Anti-phage defense system and application thereof

Abstract

The invention discloses a novel anti-bacteriophage defense system which is named as an HXS system, and the nucleotide sequence of the novel anti-bacteriophage defense system is shown as SEQ ID NO.1. According to the invention, an anti-phage engineering bacterium is successfully constructed by using the system, and the modified strain has strong and wide defense capability to a plurality of phages. The invention provides a new technical approach for solving the problem of bacteriophage pollution of bacteria in industrial fermentation and biological pharmacy processes, and has wide application prospects in the aspects of bacteriophage pollution resistance in probiotics and zymophyte, domestication of efficient bacteriophage treatment preparations and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a novel anti-phage defense system and application thereof. Background Art

[0002] In the field of industrial fermentation and biopharmaceuticals, bacteriophage contamination of bacteria is a serious problem that can lead to reduced product quality, interruptions in production processes, and economic losses. Bacteriophages are viruses that can specifically infect and lyse bacteria, which can have a serious impact on bacterial populations. Therefore, it is crucial to find a method to effectively improve the resistance of bacteria to bacteriophages.

[0003] In recent years, in addition to the three major defense systems of RM system, CRISPR-Cas system and abortive infection system, scientists have discovered more and more new defense systems, such as BREX (Bacteriophage Exclusion), Zorya, Septu, pVips (prokaryotic Viperin homologues), Gabija, Thoeris, Shedu, CBASS (cyclic oligonucleotide-based antiphage signaling system), Retron, Druantia, qatABCD, STAND, SspABCD-SspE, mzaABCD, etc. Through the integration and research of laboratory resources, we discovered a new anti-phage defense system, which consists of 4 proteins (A, B, C, D), of which proteins B and C belong to the SAM protein superfamily, A protein contains a histidine and serine domain, and D protein has no annotated functional domain. We cloned one of the systems containing the above domains and named it the HXS system. The experiment found that the HXS system is a new bacterial defense system with strong defense effect and broad defense spectrum. Therefore, integrating the HXS defense system into production strains by means of gene editing can be used as one of the methods to prevent phage contamination. Our research aims to solve the common bacterial contamination problems in industrial fermentation and biopharmaceutical processes and obtain engineered strains that can be used in actual production. At the same time, the discovery of this type of new system will also provide new technical paths and application prospects for the future in the fields of anti-phage contamination in probiotics and fermentation bacteria, and the development of efficient phage therapeutic preparations. Summary of the invention

[0004] The purpose of the present invention is to provide a novel anti-phage defense system and to provide the application of the system in constructing anti-phage engineered bacteria. The present invention also provides an anti-phage engineered bacteria and a construction method thereof.

[0005] To achieve the above objectives, the present invention utilizes λ-Red homologous recombination technology to integrate the coding gene of the HXS defense system into the genome of BL21(DE3) to obtain the BL21(DE3)-HXS recombinant engineered strain, and then determines its growth performance, passage stability and ability to defend against bacteriophages. The results show that the ability of the engineered bacteria to resist bacteriophage infection is significantly improved.

[0006] The nucleotide sequence of the HXS defense system is shown in SEQ ID NO.1. It consists of four proteins A, B, C, and D, among which proteins B and C belong to the SAM protein superfamily. Protein A contains a histidine and serine domain, and protein D has no annotated functional domain. We named it the HXS system, and the sequences of the four proteins are shown in SEQ ID NO.2-5.

[0007] The anti-phage defense system is integrated into the bacterial genome using λ-Red homologous recombination technology to obtain a recombinant engineered bacterium with a knock-in target gene. The specific steps of the knock-in method are as follows: S1. Connect the DNA fragment (HXS) with the sequence shown in SEQ ID NO.1 with the linearized fragment of pKD4 by one-step cloning method, transform the product into competent cells, and obtain the bacterial gene knock-in plasmid (pKD4-HXS) containing the target gene; S2. Design primers and use bacterial genomic DNA as a template to amplify the upstream and downstream homologous arms of the knock-in target gene (aceB-Larm and aceB-Rarm). Use the bacterial gene knock-in plasmid obtained in step S1 as a template to amplify the DNA fragment containing Red recombinase and the target gene (Red-HXS). Perform overlap extension PCR on the three obtained fragments to obtain a targeting fragment with homology to the target gene at both ends and the target gene in the middle (aceB-Larm-Red-HXS-aceB-Rarm). S3. The targeting fragment obtained in step S2 is electroporated into bacterial electroporation competent cells containing the pKD46 plasmid to obtain a strain with the target gene knocked in (BL21(DE3)-HXS-KanR); S4. The pCP20 plasmid was electroporated into the strain obtained in step S3 to obtain a strain with resistance gene eliminated, and cultured at 42°C to obtain a phage-resistant engineered bacterium (BL21(DE3)-HXS) with pKD46 and pCP20 plasmids eliminated.

[0008] Wherein, the bacterium is Escherichia coli, but the present invention is not limited to Escherichia coli. Integrating the defense system obtained by the present invention into the genome of other bacteria can also achieve the purpose of resisting phage infection.

[0009] Wherein, the knock-in target gene is aceBGene, but the present invention is not limited to aceB Gene, using other genes in the bacterial genome as knock-in target genes can also achieve the knock-in of target genes. Experiments have shown that inserting HXS into aceB Genes do not affect the growth and proliferation of bacteria.

[0010] The sequence of the pKD4 linearized fragment is shown in SEQ ID NO.6, which contains two FRTs and the kanamycin resistance gene KanR, and has a size of 1457 bp.

[0011] The sequence of the upstream homology arm is shown in SEQ ID NO.7, the sequence of the downstream homology arm is shown in SEQ ID NO.8, and the target gene is aceB Gene.

[0012] Wherein, the sequence containing Red recombinase and target gene DNA fragment is shown as SEQ ID NO.9.

[0013] A genetically recombinant engineered bacterium is an engineered Escherichia coli bacterium, wherein aceB The gene is inserted into a DNA fragment with a nucleotide sequence such as SEQ ID NO.1, resulting in the strain having the ability to resist bacteriophage.

[0014] 1) The growth characteristics of the BL21(DE3)-HXS strain before and after transformation were evaluated using an automatic growth curve analyzer, and no significant difference was found between the two strains; 2) The recombinant strain BL21(DE3)-HXS was continuously liquid-cultured for 30 generations, and the stability of the inserted sequence was detected by PCR, and it was found that the inserted sequence could be stably propagated; 3) Prepare agar plates with BL21(DE3)-HXS strain, select phages that can infect BL21(DE3) strains and spot them on the plates, and culture them overnight. Use BL21(DE3) strain as a control, and evaluate the ability of BL21(DE3)-HXS to defend against phage infection by plaque formation. It was found that 39 strains of BL21(DE3)-HXS had an efficiency of 100 times that of phage inhibition, accounting for 72% of the tested strains, and had a strong ability to resist phages; 4) After mixing BL21(DE3)-HXS culture medium with phage solutions of different MOI, the ability of BL21(DE3)-HXS strain to resist phage was evaluated using an automatic growth curve analyzer. It was found that during the liquid proliferation process, the growth rate of BL21(DE3)-HXS slowed down only under the infection of high MOI (>1) phage, while it could still maintain a growth state comparable to that of the control BL21(DE3) under the infection of low MOI phage; 5) EGFP protein was expressed in BL21(DE3)-HXS and the strain before transformation, and the production performance of the two was evaluated. It was found that when the recombinant strain BL21(DE3)-HXS was used to express the protein, the production capacity and biochemical activity of the EGFP protein were not affected, and it could be used normally for protein expression.

[0015] The beneficial effects of the present invention are: The present invention has developed a novel anti-phage defense system for the first time, and successfully constructed an anti-phage engineered bacterium using the system. The present invention provides a new technical approach to solve the problem of bacteriophage contamination of bacteria in industrial fermentation and biopharmaceutical processes. When using recombinant engineered bacteria for protein expression, the strain can tolerate multiple bacteriophage infections, thereby improving the yield and quality of the protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Plasmid map of pKD4-HXS.

[0017] Figure 2 :PCR verification of the construction of the recombinant strain BL21(DE3)-HXS. M:DL 15,000 Marker; 1:fragment length when the strain is not edited; 2:fragment length after HXS and KanR sequences are inserted; 3:fragment length after KanR sequence is removed.

[0018] Figure 3 : Growth curve of the recombinant strain BL21(DE3)-HXS.

[0019] Figure 4 : Stability of HXS insertion sequence in host bacteria.

[0020] Figure 5 :The recombinant strain BL21(DE3)-HXS is resistant to phages with different MOIs. DETAILED DESCRIPTION

[0021] The present invention will be further described in detail below in conjunction with specific examples. The following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention.

[0022] The HXS system was discovered serendipitously when we were trying to identify homologs of the viperin protein from Escherichia coli by sequence and domain comparison. Mammalian viperin is an interferon-induced antiviral protein that belongs to the SAM superfamily. It produces the ribonucleotide analog ddhCTP, which acts as a chain terminator during viral RNA synthesis, thereby inhibiting viral replication. A recent study showed that prokaryotic viperin also has anti-phage activity and protects against infection by T7 phage by inhibiting viral polymerase-dependent transcription, similar to mammalian viperin.

[0023] While identifying viperin homologs in the Escherichia coli genome, we identified 114 proteins belonging to the COG0535 radical SAM superfamily. Interestingly, we found that nearly half of these proteins were located in a unique operon structure. The operon contained two adjacent radical SAM-containing proteins (proteins B and C) accompanied by two other proteins (proteins A and D), of which protein A contained five His-Xaa-Ser (HXS) amino acid repeats, which we named the HXS bacterial anti-phage defense system, or HXS system for short.

[0024] Key material description: Escherichia coli containing the HXS defense system: isolated and preserved by our laboratory earlier.

[0025] pKD4 vector, pKD46 vector and pCP20 vector: all are commercial vectors well known in the art, modified and preserved by this laboratory.

[0026] pET28a-EGFP plasmid: EGFP is a fluorescent protein well known in the art and is often used as a reporter gene to study gene expression and regulation. The EGFP expression plasmid was prepared and preserved by our laboratory.

[0027] Escherichia coli BL21 (DE3) competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0028] 54 strains of bacteriophages infecting Escherichia coli BL21 (DE3): isolated and preserved by our laboratory in the early stage.

[0029] Other materials not described are conventional materials in this field.

[0030] Example 1: Construction of pKD4-HXS plasmid 1. Use PCR technology to amplify the linearized fragment pKD4 and HXS fragment 1) Use primers pKD4-F / pKD4-R and plasmid pKD4 as template to amplify the linearized fragment of pKD4 (the sequence of the fragment is shown in SEQ ID NO.6). After the PCR product is identified by agarose gel electrophoresis, it is excised and recovered using the OMEGA gel recovery kit for later use.

[0031] PCR amplification system for pKD4 linearized fragment: Template pKD41.0uL Primer pKD4-F1.0uL Primer pKD4-R1.0uL 2×PrimerStarMix20.0uL (Company: Novozymes, Product No. P510) ddH2O17.0uL Primer sequences: pKD4-F:5'-CATATGAATATCCTCCTTAGTTCC-3' pKD4-R:5'-GAAGTTCCTATACTTTCTAGAGAA-3' PCR amplification conditions for the linearized fragment of pKD4: 98℃ for 5 min, 98℃ for 15 sec, 57℃ for 15 sec, 72℃ for 1.5 min, 30 cycles, extension at 72℃ for 5 min.

[0032] 2) Use primers HXS-F / HXS-R and Escherichia coli containing the HXS defense system as a template to amplify the HXS fragment (the sequence of the fragment is shown in SEQ ID NO.1). After the PCR product is identified by agarose gel electrophoresis, it is excised and recovered using the OMEGA gel recovery kit for later use.

[0033] PCR amplification system for HXS fragments: Template 1.0uL Primer HXS-F1.0uL Primer HXS-R1.0uL 2×PrimerStarMix20.0uL (Company: Novozymes, Product No. P510) ddH2O17.0uL Primer sequences: HXS-F:5'-TTCTCTAGAAAGTATAGGAACTTCATGGCGATGTGGGAAAAGATAT-3' HXS-R:5'-GGAACTAAGGAGGATATTCATATGTTACCGAGCCAATATATTCAAT-3' PCR amplification conditions for HXS fragment: 98℃ 5min, 98℃ 15sec, 57℃ 15sec, 72℃ 1min, 30 cycles, extension at 72℃ for 5min.

[0034] 2. Fragment ligation The pKD4 linearized fragment and the HXS fragment were spliced ​​together in vitro using the one-step cloning kit (Cat. No. C116) from Nanjing Novozymes to obtain the pKD4-HXS recombinant product. The in vitro recombination reaction system and reaction conditions are as follows: Reaction system (10uL): Fragment A1uL Fragment B1uL Enzyme 5uL ddH2O3uL Reaction conditions: The mixture was incubated at 50°C for 10 min and then immediately placed on ice.

[0035] 3. Plasmid transformation and identification The above recombinant product was transformed into E. coli DH5α and cultured overnight at 37°C. The next day, a single colony was picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin. After culturing at 37°C for 12 hours, PCR identification of the bacterial solution was performed. PCR positive clones were selected, plasmids were extracted and sent to the company for sequencing, and plasmids were extracted from positive clones with correct sequencing to obtain the bacterial gene knock-in plasmid pKD4-HXS.

[0036] The four protein sequences of the HXS defense system are shown in SEQ ID NO. 2-5, respectively. The plasmid map of pKD4-HXS is shown in Figure 1 shown.

[0037] Example 2: Construction of a targeting fragment LHR containing the HXS defense system for knocking into Escherichia coli BL21 (DE3) 1. PCR amplification of fragments aceB-Larm, aceB-Rarm and Red-HXS aceB The NCBI accession number of the gene is NP_418438.1, and the 5' end specific homology arm amplification primers (Larm-F and Larm-R) were used to amplify aceB The left homology arm DNA fragment of the gene, i.e., fragment aceB-Larm (SEQ ID NO.7); 3' end specific homology arm amplification primers (Rarm-F and Rarm-R) were used to amplify aceB The right homologous arm DNA fragment of the gene, i.e., fragment aceB-Rarm (SEQ ID NO.8); using primers Red-F / Red-R, plasmid pKD4-HXS was used as a template to amplify fragment Red-HXS (SEQ ID NO.9). The above PCR products were identified by agarose gel electrophoresis, and the OMEGA gel recovery kit was used to obtain the recovered DNA fragment for use.

[0038] The amplification system and conditions involved are as follows: PCR amplification system for fragment aceB-Larm: Template E. coli BL21 (DE3) genome 1.0uL Primer Larm-F 1.0uL Primer Larm-R 1.0uL 2×PrimerStarMix20.0uL (Company: Novozymes, Product No. P510) ddH2O17.0uL Primer sequences: Larm-F:5'-ggactagacccggatgtaccg-3' Larm-R:5'-tgctcgccatacggccttgt-3' PCR amplification conditions for fragment aceB-Larm: 98°C for 5 min, 98°C for 15 sec, 57°C for 15 sec, 72°C for 30 sec, for 30 cycles, and extension at 72°C for 5 min.

[0039] PCR amplification system for fragment aceB-Rarm: Template E. coli BL21 (DE3) genome 1.0uL Primer Rarm-F1.0uL Primer Rarm-R1.0uL 2×PrimerStarMix20.0uL (Company: Novozymes, Product No. P510) ddH2O17.0uL Primer sequences: Rarm-F:5'-cacgatggcacatggatcgc -3' Rarm-R:5'-ggcagggtcaggaaatca-3' PCR amplification conditions for fragment aceB-Rarm: 98°C for 5 min, 98°C for 15 sec, 57°C for 15 sec, 72°C for 30 sec, for 30 cycles, and extension at 72°C for 5 min.

[0040] PCR amplification system for fragment Red-HXS: Template plasmid pKD4-HXS1.0uL Primer HXS-F1.0uL Primer HXS-R1.0uL 2×PrimerStarMix20.0uL (Company: Novozymes, Product No. P510) ddH2O17.0uL Primer sequences: Red-F:5'-acaaggccgtatggcgagcaGAAGTTCCTATTCCGAAGTT-3' Red-R:5'-GCGATCCATGTGCCATCGTGTTACCGAGCCAATATATTCA -3' PCR amplification conditions for fragment Red-HXS: 98℃ for 5 min, 98℃ for 15 sec, 57℃ for 15 sec, 72℃ for 1.5 min, 30 cycles, extension at 72℃ for 5 min.

[0041] 2. Obtaining the targeting fragment LHR containing the HXS defense system using SOE-PCR technology Using aceB-Larm, aceB-Rarm and Red-HXS fragments as templates and Larm-F and Rarm-R as primers, the SOE-PCR technology was used to obtain the fusion fragment LHR, the order of which was aceB-Larm-Red-HXS-aceB-Rarm, which is the targeting fragment containing the HXS defense system used to knock into Escherichia coli BL21 (DE3).

[0042] PCR amplification system of fragment LHR: Template (aceB-Larm / Red-HXS / aceB-Rarm) 1.0uL each Primer Larm-F 1.0uL Primer Rarm-R1.0uL 2×PrimerStarMix20.0uL (Company: Novozymes, Product No. P510) ddH2O15.0uL Primer sequences: Larm-F:5'-ggactagacccggatgtaccg-3' Rarm-R:5'-ggcagggtcaggaaatca-3' PCR amplification conditions for fragment LHR: 98°C for 5 min, 98°C for 15 sec, 57°C for 15 sec, 72°C for 2 min, 30 cycles, and extension at 72°C for 5 min.

[0043] Example 3: Construction of recombinant strain BL21(DE3)-HXS 1. Preparation of electrocompetent cells of E. coli BL21(DE3) carrying plasmid pKD46 1) Transformation of plasmid pKD46 in E. coli BL21(DE3) The Red homologous recombination plasmid pKD46 was transformed into Escherichia coli BL21 (DE3) competent cells and cultured at 30°C overnight. The next day, a single colony was picked and inoculated into LB liquid culture medium containing 100 μg / mL ampicillin. After culturing at 30°C for 24 hours, Escherichia coli BL21 (DE3) containing plasmid pKD46 was obtained.

[0044] 2) Preparation of electroporated E. coli BL21(DE3) competent cells containing plasmid pKD46 E. coli BL21 (DE3) carrying plasmid pKD46 was streaked three times on LB solid medium containing 100 μg / mL ampicillin for full activation. Single clones of the activated strain were directly picked into fresh LB liquid medium containing 100 μg / mL ampicillin and cultured overnight at 30°C. The next day, it was transferred to fresh LB liquid medium containing 100 μg / mL ampicillin at a ratio of 1:100. When the OD600 of the bacterial solution was 0.2, 0.2%-0.5% L-arabinose was added and induced for about 2 hours. When the OD600 of the bacterial solution was 0.4-0.5, the bacteria were collected and washed with 10% glycerol to prepare for electroporation.

[0045] 2. Transformation of the targeting fragment LHR 1) Electroporation of DNA targeting fragments The targeting fragment LHR constructed in Example 2 (a fragment containing the HXS defense system for knocking into E. coli BL21 (DE3)) was thoroughly mixed with the electroporation competent cells of E. coli BL21 (DE3) carrying the plasmid pKD46, and placed in an electroporation cuvette for electroporation. The parameters of the electroporation instrument were voltage 2500V, resistance 200, and capacitance 25uF. At least 1ug of the fragment LHR was prepared.

[0046] 2) Recovery culture of bacterial solution after electroporation After the electroporation, 1 mL of LB liquid culture containing 0.2%-0.5% L-arabinose was quickly added, and then the tube was transferred to a 2 mL centrifuge tube and placed in a 30°C shaker for 2 hours. Centrifuge at 5000 rpm for 3 minutes, discard 900 μL of supernatant, resuspend the precipitate and spread it on LB solid culture medium containing 50 μg / mL kanamycin, and placed in a 30°C incubator for 24 hours.

[0047] 3. Positive identification of the recombinant strain BL21(DE3)-HXS-KanR Pick a single colony from the resistant plate and inoculate it into LB liquid medium containing 50μg / mL kanamycin. After culturing at 37℃ for 12h, use identification primers JD-F / JD-R to perform PCR identification of the bacterial liquid. Select PCR positive clones, amplify the DNA target fragment and send it to the company for sequencing. The positive strain with correct sequencing is the strain with successful recombination, and it is named BL21(DE3)-HXS-KanR. The results are as follows Figure 2 As shown, the length of the amplified fragment after the HXS system fragment and the KanR resistance gene were inserted into the genome was 6,238 bp, which was consistent with expectations.

[0048] PCR amplification system: Template bacterial solution 1.0uL Primer JD-F1.0uL Primer JD-R1.0uL 2×PrimerStarMix10.0uL (Company: Novozymes, Product No. P510) ddH2O7.0uL Primer sequences: JD-F:5'-gcgtggctttgatgattcattcctgg-3' JD-R:5'-ctgaaccgcgtaatttcaccacatct-3' PCR amplification conditions: 98°C for 5 min, 98°C for 15 sec, 57°C for 15 sec, 72°C for 2.0 min, 30 cycles were performed, and extension was performed at 72°C for 5 min.

[0049] 4. Eliminate the resistance gene of the recombinant strain BL21(DE3)-HXS-KanR and construct the recombinant strain BL21(DE3)-HXS 1) Preparation of electroporation competent recombinant strain BL21(DE3)-HXS-KanR The recombinant strain BL21(DE3)-HXS-KanR successfully identified above was inoculated into LB solid medium containing 50μg / mL kanamycin and streaked 3 times for full activation. The activated strain was directly picked into a single clone in fresh LB liquid medium containing 50μg / mL kanamycin and cultured overnight at 30°C. The next day, it was transferred to fresh LB medium containing 50μg / mL kanamycin at a ratio of 1:100. When the bacterial liquid OD600 = 0.4-0.5, the cells were collected and washed with 10% glycerol to prepare for electroporation.

[0050] 2) Transformation of plasmid pCP20 The pCP20 plasmid used to eliminate the gene knockout scar was thoroughly mixed with the recombinant strain BL21(DE3)-HXS-KanR competent cells and placed in an electroporation cup for electroporation. The parameters of the electroporation instrument are voltage 2500V, resistance 200, and capacitance 25uF. At least 0.1ug of pCP20 plasmid was prepared.

[0051] 3) Recovery culture of bacterial solution after electroporation After electroporation, 1 mL of LB liquid culture medium was quickly added, then transferred to a 2 mL centrifuge tube and placed in a 30°C incubator for 2 hours to recover, centrifuged at 5000 rpm for 3 minutes, 900 μL of supernatant was discarded, the precipitate was resuspended and spread on LB solid culture medium containing 34 μg / mL chloramphenicol, and cultured at 30°C for 24 hours.

[0052] 4) Positive identification of the recombinant strain BL21(DE3)-HXS with the KanR resistance gene eliminated Take a single colony from the resistance plate and inoculate it into LB liquid medium containing 34μg / mL chloramphenicol. After culturing at 30℃ for 12h, use identification primers JD-F / JD-R to perform PCR identification of the bacterial liquid. The amplification system and procedure are the same as those for identifying the recombinant strain BL21(DE3)-HXS-KanR mentioned above. Select PCR positive clones and send them to the company for sequencing. The positive strain with correct sequencing is the recombinant strain that eliminates the KanR resistance gene and is named: BL21(DE3)-HXS. The results are as follows Figure 2 As shown, the length of the amplified fragment after removing the KanR resistance gene from the insert was 4,845 bp, which was consistent with the expectation.

[0053] 5) Elimination of plasmids pKD46 and pCP20 The above BL21(DE3)-HXS was inoculated into liquid LB medium and cultured overnight at 42°C. The next day, it was spread on LB solid medium containing no antibiotics, 50 μg / mL kanamycin, and 100 μg / mL ampicillin, respectively, and streaked for culture. If no colonies grew on the plate containing resistance, but colonies grew on the plate without resistance, it proved that plasmids pKD46 and pCP20 had been eliminated, and the colonies on the plate without resistance were the recombinant strain BL21(DE3)-HXS without resistance.

[0054] Example 4: Growth characteristics and passage stability of the recombinant strain BL21(DE3)-HXS 1. Evaluation of the growth characteristics of BL21(DE3)-HXS strain using an automated growth curve analyzer Take 100 mL of the overnight cultured recombinant strain BL21(DE3)-HXS bacterial solution and the standard strain BL21(DE3), transfer them to 10 mL of LB liquid medium respectively, and culture them at 37°C for 12 h. Then take 200 uL of LB liquid medium and mix it with 2μL of bacterial solution and transfer it to a 100-well plate. Then place the 100-well plate in a fully automatic growth curve analyzer, and set the conditions to: 37°C, measure OD600 every 10 min (repeat three times). Finally, use GraphPad Prism software to draw the bacterial growth curve with growth time as the horizontal axis and OD600 as the vertical axis. The results are shown in the figure. Figure 3 As shown, the recombinant strain BL21(DE3)-HXS has the same growth trend as the standard strain BL21(DE3), and their population growth patterns are consistent.

[0055] 2. Stability of recombinant strain BL21(DE3)-HXS The recombinant strain BL21(DE3)-HXS was continuously liquid-cultured for 30 generations, and the stability of the inserted sequence was detected by PCR. The identification primers were JD-F / JD-R, and the PCR amplification system and procedure were the same as those used to identify the recombinant strain BL21(DE3)-HXS in Example 3 above. The results of PCR amplification of the insertion site length are shown in Figure 4 As shown: the amplification length near the insertion site has been maintained at 4,845 bp, there is no weakening or disappearance of the positive band, and there is no loss of the insertion sequence, which proves that the insertion sequence of the recombinant strain has good stability in generation.

[0056] Example 5: Test of the defensive effect of the recombinant strain BL21(DE3)-HXS 1. Observation of plaque formation on solid plates to evaluate the protective effect of BL21(DE3)-HXS In the phage library established in the laboratory, 54 phages that can infect BL21 (DE3) were screened. Before testing, each phage was adjusted to 10 7PFU, 10-fold gradient dilution was performed using Pi-Mg buffer (26 mM Na2HPO4, 68 mMNaCl, 22 mM KH2PO4, 1 mM MgSO4, pH 7.5). The recombinant strain BL21(DE3)-HXS was inoculated into 10 mL of fresh LB liquid medium at a ratio of 1:100, and then placed in a shaker and cultured at 37°C overnight. The next day, the semi-solid TSB medium was preheated and melted and then cooled to 60°C. Take 300 uL of bacterial solution and mix with 6 mL of semi-solid TSB medium and pour it on the agar plate. After it solidifies, 2 uL of the gradient diluted phage is spotted on the semi-solid surface in sequence (they do not dissolve each other and keep a certain distance). After the droplets are dry, they are placed in a 37°C incubator and incubated overnight. Using the BL21(DE3) strain as a control, the plaque efficiency of the recombinant strain BL21(DE3)-HXS was counted. The results showed that BL21(DE3)-HXS had an efficiency of 100-fold in inhibiting phages in 39 strains, accounting for 72% of the tested strains, and had a strong ability to resist phages.

[0057] 2. Evaluation of the protective effect of phage infection in BL21(DE3)-HXS at different MOI (multiplicity of infection) Take 100 μL of the overnight cultured recombinant strain BL21(DE3)-HXS and standard strain BL21(DE3), inoculate into 10 mL of LB liquid medium, and culture at 37°C until the logarithmic phase (2×10 8 CFU / mL). Dilute long-tailed phage T7 and long-tailed phage #41 to the expected concentration. Take 180 uL of the above host bacterial culture solution and mix with 20 uL of phage dilutions with different MOIs and transfer to a 100-well plate. Then place the 100-well plate in an automatic growth curve analyzer, set the conditions to: 37°C, and measure OD600 every 10 min. Finally, use GraphPad Prism software to draw a curve with infection time as the horizontal axis and OD600 as the vertical axis. Figure 5 As shown in the figure, during the liquid proliferation process, the growth rate of BL21(DE3)-HXS slowed down only under the infection of high MOI (>1) phage, while it was still able to maintain a growth state comparable to that of the control BL21(DE3) under the infection of low MOI phage, indicating that BL21(DE3)-HXS has a strong ability to resist phage.

[0058] Example 6: EGFP protein expression ability of the recombinant strain BL21(DE3)-HXS The pET28a-EGFP plasmid was transformed into BL21(DE3)-HXS and the strain before transformation. Before IPTG induction, the expression level of EGFP protein in the standard strain BL21(DE3) was used as the control. The expression level of EGFP protein in the recombinant strain BL21(DE3)-HXS was not significantly different from that in the standard strain BL21(DE3). Similarly, by observing the expression levels of the supernatant and precipitate of the strain before and after the recombination, it was found that there was no significant difference in the proportion of soluble protein. After that, the EGFP protein was purified by Ni-NTA column and it was found that there was no significant difference in protein yield. At the same time, the EGFP protein purified from the strain before and after the recombination was adjusted to the same concentration, and the protein fluorescence intensity was compared by a microplate reader. It was found that their change trends were consistent. This shows that when the recombinant strain BL21(DE3)-HXS is used to express proteins, it does not affect the biochemical activity of the EGFP protein. Therefore, this strain can also be used for the expression of other proteins.

[0059] Appendix: Description of Sequence Listing SEQ ID NO.1: Nucleotide sequence of the HXS system; SEQ ID NO.2: Amino acid sequence of protein A in the HXS system; SEQ ID NO.3: Amino acid sequence of protein B in the HXS system; SEQ ID NO.4: Amino acid sequence of protein C in the HXS system; SEQ ID NO.5: Amino acid sequence of protein D in the HXS system; SEQ ID NO.6: DNA sequence of the linearized fragment of pKD4; SEQ ID NO.7: upstream homology arm sequence of the HXS insertion site; SEQ ID NO.8: downstream homology arm sequence of the HXS insertion site; SEQ ID NO.9: DNA sequence of Red-HXS fragment.

Claims

1. An anti-phage defense system, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. Use of the defense system according to claim 1 in constructing phage-resistant engineered bacteria.

3. A method for constructing phage-resistant engineered bacteria, characterized in that: The anti-phage defense system of claim 1 is integrated into the bacterial genome using λ-Red homologous recombination technology to obtain a recombinant engineered bacterium with a knock-in target gene.

4. The method for constructing phage-resistant engineered bacteria according to claim 3, characterized in that: The specific steps of this method are as follows: S1. The DNA fragment with the sequence shown in SEQ ID NO.1 was connected with the linearized fragment of pKD4 by one-step cloning method, and the product was transformed into competent cells to obtain a bacterial gene knock-in plasmid containing the target gene; S2. Design primers and use bacterial genomic DNA as a template to amplify the upstream and downstream homology arms of the knock-in target gene respectively; use the bacterial gene knock-in plasmid obtained in step S1 as a template to amplify the DNA fragment containing Red recombinase and the target gene; perform overlap extension PCR on the three obtained fragments to obtain a targeting fragment with homology to the target gene at both ends and containing the target gene in the middle; S3. The targeting fragment obtained in step S2 is electroporated into a bacterial electroporation competent cell containing a pKD46 plasmid to obtain a strain in which the target gene is knocked in; S4. The pCP20 plasmid was electroporated into the strain obtained in step S3 to obtain a strain with resistance gene eliminated, and cultured at 42° C. to obtain a phage-resistant engineered bacterium with pKD46 and pCP20 plasmids eliminated.

5. The method for constructing phage-resistant engineered bacteria according to claim 4, characterized in that: The bacteria is Escherichia coli.

6. The method for constructing phage-resistant engineered bacteria according to claim 4, characterized in that: The knock-in target gene is aceB Gene.

7. The method for constructing phage-resistant engineered bacteria according to claim 4, characterized in that: The sequence of the pKD4 linearized fragment is shown as SEQ ID NO.

6.

8. The method for constructing phage-resistant engineered bacteria according to claim 4, characterized in that: The sequence of the upstream homology arm is shown in SEQ ID NO.7, and the sequence of the downstream homology arm is shown in SEQ ID NO.

8.

9. The method for constructing phage-resistant engineered bacteria according to claim 4, characterized in that: The sequence of the DNA fragment containing Red recombinase and target gene is shown in SEQ ID NO.

9.

10. The phage-resistant engineered bacteria constructed by the method according to any one of claims 3 to 9.

Images