A gene deletion attenuated mutant of wild strain of proteus vulgaris, construction method and application thereof

Abstract

The present application relates to the technical field of microbiology and animal vaccine cross, and particularly relates to a gene deletion attenuated mutant of wild strain of Pseudomonas plecoglossicida, a construction method and application thereof, and provides the gene deletion attenuated mutant based on deletion of the coding genes of proteins participating in maintaining cell membrane lipid asymmetry of Pseudomonas plecoglossicida strain on the wild strain of Pseudomonas plecoglossicida mlaE , the coding gene of response regulator protein of envZ / ompR two-component system ompR , and the coding gene of DeoR family transcriptional regulator deoR The mutant strain has obvious low toxicity, can effectively protect the test fish from the invasion of Pseudomonas plecoglossicida causing the disease of Pseudomonas plecoglossicida visceral white spot disease, and researches show that the vaccine prepared based on the gene deletion attenuated mutant has significant immunization effect and excellent prevention and treatment effect on the disease of Pseudomonas plecoglossicida visceral white spot disease in aquaculture.

Description

Technical Field

[0001] This invention relates to the interdisciplinary fields of microbiology and animal vaccines, and in particular to a gene-deleted attenuated mutant strain of wild-type Pseudomonas proteus, its construction method, and its application. Background Technology

[0002] Large yellow croaker ( Larimichthys crocea Large yellow croaker (VGD) is mainly distributed in the southern Yellow Sea and East China Sea of ​​China. Since the 1980s, its artificial aquaculture industry has gradually emerged, quickly becoming one of the leading marine aquaculture species in provinces such as Fujian and Zhejiang, and even nationwide. Rich in nutrients and with delicious meat, it is widely favored by consumers. With the development of mature intensive and large-scale aquaculture models, the scale of large yellow croaker farming has continued to grow. However, due to intensive farming, visceral white spot disease has broken out in large yellow croaker farming areas year after year. Visceral Granulomas Disease (VGD) is a disease caused by *Pseudomonas proteus* (…). Pseudomonas plecoglossicida This bacterial disease, caused by white spot disease, poses a serious threat to the large yellow croaker aquaculture industry. The main characteristic of this disease is the appearance of white nodules about 1 mm in size on the spleen, kidneys, liver, and other internal organs of the large yellow croaker. In the natural state, the mortality rate of infected fish can reach up to 80%, causing huge economic losses to fish farmers. Large yellow croakers suffering from visceral white spot disease may not show obvious external symptoms in the early stages, but as the disease progresses, the fish will exhibit emaciation, lethargy, abnormal swimming, and loss of appetite. Necropsy reveals numerous white nodules on the internal organs, enlarged spleen and kidneys, and ascites. Pathological histological observation shows significant lesions in the internal organs, with severe inflammation and cell necrosis in tissues such as the spleen and kidneys.

[0003] The occurrence of visceral white spot disease in large yellow croaker caused by *Pseudomonas proteus* infection is closely related to water temperature. Researchers have compared the virulence factors expressed by *P. proteus* at different temperatures, finding that the types and quantities of proteins secreted by the bacteria were highest at 20°C, suggesting that temperature may be one of the important factors controlling the expression of key virulence factors in this bacterium. Currently, the control of visceral white spot disease in large yellow croaker mainly relies on the use of antibiotics, such as enrofloxacin and doxycycline hydrochloride. However, long-term use of antibiotics not only easily leads to the emergence of drug-resistant strains but may also pose potential threats to the aquaculture environment and food safety. Therefore, developing a green and efficient control method is particularly important. Live attenuated vaccines, as a novel control measure, have advantages such as strong immunogenicity and good immune persistence, and are expected to provide a new solution for the control of visceral white spot disease. Summary of the Invention

[0004] To address the aforementioned problems, the present invention aims to provide a gene-deleted attenuated mutant strain of wild-type *Pseudomonas proteus*, its construction method, and its application. This attenuated mutant strain or related preparation reduces the product safety risks commonly associated with traditional attenuated live vaccines and is an economical and effective vaccine against visceral white spot disease in farmed large yellow croaker.

[0005] The objective of this invention can be achieved through the following technical solutions: The first objective of this invention is to provide a gene-deleted attenuated mutant of a wild-type *Pseudomonas proteus* strain, which is based on the deletion of a gene encoding a protein involved in maintaining cell membrane lipid asymmetry in the wild-type *Pseudomonas proteus* strain. melE envZ / ompR two-component system response regulatory protein encoding genes ompR and DeoR family transcription factor encoding genes deoR The resulting mutant strain; Among them, encoding melE The nucleotide sequence is shown in SEQ ID NO.21, encoding the gene. oh my The nucleotide sequence is shown in SEQ ID NO.22, encoding the gene. deoR The nucleotide sequence is shown in SEQ ID NO.23.

[0006] In one embodiment of the present invention, the gene-deleted attenuated mutant strain does not carry any antibiotic resistance markers or other markers, and has no exogenous gene fragments.

[0007] In one embodiment of the present invention, the *Pseudomonas proteus* strain is *Pseudomonas proteus* subspecies ND2304.

[0008] The second objective of this invention is to provide a method for constructing a gene-deleted attenuated mutant of a wild-type Pseudomonas proteus strain, comprising the following steps: (S1) Genes melE ,Gene melE and genes melE Recombinant plasmids Δ were obtained by inserting them into plasmid vectors. melE Recombinant plasmid Δ ompR Recombinant plasmid Δ deoR ; (S2) The recombinant plasmid Δ obtained in step (S1) melE The recombinant strain ND2304Δ was obtained by conjugation into *Pseudomonas proteus* ND2304. melE ; (S3) The recombinant strain ND2304Δ obtained in step (S2) melE Based on this, recombinant plasmid Δ was introduced via conjugation. ompR Recombinant plasmid Δ deoR The recombinant strain LAV-1 was obtained, which is the gene-deleted attenuated mutant strain of the wild-type Pseudomonas proteus strain.

[0009] In one embodiment of the present invention, during the bonding process, the volume ratio of the donor to the recipient is 1:2~3; Preferably, during the conjugation process, the volume ratio of the donor to the recipient is 1:3.

[0010] A third objective of this invention is to provide a method for culturing a gene-deleted attenuated mutant of wild-type *Pseudomonas proteus*, comprising the following steps: After activation and resuscitation, a gene-deleted attenuated mutant of wild-type Pseudomonas proteus was inoculated into TSB culture medium.

[0011] In one embodiment of the present invention, during the cultivation process, the temperature is 28~37 ℃ (preferably, the temperature is 30 ℃), the rotation speed is 180~220 rpm, and the time is 6~12 h.

[0012] In one embodiment of the present invention, the TSB culture medium consists of 17 g / L tryptone, 3 g / L soybean papain hydrolysate, 5 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, and 2.5 g / L glucose.

[0013] The fourth objective of this invention is to provide the application of a gene-deleted attenuated mutant strain of wild-type Pseudomonas proteus in the preparation of a vaccine for the prevention and / or treatment of visceral white spot disease in farmed fish.

[0014] The fifth objective of this invention is to provide a vaccine for the prevention and / or treatment of visceral white spot disease in farmed fish, comprising a gene-deleted attenuated mutant strain of the above-mentioned wild-type Pseudomonas proteus strain.

[0015] In one embodiment of the invention, the visceral white spot disease vaccine contains a pharmaceutically acceptable carrier.

[0016] In one embodiment of the present invention, the carrier is selected from solvents, dispersion media, coating agents, antibacterial agents, antifungal agents, isotonic agents, absorption delay agents, preservatives, sweeteners for oral administration, thickeners, buffers, liquid carriers, wetting agents, solubilizers, or emulsifiers; acidifiers, antioxidants, alkalizing agents, carriers, chelating agents, colorants, complexing agents, suspending agents or thickeners, flavoring agents or fragrances, oils, penetration enhancers, polymers, hardening agents, proteins, carbohydrates, fillers, and lubricants.

[0017] In one embodiment of the present invention, the application dose of the gene-deleted attenuated mutant strain of *Pseudomonas proteus* wild-type strain in the vaccine is 10.2 ~10 3 CFU / tail.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) The gene-deleted attenuated mutant strain of Pseudomonas proteus wild-type strain provided by the present invention lacks the gene encoding a protein involved in maintaining cell membrane lipid asymmetry. melE envZ / ompR two-component system response regulatory protein encoding genes ompR and DeoR family transcription factor encoding genes deoR It has significant low toxicity and can effectively protect experimental fish from the pathogenic strain of Pseudomonas proteus that causes white spot disease in large yellow croaker. Studies have shown that the vaccine prepared based on this gene-deleted attenuated mutant strain has significant immunization effect and excellent prevention and control effect of white spot disease in large yellow croaker in aquaculture. (2) The gene deletion attenuated mutant strain of wild-type Pseudomonas proteus provided by the present invention does not contain any exogenous gene fragments. It adopts gene deletion mutation (especially multi-gene deletion) to delete large fragments of virulence-related genes. Theoretically, the virulence is considered irreversible, which greatly eliminates the possibility of spreading a large number of toxic pathogens to the environment. It has technical environmental and product safety and has practical commercial development and application value. (3) The gene deletion attenuated mutant strain of Pseudomonas proteus wild-type strain provided by the present invention has the advantages of clear genetic background of mutation, clear attenuation mechanism, easy to distinguish between vaccine strain and wild strain, easy to monitor the environment, and can improve the environmental safety and controllability of vaccine. (4) The gene deletion attenuated mutant strain of Pseudomonas proteus wild-type virus provided by the present invention provides practical development significance for the commercialization of deletion attenuated live vaccines and has broad application prospects. Detailed Implementation

[0019] In this invention, the *Pseudomonas proteus* ND2304 is the *Pseudomonas aeruginosa* strain ND2304 described in patent application CN119410545A. This strain was deposited at the China Center for Type Culture Collection on March 4, 2024, with accession number CCTCC M 2024372ND2304. Pseudomonas plecoglossicida The address of the China Center for Type Culture Collection is Wuhan University, Wuhan, China.

[0020] The present invention will now be described in detail with reference to specific embodiments.

[0021] Unless otherwise specified, all reagents used in the following embodiments are commercially available reagents, and all detection methods and techniques used are conventional detection methods and techniques in the art.

[0022] Example 1 This embodiment provides the construction of a gene-deleted attenuated mutant of wild-type Pseudomonas proteus, as detailed below: (1) melE , ompR , deoR Construction of gene-deleted strains 1) PCR amplification to obtain homologous fragments Table 1 Primer Summary Table Using the genome of wild-type Pseudomonas proteus ND2304 as a template, the genomes of *Pseudomonas proteus* were amplified using mLaE-P1 and mLaE-P2. melE upstream homologous arm of gene, obtained melE Gene fragment 1; amplified using mlaE-P3 and mlaE-P4 melE Downstream homologous arm of the gene, obtained melE Gene fragment 2; Amplification was performed using ompR-P1 and ompR-P2. ompR upstream homologous arm of gene, obtained ompR Gene fragment 1; amplified using ompR-P3 and ompR-P4 ompR Downstream homologous arm of the gene, obtained ompR Gene fragment 2; Amplification was performed using deoR-P1 and deoR-P2. deoR upstream homologous arm of gene, obtained deoR Gene fragment 1; amplified using deoR-P3 and deoR-P4 deoR Downstream homologous arm of the gene, obtained deoR Gene fragment 2; After the amplification products were verified by agarose gel electrophoresis, they were recovered and purified using a commercial gel electrophoresis recovery kit.

[0023] To achieve efficient ligation with plasmid pDMK (https: / / www.addgene.org / ), the overlap region sequence of the pDMK vector was introduced at both ends during the primer design stage to facilitate subsequent seamless cloning reactions.

[0024] Among them, encoding melE The nucleotide sequence is shown in SEQ ID NO.21, encoding the gene. oh my The nucleotide sequence is shown in SEQ ID NO.22, encoding the gene. deoR The nucleotide sequence is shown in SEQ ID NO.23.

[0025] SEQ ID NO.21 (5'-3') is as follows: atgcgtagaaaatccttactggagcgtattcgcctgatgggccgttcggctatcgacgtgctggcggtgctggggcgctcctgcctgttcctgttccatgccctggtcggccgtggcggtatcggcggcggtttccagttgctgaccaagcagctgtattcggtgggcgtgctgtcgctggcgatcgtggtcgtgtcgggtgtgttcatcggcatggtgctggcgctgcagggctacagcatcctgaccaagtacggttccgagcaggcggtcgggcagatggtggccttgaccctgctgcgtgaactgggcccggtggtcactgcgttgctgttcgccgggcgtgcgggttctgcgctgacggccgaaatcggcaacatgaagtccaccgagcagctgtcgagccttgaaatgatcggcgtcgacccgctcaagtacatcgtcgcgccgcgcttgtgggccggtttcatttccctgccgttgctggcactgatcttcagtgtggtcggcatctggggtggttcgtgggtcgcggtggactggctgggcgtctacgaaggctccttctgggccaacatgcagaacagtgtttcctttaccgacgacgtgctcaacgggctggtcaagagcctggtgttcgccttcgtctcgacctggatcgccgtattccaggggtacgactgcgagcccacctcggaagggatcagccgtgccaccaccaagaccgtggtctatgcctcattggcagtactgggtctggactttattctgaccgccttgatgtttggagatttctga SEQ ID NO. 22 (5'-to-3') is as follows: atgactggcacccccaacaccgctgaaggtgacaagattctcatcgtcgatgacgacccggggctcagcagcctgctggaacgtttcttcaccagcaagggctatcgcgcccgtgcggtccccaacaccgaacagatggaccgcctgctgcagcgcgaggtcttcaacctggtggtgctcgacctgatgctgccgggtgaggacggcctgtccgcgtgcaagcgcctgcgccagtcgaacaaccagattccgatcatcatgctcaccgccaagggcgacgaactcagccgcatcaagggcctggaactgggcgccgacgactacctcggcaagccgttcaacccggacgagctgatggcgcgggtcaaggccgtgctgcgccgccaggctcccagcgtgccgggtgcaccgggcagcgaggacgagtcggtcactttcggcgactacgagctgtcgctggccacccgcgagctcaagcgcggtgacgaagtgcacatgctcaccaccggtgaattcgccgtgctcaaggcgctggtgatgcatgcacgcgagccgttgacccgcgacaagctgatgagcctggcccgcggtcgcgaatgggatgccctggagcgctccatcgacgtacagatttcgcgcctgcgtcgcatgatcgagcctgatccgtccaagccacgctacatccagacggtctggggcgtgggttacgtgttcgttccggacggaaacgccggtaaatga SEQ ID NO.23 (5'-3') is as follows: atgtcgaaacgaaacacgccccaacgccgccacaacatcctggccctgctcagcgaacagggcgaggtcagtgtggacgccttggccaagcgcttcg aaacgtcggaagtcaccattcgcaaggacctcgccgcgctcgaggccaacggtctgttgctgcgtcgctatggcggcgcggtgcccgtgcctcagga aatgctgggcgagtctgcgcagccggtgtcggcttataaaaaagccatcgcccgcgccgccgtcggccgcatccgcgagcatgcacgcatcatcatc gacagcggcagcacgacggccgccatgatcccacagctagggcgccagccgggtctggtggtgatgaccaactcgctgaacgtggcgcgggccatca gcgaactcgagcacgagcccgtgctgttgatgacgggcggcacctgggacccgcactccgaatcgttccagggccaggtcgcggagcaggtactacg ctcctacgatttcgaccagctgttcattggcgccgatggcatcgacctcagccgcggcaccactaccttcaatgagttgctcggcttgagccgggtg atggccgaggtggcccgcgaagtgatcgtgatggtcgagtctgacaaggtcggtcgcaagatccccaacctcgagctgccctggggcagcgtgaata cccttattacagatgaacgcctccccgcagaggcgcgtgaacatattcaagcccgcggcatcaacctgatctgtgccgcgatcagccaggagcaataa 2) Construction of suicide vector and PCR validation After linearization by XbaI single enzyme digestion, the pDMK plasmid was combined with... melE Gene fragment 1 and melE Gene fragment 2 was ligated using a commercial recombinase (ABclonal 2×MultiF Seamless Assembly Mix, RK21020; the same applies below) at 37 ℃ for 30 min to obtain the recombinant plasmid pDMK-Δ. melE ; After linearization by XbaI single enzyme digestion, the pDMK plasmid was combined with... ompR Gene fragment 1 and ompR Gene fragment 2 was ligated using a commercial recombinase at 37 °C for 30 min to obtain the recombinant plasmid pDMK-Δ. ompR ; After linearization by XbaI single enzyme digestion, the pDMK plasmid was combined with... deoR Gene fragment 1 and deoR Gene fragment 2 was ligated using a commercial recombinase at 37 °C for 30 min to obtain the recombinant plasmid pDMK-Δ. deoR .

[0026] The above recombinant plasmid (recombinant plasmid pDMK-Δ) melE Recombinant plasmid pDMK-Δ ompR Recombinant plasmid pDMK-Δ deoR ) respectively converted into E. coli SM10 λpir competent cells were cultured overnight on LB plates containing 30 μg / mL chloramphenicol for selection.

[0027] Single colonies were picked and amplified by PCR using primers pDMK-F and pDMK-R. The correct suicide vector was verified by gel electrophoresis and sequencing.

[0028] 3) Conjugation transfer and reverse screening ①Recombinant strain ND2304Δ melE Preparation: The recombinant plasmid (recombinant plasmid pDMK-Δ) was conjugated separately. melE The cells were transferred into *Pseudomonas proteus* ND2304 at a conjugation ratio of 1:3 (recipient to donor volume ratio). After conjugation, the cells were washed with sterile PBS and spread onto TSB plates containing 100 μg / mL carbenicillin and 50 μg / mL kanamycin to obtain the recombinant plasmid pDMK-Δ. melE Inserted strain; The recombinant plasmid pDMK-Δ was used separately. melE The inserted strain was cultured in TSB liquid medium containing 100 μg / mL carbenicillin and 50 μg / mL kanamycin at 30 °C and 200 rpm for 12 hours. The cells were then collected by centrifugation (8000 × G, 5 min, 20 °C), washed twice with 1 mL sterile PBS, and resuspended. The resuspended bacterial solution was then inoculated into TSB liquid medium containing 5% sucrose and cultured at 30 °C for 3 h. Finally, the culture was plated on TSB semi-solid medium containing 5% sucrose and analyzed using pDMK. sacBThe sucrose lethality of the gene was used for reverse screening; the correctness of the deleted fragment was verified using mlaE-out-F and mlaE-out-R as verification primers, and the recombinant strain ND2304Δ was obtained after sequencing confirmation. melE Store at -80 ℃.

[0029] ②Recombinant strain ND2304Δ ompR Preparation: The recombinant plasmid (recombinant plasmid pDMK-Δ) was conjugated separately. ompR The cells were transferred into *Pseudomonas proteus* ND2304 at a conjugation ratio of 1:3 (recipient to donor volume ratio). After conjugation, the cells were washed with sterile PBS and spread onto TSB plates containing 100 μg / mL carbenicillin and 50 μg / mL kanamycin to obtain the recombinant plasmid pDMK-Δ. ompR Inserted strain; The recombinant plasmid pDMK-Δ was used separately. ompR The inserted strain was cultured in TSB liquid medium containing 100 μg / mL carbenicillin and 50 μg / mL kanamycin at 30 °C and 200 rpm for 12 hours. The cells were collected by centrifugation (8000 × G, 5 min, 20 °C), washed twice with 1 mL sterile PBS, and resuspended. The resuspended bacterial solution was then inoculated into TSB liquid medium containing 5% sucrose and cultured at 30 °C for 3 h. Finally, it was plated onto TSB semi-solid medium plates containing 5% sucrose and analyzed using pDMK. sacB The sucrose lethality of the gene was used for reverse screening; and the correctness of the deleted fragment was verified using ompR-out-F and ompR-out-R as verification primers. After confirmation by sequencing, the recombinant strain ND2304Δ was obtained. ompR Store at -80 ℃.

[0030] ③ Recombinant strain ND2304Δ deoR Preparation: The recombinant plasmid (recombinant plasmid pDMK-Δ) was conjugated separately. deoR The cells were transferred into *Pseudomonas proteus* ND2304 (Genetic Resource Disclosure Form) at a conjugation ratio of 1:3 (recipient to donor volume ratio). After conjugation, the cells were washed with 100 μL of sterile PBS and then plated onto TSB plates containing 100 μg / mL carbenicillin and 50 μg / mL kanamycin to obtain the recombinant plasmid pDMK-Δ. deoR Inserted strain; The recombinant plasmid pDMK-Δ was used separately. deoRThe inserted strain was cultured in TSB liquid medium containing 100 μg / mL carbenicillin and 50 μg / mL kanamycin at 30 °C and 200 rpm for 12 hours. The cells were collected by centrifugation (8000 × G, 5 min, 20 °C), washed twice with 1 mL sterile PBS, and resuspended. The resuspended bacterial solution was then inoculated into TSB liquid medium containing 5% sucrose and cultured at 30 °C for 3 h. Finally, it was plated onto TSB semi-solid medium plates containing 5% sucrose and analyzed using pDMK. sacB The sucrose lethality of the gene was used for reverse screening; and the correctness of the deleted fragment was verified using deoR-out-F and deoR-out-R as verification primers. After confirmation by sequencing, the recombinant strain ND2304Δ was obtained. deoR Store at -80 ℃.

[0031] ④ Recombinant strain ND2304Δ melE Δ ompR Preparation: The recombinant plasmid (recombinant plasmid pDMK-Δ) was conjugated separately. ompR Transformed into recombinant strain ND2304Δ melE In the conjugation, the ratio of recipient to donor was 1:3. After conjugation, the bacterial cells were washed with sterile PBS and then spread onto TSB plates containing 100 μg / mL carbenicillin and 50 μg / mL kanamycin to obtain the recombinant plasmid pDMK-Δ. melE and pDMK-Δ ompR Inserted strain; The recombinant plasmid pDMK-Δ was used separately. melE and pDMK-Δ ompR The inserted strain was cultured in TSB liquid medium containing 100 μg / mL carbenicillin and 50 μg / mL kanamycin at 30 °C and 200 rpm for 12 hours. The cells were collected by centrifugation (8000 × G, 5 min, 20 °C), washed twice with 2 mL of sterile PBS, and resuspended. The resuspended bacterial solution was then inoculated into TSB liquid medium containing 5% sucrose and cultured at 30 °C for 3 h. Finally, it was plated onto TSB semi-solid medium containing 5% sucrose and analyzed using pDMK. sacB The sucrose lethality of the gene was used for reverse screening; the correctness of the deleted fragment was verified using mlaE-out-F and mlaE-out-R, ompR-out-F and ompR-out-R as verification primers, and the recombinant strain ND2304Δ was obtained after sequencing confirmation. melE Δ ompR Store at -80 ℃.

[0032] ⑤ Recombinant strain ND2304Δ melE Δ deoRPreparation: The recombinant plasmid (recombinant plasmid pDMK-Δ) was conjugated separately. deoR Transformed into recombinant strain ND2304Δ melE In the conjugation, the ratio of recipient to donor was 1:3. After conjugation, the bacterial cells were washed with sterile PBS and then spread onto TSB plates containing 100 μg / mL carbenicillin and 50 μg / mL kanamycin to obtain the recombinant plasmid pDMK-Δ. melE and pDMK-Δ deoR Inserted strain; The recombinant plasmid pDMK-Δ was used separately. melE and pDMK-Δ deoR The inserted strain was cultured in TSB liquid medium containing 100 μg / mL carbenicillin and 50 μg / mL kanamycin at 30 °C and 200 rpm for 12 hours. The cells were collected by centrifugation (8000 × g, 5 min, 20 °C), washed twice with 2 mL of sterile PBS, and resuspended. The resuspended bacterial solution was then inoculated into TSB liquid medium containing 5% sucrose and cultured at 30 °C for 3 h. Finally, it was plated onto TSB semi-solid medium containing 5% sucrose and analyzed using pDMK. sacB The sucrose lethality of the gene was used for reverse screening; the correctness of the deleted fragment was verified using mlaE-out-F and mlaE-out-R, deoR-out-F and deoR-out-R as verification primers, and the recombinant strain ND2304Δ was obtained after sequencing confirmation. melE Δ ompR Store at -80 ℃.

[0033] ⑥ Recombinant strain ND2304Δ ompR Δ deoR Preparation: The recombinant plasmid (recombinant plasmid pDMK-Δ) was conjugated separately. deoR Transformed into recombinant strain ND2304Δ ompR In the conjugation, the ratio of recipient to donor was 1:3. After conjugation, the bacterial cells were washed with sterile PBS and then spread onto TSB plates containing 100 μg / mL carbenicillin and 50 μg / mL kanamycin to obtain the recombinant plasmid pDMK-Δ. ompR and pDMK-Δ deoR Inserted strain; The recombinant plasmid pDMK-Δ was used separately. ompR and pDMK-Δ deoRThe inserted strain was cultured in TSB liquid medium containing 100 μg / mL carbenicillin and 50 μg / mL kanamycin at 30 °C and 200 rpm for 12 hours. The cells were collected by centrifugation (8000 × G, 5 min, 20 °C), washed twice with 2 mL of sterile PBS, and resuspended. The resuspended bacterial solution was then inoculated into TSB liquid medium containing 5% sucrose and cultured at 30 °C for 3 h. Finally, it was plated onto TSB semi-solid medium containing 5% sucrose and analyzed using pDMK. sacB The sucrose lethality of the gene was used for reverse screening; and the correctness of the deleted fragment was verified using ompR-out-F and ompR-out-R, deoR-out-F and deoR-out-R as verification primers. After confirmation by sequencing, the recombinant strain ND2304Δ was obtained. ompR Δ deoR Store at -80 ℃.

[0034] ⑦ Recombinant strain LAV-1 (also known as: Pseudomonas proteus ND2304Δ) melE Δ ompR Δ deoR , Pseudomonas plecoglossicida ND2304Δ melE Δ ompR Δ deoR Preparation of recombinant plasmid (pDMK-Δ) by conjugation: deoR Transformed into recombinant strain ND2304Δ melE Δ ompR In the conjugation, the ratio of recipient to donor was 1:3. After conjugation, the bacterial cells were washed with sterile PBS and then spread onto TSB plates containing 100 μg / mL carbenicillin and 50 μg / mL kanamycin to obtain the recombinant plasmid pDMK-Δ. melE and pDMK-Δ ompR and pDMK-Δ deoR and inserted strains; The recombinant plasmid pDMK-Δ was used separately. melE and pDMK-Δ ompR and pDMK-Δ deoR The inserted strain was cultured in TSB liquid medium containing 100 μg / mL carbenicillin and 50 μg / mL kanamycin at 30 °C and 200 rpm for 12 hours. The cells were collected by centrifugation (8000 × G, 5 min, 20 °C), washed twice with 2 mL of sterile PBS, and resuspended. The resuspended bacterial solution was then inoculated into TSB liquid medium containing 5% sucrose and cultured at 30 °C for 3 h. Finally, it was plated onto TSB semi-solid medium plates containing 5% sucrose and analyzed using pDMK. sacBThe sucrose lethality of the gene was used for reverse screening; the correctness of the deleted fragment was verified using mlaE-out-F and mlaE-out-R, ompR-out-F and ompR-out-R, and deoR-out-F and deoR-out-R as verification primers. After confirmation by sequencing, the recombinant strain LAV-1 was obtained and stored at -80 ℃.

[0035] Example 2 This embodiment provides a method for culturing the gene-deleted attenuated mutant strain of Pseudomonas proteus wild-type obtained in Example 1, as follows: 1) Composition of culture medium and PBS TSB medium: tryptone 17 g / L, soybean papain hydrolysate 3 g / L, sodium chloride 5 g / L, dipotassium hydrogen phosphate 2.5 g / L, glucose 2.5 g / L, pH 7.4; PBS (pH 7.4): NaCl 8 g / L, KCl 0.2 g / L, Na2HPO4 1.44 g / L, KH2PO4 0.24 g / L, sterilized at 121℃ for 20 min.

[0036] 2) Cultivation A small amount of ice residue containing the gene-deleted attenuated mutant strain of Pseudomonas proteus wild-type virus prepared in Example 1 was taken out from the -80 ℃ storage tube and immediately streaked on TSB solid plates to isolate and revive the strain. After being cultured at 30 ℃ for 24 h, single colonies of the corresponding strains were obtained. Single colonies of the corresponding strains were inoculated into 5 mL of TSB liquid medium and cultured at 30 ℃ and 200 rpm with shaking.

[0037] Example 3 This embodiment provides an immunoprotection test and a challenge experiment on the gene-deleted attenuated mutant strain of Pseudomonas proteus prepared in Example 1, as detailed below: 1) Laboratory animals and grouping Select large yellow croakers weighing 100 ± 10 g. Larimichthys Crocea Before the experiment, the fish were acclimatized under SPF conditions for 7 days, and abnormal individuals were removed to serve as test fish.

[0038] 2) Dilution of bacterial culture The bacterial culture of the corresponding strains after Example 2 was centrifuged and the bacterial cells were collected. The cells were then washed three times with sterile PBS (pH 7.4) and resuspended to prepare 10% concentrations of [missing information]. 6 Prepare the corresponding bacterial solution at CFU / mL and dispense it into 10 sterile 1 mL syringes (if dilution is required, dilute with sterile PBS).

[0039] Among them, 1×10 3 CFU is the injected bacterial load, 1×10⁻⁶. 4 CFU / mL is the concentration of the soaking bacterial solution. The bacterial solution is serially diluted and then dropped onto a plate for viable cell counting.

[0040] 3) Infection dosage and method The experimental fish were randomly divided into groups of 75 each, as follows: ① Inject recombinant strain ND2304Δ melE Δ melE Group: Each fish was injected intraperitoneally with recombinant strain ND2304Δ melE Bacterial solution, 1×10 3 CFU / tail; ② Inject recombinant strain ND2304Δ ompR Δ ompR Group: Each fish was injected intraperitoneally with recombinant strain ND2304Δ ompR Bacterial solution, 1×10 3 CFU / tail; ③ Inject recombinant strain ND2304Δ deoR Δ deoR Group: Each fish was injected intraperitoneally with recombinant strain ND2304Δ deoR Bacterial solution, 1×10 3 CFU / tail; ④ Inject recombinant strain ND2304Δ melE Δ ompR Δ melE Δ ompR Group: Each fish was injected intraperitoneally with recombinant strain ND2304Δ melE Δ ompR Bacterial solution, 1×10 3 CFU / tail; ⑤ Inject recombinant strain ND2304Δ melE Δ deoR Δ melE Δ deoR Group: Each fish was injected intraperitoneally with recombinant strain ND2304Δ melE Δ deoR Bacterial solution, 1×10 3 CFU / tail; ⑥ Inject recombinant strain ND2304Δ ompR Δ deoR Δ ompR Δ deoR Group: Each fish was injected intraperitoneally with recombinant strain ND2304Δ melE Δ deoR Bacterial solution, 1×10 3 CFU / tail; ⑦ Inject recombinant strain LAV-1 Δ melE Δ ompR Δ deoR Group: Each fish was injected intraperitoneally with 1×10⁻⁶ recombinant strain LAV-1 bacterial suspension. 3 CFU / tail; ⑧ WT group injected with wild-type Pseudomonas proteus ND2304: Each animal was injected intraperitoneally with 1×10⁻⁶ wild-type Pseudomonas proteus ND2304 bacterial suspension. 3 CFU / tail; ⑨ Recombinant strain LAV-1 immersion group (Imm group): The test fish were immersed in 200 L of a solution with a concentration of 1×10 4 Immerse in a CFU / mL recombinant strain LAV-1 bacterial solution for 15 min, then rinse with seawater (East China Sea); ⑩ Immunocontrol group: Injected with 100 μL PBS (pH 7.4); Wait 28 days for the fish to complete the immunization, and record the mortality rate at the same time (see Table 2).

[0041] Table 2 Mortality rates during the immunization phase in each group 4) Intraperitoneal injection immunization Step 3) Four weeks after immunization, use Pseudomonas proteus ND2304 wild-type strain (10 3 CFU / tail (the pathogen causing white spot disease in the viscera of large yellow croaker) was used to challenge the fish; the challenge was also performed by intraperitoneal injection. The mortality rate of each group of experimental fish was recorded within 21 days and the relative protection rate was calculated. The results are shown in Table 3.

[0042] Table 3 Mortality and relative protection rate of each group during the challenge phase Note: In Table 3, "-" indicates that no experiment was conducted.

[0043] Results and Analysis: At the same infectious dose, the recombinant strain LAV-1 showed a significantly reduced lethal effect on large yellow croaker after intraperitoneal injection, and its virulence was significantly lower than that of the wild strain. Challenge experiments 4 weeks after immunization showed that it had significantly greater anti-infective ability than the challenge control group, indicating that this multi-gene deletion strain (recombinant strain LAV-1) can serve as a safe and effective vaccine candidate for visceral white spot disease in large yellow croaker.

[0044] In addition, after 20 consecutive passages of the recombinant strain LAV-1, no virulence-related gene reversion or vector fragment reorganization was detected, indicating that the attenuated phenotype has genetic stability and is suitable for subsequent vaccine development and industrial application.

[0045] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the interpretation of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A gene-deleted attenuated mutant strain of wild-type *Pseudomonas proteus*, characterized in that, This gene-deleted attenuated mutant strain is based on the deletion of the gene encoding a protein involved in maintaining cell membrane lipid asymmetry in wild-type Pseudomonas proteus strains. mlaE envZ / ompR two-component system response regulatory protein encoding genes ompR and DeoR family transcription factor encoding genes deoR The resulting mutant strain; Among them, encoding mlaE The nucleotide sequence is shown in SEQ ID NO.21, encoding the gene. omp The nucleotide sequence is shown in SEQ ID NO.22, encoding the gene. deoR The nucleotide sequence is shown in SEQ ID NO.

23.

2. A method for constructing a gene-deleted attenuated mutant of the wild-type *Pseudomonas proteus* strain as described in claim 1, characterized in that, Includes the following steps: (S1) Genes mlaE ,Gene mlaE and genes mlaE Recombinant plasmids Δ were obtained by inserting them into plasmid vectors. mlaE Recombinant plasmid Δ ompR Recombinant plasmid Δ deoR ; (S2) The recombinant plasmid Δ obtained in step (S1) mlaE The recombinant strain ND2304Δ was obtained by conjugation into *Pseudomonas proteus* ND2304. mlaE ; (S3) The recombinant strain ND2304Δ obtained in step (S2) mlaE Based on this, recombinant plasmid Δ was introduced via conjugation. ompR Recombinant plasmid Δ deoR The recombinant strain LAV-1 was obtained, which is the gene-deleted attenuated mutant strain of the wild-type Pseudomonas proteus strain.

3. The method for preparing a gene-deleted attenuated mutant strain of *Pseudomonas proteus* wild-type strain according to claim 2, characterized in that, During the conjugation process, the volume ratio of donor to recipient is 1:2~3.

4. A method for culturing a gene-deleted attenuated mutant of the wild-type *Pseudomonas proteus* strain as described in claim 1, characterized in that, Includes the following steps: After activation and resuscitation, a gene-deleted attenuated mutant of wild-type Pseudomonas proteus was inoculated into TSB culture medium.

5. The method for culturing a gene-deleted attenuated mutant of a wild-type *Pseudomonas proteus* strain according to claim 4, characterized in that, During the cultivation process, the temperature was 28~37 ℃, the rotation speed was 180~220 rpm, and the time was 6~24 h.

6. The use of a gene-deleted attenuated mutant strain of wild-type Pseudomonas proteus as described in claim 1 in the preparation of a vaccine for the prevention and / or treatment of visceral white spot disease in farmed fish.

7. A vaccine for the prevention and / or treatment of visceral white spot disease in farmed fish, characterized in that, A gene-deleted attenuated mutant strain of the wild-type Pseudomonas proteus strain as described in claim 1.

8. A vaccine for the prevention and / or treatment of visceral white spot disease in farmed fish according to claim 7, characterized in that, The visceral white spot disease vaccine contains a pharmaceutically acceptable carrier.

9. A vaccine for the prevention and / or treatment of visceral white spot disease in farmed fish according to claim 8, characterized in that, The carrier is selected from the following: solvent, dispersion medium, coating agent, antibacterial agent, antifungal agent, isotonic agent, absorption delay agent, preservative, sweetener for oral administration, thickener, buffer, liquid carrier, wetting agent, solubilizer, or emulsifier; acidifier, antioxidant, alkalizing agent, carrier, chelating agent, colorant, complexing agent, suspending agent or thickener, flavoring agent or fragrance, oil, penetration enhancer, polymer, hardening agent, protein, carbohydrate, filler, and lubricant.

10. A vaccine for the prevention and / or treatment of visceral white spot disease in farmed fish according to claim 7, characterized in that, The application dose of the gene-deleted attenuated mutant strain of *Pseudomonas proteus* in the vaccine is 10. 2 ~10 3 CFU / tail.

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