGCTGGTGCGGGT wherein the italicized portion is the coding sequence for the linker AGAGAG connecting the antibacterial peptides.

[0036] The nucleotide sequences encoding the 40 cell-penetrating peptides are shown in Table 1.

[0037] Table 1 Nucleotide sequences of 40 cell-penetrating peptides

[0038]

[0039]

[0042] To facilitate the fusion of lysozyme E8 and transmembrane peptides, the homologous sequence ACCCGCACCAGCACCCGC was added at the 5' end of each transmembrane peptide nucleotide sequence, and the TAA termination codon sequence and the homologous sequence CGGAGCTCGAATTCG in the pET-28a vector were added at the 3' end when synthesizing each transmembrane peptide nucleotide sequence.

[0043] Example 2 Fusion expression of phage lysozyme E8 and transmembrane peptides The nucleic acid sequence (SEQ ID NO: 1) encoding lysozyme E8 was used as a template for amplification.

[0044] The reaction system was as follows: 25 μL 2×Phanta Max Master Mix (Nanjing Novozyme BioTech Co., Ltd.); 2 μL upstream primer E8_F (AAGAAGGAGATATACATGAAAACGAGCAACCCG); 2 μL downstream primer E8_R (ACCCGCACCAGCACCCGCCAGT); 2 μL nucleic acid sequence encoding lysozyme E8; 19 μL ddH2O.

[0045] The PCR reaction program was as follows: 95 ℃ pre-denaturation for 3 min; 95 ℃ denaturation for 15 s; 56 ℃ annealing for 15 s; 72 ℃ extension for 1 min; 35 cycles, and finally 72 ℃ extension for 5 min. The PCR product of phage lysozyme E8 was obtained.

[0046] The 40 nucleotide sequences encoding transmembrane peptides were mixed with the PCR product of phage lysozyme E8 in equal proportions. The PCR product of phage lysozyme E8 and the mixture of 40 transmembrane peptides were seamlessly connected to the Nco I and Hind III enzyme cutting sites of the expression vector pET 28a (+) in a 1:1 ratio using ClonExpress Ultra One Step Cloning Kit (Nanjing Novozyme BioTech Co., Ltd.), obtaining a series of recombinant expression plasmids pET28a-E8-OMPs, the map of which is shown in Figure 1 The recombinant plasmids were transformed into E. coli BL21 (DE3) competent cells to obtain engineering strains expressing different types of E8-OMPs fusion proteins.

[0047] Example 3 Screening of recombinant phage lysozyme E8-OMPs Acinetobacter baumannii (ATCC 19606) was cultured overnight at 37 °C, 200 rpm in a shaker, and the bacterial solution was collected by centrifugation at 5000 rpm, washed once with sterile water, and diluted to a final concentration of 5 OD / mL. After sterilization, 2x fermentation medium (2.5% tryptone, 1.25% yeast extract, 0.75% NaCl, 0.6% ammonium chloride, 0.6% lactose, 2% agar) and the Acinetobacter solution were mixed at a ratio of 1:1, and poured into plates, about 20 mL per plate, as enzyme activity screening plates for recombinant bacteriophage lyase E8-OMPs. The BL21 (DE3) transformant colonies containing the E8-OMPs gene were picked onto the screening plates and incubated overnight at 37 °C. The lytic activity was evaluated by the plate hydrolysis ring method, and the formation of hydrolysis rings around the colonies was observed. If hydrolysis rings appear, it indicates that the recombinant bacteriophage lyase E8-OMPs produced by the transformant can act on the cell wall of Acinetobacter baumannii and cause lysis; at the same time, the recombinant bacteriophage lyase E8-OMPs can be detected in the environment outside the colony, suggesting that it can be released from the cell and spread into the plate matrix. The results of the hydrolysis ring are shown in Figure 2

[0048] Example 4 Shake flask fermentation of recombinant bacteriophage lyase E8-OMPs and initial screening of antibacterial activity The transformants (E8-1 to E8-8) with larger hydrolysis rings on the screening plates were inoculated into 5 mL of LB medium (composition: 1% tryptone, 0.5% yeast extract, 0.5% NaCl) and cultured at 37 °C in a shaker at 180 rpm for 5 hours, then transferred to 50 mL of fermentation medium (2.5% tryptone, 1.25% yeast extract, 0.75% NaCl, 0.6% ammonium chloride, 0.6% lactose) at a ratio of 1%. The overnight culture was incubated at 30 °C in a shaker at 200 rpm for about 16 hours, and lactose was used to induce the expression of the target protein.

[0049] 5 mL of the fermentation broth was centrifuged to collect the supernatant, which was filtered through a 0.22 μm filter to remove bacteria.

[0050] Acinetobacter baumannii, Klebsiella pneumoniae, Salmonella typhi, and Helicobacter pylori cultured overnight were diluted to 1.0 OD / mL with LB medium (for Helicobacter pylori, BHI medium: CM1135 medium 37 g, distilled water 500 mL, sterilized and added with 10% calf serum), and then diluted to 10 4 ​CFU / mL, standby. Take 100 μL of the test bacteria solution into a 96-well cell culture plate, take 100 μL of the filtered E8-OPMs fermentation supernatant into the 96-well plate, mix well with the bacteria to be tested, and then place in a 37 ℃ incubator for 48 h (H. pylori is placed in an anaerobic incubator for culture). LB medium and BHI medium are used as negative controls. Three parallel samples are set for each well.

[0051] The results are shown in Table 2. The E8-3 transformant showed antibacterial activity against A. baumannii and H. pylori.

[0052] Table 2. Antibacterial activity of E8-OMPs against different bacteria

[0053] Note: - indicates that the bacteria cannot be inhibited; + indicates that the bacteria can be inhibited.

[0054] Example 5. Gene sequencing of recombinant bacteriophage lyase E8-3 The plasmid in the E8-3 transformant was extracted and sent to Suzhou Jinweizhi Biological Technology Co., Ltd. for sequencing. The coding nucleotide sequence of the recombinant bacteriophage lyase E8-3 is shown in SEQ ID NO: 42, wherein the amino acids at positions 1-243 encode E8; the amino acids at positions 244-249 encode the linker AGAGAG; and the amino acids at positions 250-261 encode the transmembrane peptide. The amino acid sequence of the recombinant bacteriophage lyase E8-3 is shown in SEQ ID NO: 43.

[0055] Coding nucleotide sequence of recombinant bacteriophage lyase E8-3 (SEQ ID NO: 42): ATGAAAACGAGCAACCCGGGCGTGGATCTGATTAAAGGCTTTGAAGGCCTGCGCCTGAAAGCGTATGATGATGGCGTGGGCGTGTGGACCATTGGCTTTGGCACCATTAAATATCCGAACGGCGTGCGCGTGAAAAAAGGCGATACCTGCACCGAAAGCCAAGCGGAAGAATATCTGCGCAACGATCTGGTGGTGTTTGAAAGCGCGATTAACCGCCTGGTGAAAGTGCCGCTGAATCAGAATCAGTTTGATGCGCTGGCGAGCTTTACCTATAACCTGGGCGAAGGCAACCTGAGCATTAGCACCCTGCTGAAAAAACTGAACGCGAAAGACTATAAAGGCGCGGCCGCGGAATTTCCGAAATGGAACAAAGCGGGCGGCCGCGTGCTGGCGGGCCTGGTGAAACGCCGCAAAGCGGAAATGGAACTGTTTCTGAAAAATAGTACCTCTAATAGTAGCACCAATTCAGGCTCTACCGGTAAAGTGAGTCTGCCGAATCGTGTGATTCGCGTGACCAAACCGATTGTTCATGGTAGCGATGTGCTGGCAATTCAGAAAGCACTGTCAAGCCTGTATTTTTATCCGGAAAAAGGTGCCAAAGATAATGGTTCCGATAGCTATTATGGTCCGAAAACCGCCAATGCCGTTAAACGCTTTCAGTCTGTTAATGGCTTAGTTGCAGATGGCGTGTATGGCCCGAAAACCCGCGCAGCCATTCTGAAAAAACTGGCGGGTGCTGGTGCGGGTAAATTCCATGAAAAACATCACTCCCACCGTGGTTACTAA Amino acid sequence of recombinant bacteriophage lytic enzyme E8-3 (SEQ ID NO: 43): MKTSNPGVDLIKGFEGLRLKAYDDGVGVWTIGFGTIKYPNGVRVKKGDTCTESQAEEYLRNDLVVFESAINRLVKVPLNQNQFDALASFTYNLGEGNLSISTLLKKLNAKDYKGAAAEFPKWNKAGGRVLAGLVKRRKAEMELFLKNSTSNSSTNSGSTGKVSLPNRVIRVTKPIVHGSDVLAIQKALSSLYFYPEKGAKDNGSDSYYGPKTANAVKRFQSVNGLVADGVYGPKTRAAILKKLAGAGAGKFHEKHHSHRGY Example 6 Protein purification of recombinant bacteriophage lytic enzyme E8-3 Buffer preparation: Equilibrium buffer: 20 mM phosphate buffer (pH 7.2); Regeneration buffer (1M NaCl): weigh 58.44 g of NaCl, dissolve, and dilute with purified water to 1 L; Washing buffer (0.3M NaCl): mix 700 mL of equilibrium buffer with 300 mL of 1M NaCl solution to prepare; Elution buffer (0.6M NaCl): mix 400 mL of equilibrium buffer with 600 mL of 1M NaCl solution to prepare; Regeneration buffer (0.5M NaOH): weigh 20 g of NaOH, and dilute with purified water to 500 mL; Storage buffer (20% ethanol): measure 200 mL of ethanol and dilute with purified water to 1 L.

[0056] Column loading: Pour 50 mL of CMFF weak cation gel filler into the chromatography column, stand for about 30 min, absorb the upper suspended gel, resuspend with purified water, stand for 30 min, then tighten the upper part, adjust the sample flow rate to 5 mL / min, slowly press the filler, maintain the pressure at 0.2 MPa, stop when the distributor is close to the filler, increase the flow rate, maintain the pressure at 0.2 MPa for 30 min.

[0057] Centrifuge the fermentation broth at 10 000 rpm, 4 ℃ for 30 min, collect the supernatant, dilute the supernatant with purified water, adjust the pH of the fermentation broth to 7.2, and adjust the conductivity to 7.0 S·m -1 .

[0058] The purification steps are shown in Table 3.

[0059] Table 3 Purification steps

[0060] SDS-PAGE was used to detect the purity of the protein. The gel electrophoresis spectrum of the recombinant bacteriophage lytic enzyme E8-3 is shown in Figure 2: Figure 3 As shown in Figure 2, the target band after purification is relatively single. After one primary purification, the purity of E8-3 reaches 85%, which meets the requirements of subsequent experiments.

[0061] Example 7: MIC determination of recombinant bacteriophage lytic enzyme E8-3 on H. pylori and A. baumannii The overnight cultured A. baumannii (ATCC 19606), H. pylori (ATCC 43504) and H. pylori (ATCC 26695) were diluted to 1.0 OD / mL with LB medium and BHI medium, respectively, and then diluted to 10 4 CFU / mL using a ten-fold gradient method for standby. 100 μL of the test bacterial solution was taken into a 96-well cell culture plate, and the purified E8-3 antibacterial protein was diluted to 1 mg / mL, then diluted with LB medium and BHI medium in a two-fold gradient, and 100 μL of E8-3 protein at different dilutions was taken into the 96-well plate. After mixing, the mixture was incubated at 37°C for 48 h (H. pylori was incubated in an anaerobic incubator). LB medium and BHI medium were used as negative controls. Three replicates were set for each well.

[0062] The results of the in vitro MIC determination experiment of recombinant bacteriophage lytic enzyme E8-3 are shown in Table 4: recombinant bacteriophage lytic enzyme E8-3 has good antibacterial effect on H. pylori, and the MIC is 31 μg / mL. The MIC for A. baumannii is 125 μg / mL.

[0063]

[0064] The above examples are only the preferred embodiments of the present application, and are used to explain the present application, but not to limit the present application. Changes, replacements and modifications made by those skilled in the art without departing from the spirit and essence of the present application shall fall within the protection scope of the present application.

Claims

1. A recombinant phage lysin E8-3, characterized in that, The amino acid sequence of the recombinant phage lyase E8-3 is shown in SEQ ID NO:

43.

2. A nucleic acid molecule encoding the recombinant phage lysin E8-3 of claim 1, characterized in that, The nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO:42, or is a nucleic acid sequence complementary to SEQ ID NO:

42.

3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule as described in claim 2.

4. The recombinant expression vector according to claim 3, characterized in that, The expression vector is pET-28a(+).

5. A recombinant host cell, characterized in that, The recombinant host cell comprises the recombinant expression vector according to any one of claims 3-4.

6. The recombinant host cell according to claim 5, characterized in that, The host cell was Escherichia coli BL21(DE3).

7. A method for preparing the recombinant phage lysin E8-3 according to claim 1, characterized in that, Specifically, the steps include the following: (1) Culturing the recombinant host cells according to any one of claims 5-6 and expressing the E8-3 protein; (2) Collect the expression product and separate and / or purify it to obtain recombinant phage lysin E8-3.

8. The use of the recombinant phage lyase E8-3 protein according to claim 1 in the preparation of antibacterial agents.

9. The application according to claim 8, characterized in that, The antibacterial agent is used to inhibit or kill Helicobacter pylori and / or Acinetobacter baumannii.

10. An antibacterial preparation, characterized in that, The antibacterial agent comprises the recombinant phage lysin E8-3 as described in claim 1.

Images