DeR gene knockout pseudomonas mutans attenuated vaccine

Abstract

The invention belongs to the technical field of biology, and particularly relates to a pseudomonas proteinans attenuated vaccine with a deoR gene knocked out. The pseudomonas deformation attenuated vaccine comprises a pseudomonas deformation deoR gene knockout strain. The pseudomonas deformation deoR gene knockout strain comprises pseudomonas deformation deoR gene knockout strain, the pseudomonas deformation strain deoR gene knockout strain is obtained after a deoR gene of a pseudomonas deformation strain wild type strain is knocked out, and the nucleotide sequence of the deoR gene is as shown in SEQ ID NO. 1. The virulence of the pseudomonas deformation deoR gene knockout strain is obviously weakened compared with that of a pseudomonas deformation wild strain, the pseudomonas deformation deoR gene knockout strain can effectively protect experimental pseudosciaena crocea from being infected by the pseudomonas deformation, has a good immune protection effect on the pseudosciaena crocea, further has a good prevention effect on the visceral white-spot disease of the pseudosciaena crocea, and is high in commercial development and application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an attenuated Pseudomonas aeruginosa vaccine with a deoR gene knocked out. Background Art

[0002] Large yellow croaker (Larimichthyscrocea) is an important marine economic fish in my country. According to the "China Fisheries Statistical Yearbook 2023", the total output of large yellow croaker in my country in 2021 and 2022 was 254,000 and 257,000 tons respectively, which has been ranked first among my country's marine farmed fish for many years. With the surge in aquaculture production, the continuous increase in the degree of intensiveness and the deterioration of the aquaculture environment, aquaculture diseases are becoming increasingly serious. Among them, visceral white spot disease caused by Pseudomonas plecoglossicida is a common disease of large yellow croaker cultured in cages in recent years, causing serious economic losses to the large yellow croaker aquaculture industry.

[0003] At present, there is no effective treatment for white spot disease in the internal organs of large yellow croaker. Farming practitioners mostly prevent the occurrence of diseases by feeding large yellow croaker with antibiotics such as enrofloxacin before wintering. The advantages of these drugs are mainly that they are easy to use, fast-acting and low-cost, but the environmental pollution problems, food and drug residue problems and the development of bacterial resistance they bring cannot be ignored. With people's demands for green and environmentally friendly food and environmental protection, seeking safe, green, economical and effective ways to prevent and control aquatic diseases has received more and more attention. Preventing and controlling diseases by developing aquatic pathogen vaccines is widely regarded as a green, healthy, safe and effective feasible way. As of the end of 2021, 9 fish vaccines in my country have obtained new veterinary drug certificates.

[0004] Therefore, the present invention has carried out the research and development of a live attenuated vaccine for Pseudomonas aeruginosa. Compared with wild pathogens, the virulence of the live attenuated vaccine is significantly weakened, but it still retains a certain host invasiveness. Compared with inactivated vaccines, this type of vaccine retains better immunogenicity, can reproduce within a certain limit in the host body, can effectively stimulate cellular immunity, and has a longer immune duration, showing a relatively good relative immune protection rate.

[0005] By using allele knockout technology, the present invention constructs multiple Pseudomonas proteus gene knockout strains, and screens a knockout strain with obvious attenuation activity through live infection experiments with large yellow croaker. After the attenuated strain was intraperitoneally injected to immunize large yellow croaker, ELISA detected that the anti-Pseudomonas proteus specific antibodies in the serum of large yellow croaker after immunization began to increase from the second week and reached a peak in the fifth week. In addition, the absolute fluorescence quantitative PCR method detected that the pathogen content in the spleen tissue of large yellow croaker in the immunized group after the virus attack was significantly lower than that in the control group, indicating that the vaccine can effectively reduce the proliferation of pathogens. The virus attack experiment of the attenuated vaccine group has a higher relative immune protection rate (RPS). Based on the above data, the attenuated strain can induce an efficient immune response in large yellow croaker and has the potential to be developed into a live attenuated vaccine. Summary of the invention

[0006] The purpose of the present invention is to provide an attenuated vaccine of Pseudomonas aeruginosa with a deoR gene knocked out.

[0007] To achieve the above object, the present invention adopts the following technical solution: A method for constructing a Pseudomonas aeruginosa deoR gene knockout strain comprises the following steps: S1: Linearize the pRE112 suicide plasmid using the PCR method to obtain the pRE112 plasmid linearized fragment; use the genome of the wild-type strain of Pseudomonas proteus as a template to amplify the upstream homology arm fragment of the deoR gene and the downstream homology arm fragment of the deoR gene by PCR; connect the upstream homology arm fragment of the deoR gene, the downstream homology arm fragment of the deoR gene, and the pRE112 plasmid linearized fragment using a commercial seamless cloning kit to obtain the recombinant plasmid pRE112-△deoR; S2: Transform the recombinant plasmid pRE112-△deoR into E. coli S17-1λpir to obtain the conjugative donor bacteria; S3: Conjugation transfer between the conjugation donor bacteria and the wild-type strain of Pseudomonas proteus; S4: The deoR gene knockout strain of Pseudomonas proteus was obtained by screening and identifying single-exchange clones and double-exchange clones; the nucleotide sequence of the primer pair used to linearize the pRE112 suicide plasmid was: pRE112-F: 5'-TAATACGACTCACTATAGGG-3', pRE112-R: 5'-TGCTAGTTATTGCTCAGCGG-3'; the nucleotide sequence of the primer pair used to amplify the upstream homology arm fragment of the deoR gene was: Up-F: 5'-tgattacgaattcgagctcgGCTTGTC GGCGAACAGCTCGG-3', Up-R: 5'-TGGCAAGGCAGAATAACTCCtcatgtgtggaatcgttggt-3'; the nucleotide sequences of the primer pair used to amplify the downstream homology arm fragment of the deoR gene were: Down-F: 5'-GGAGTTATTCTGCCTTGCCAtggcgcttaagtttcgattc-3', Down-R: 5'-taaaacgacggccagtgccaCCGATGGCCTGGTGGTCGCGAC-3'; The nucleotide sequence of the upstream homology arm fragment of the deoR gene is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream homology arm fragment of the deoR gene is shown in SEQ ID NO.3; Among them, the screening of single exchange clones specifically refers to screening through a plate containing 5 μg / mL tetracycline, and the identification of single exchange clones specifically refers to PCR identification through primer pair Up-F / F and Check-F2 / R2; the screening of double exchange clones specifically refers to screening through a plate containing 15wt% sucrose, and the identification of double exchange clones specifically refers to PCR identification through primer pair Up-F / F and Check-F3 / R3; the nucleotide sequence of primer pair Up-F / F is: Up-F: 5'-tgattacgaattcgagctcgGCTTGTCGGCGAACAGCTCGG-3', Down-R: 5'-taa aacgacggccagtgccaCCGATGGCCTGGTGGTCGCGAC-3'; the nucleotide sequence of the primer pair Check-F2 / R2 is: Check-F2: 5'-ACGTTCAGCGAGTTGGTCAT-3', Check-R2: 5'-TCGGAAGTCACCATTCGCAA-3'; the nucleotide sequence of the primer pair Check-F3 / R3 is: Check-F3: 5'-GTCATAGCCGCGGTACTCAA-3', Check-R3: 5'-TCGGAAGTCACCATTCGCAA-3'.

[0008] A deoR gene knockout strain of Pseudomonas proteus is constructed by the above construction method.

[0009] Use of the above-mentioned Pseudomonas proteus deoR gene knockout strain in the preparation of drugs for preventing and treating Pseudomonas proteus infections in aquatic animal breeding; Furthermore, the disease is large yellow croaker visceral white spot disease, and the medicine is a vaccine.

[0010] A Pseudomonas aeruginosa attenuated vaccine, the vaccine comprises the above-mentioned Pseudomonas aeruginosa deoR gene knockout strain; further, the vaccine dosage forms include injection vaccine and immersion vaccine.

[0011] The invention discloses an application of knocking out the deoR gene in reducing the pathogenicity of Pseudomonas aeruginosa and preparing an attenuated vaccine of Pseudomonas aeruginosa, characterized in that the nucleotide sequence of the deoR gene is shown in SEQ ID NO.1.

[0012] The significant advantages of the present invention are: The Pseudomonas proteans deoR gene knockout strain of the present invention has significantly lower toxicity than the wild strain of Pseudomonas proteans, and can effectively protect experimental large yellow croaker from infection by Pseudomonas proteans. Subsequent experiments show that the Pseudomonas proteans deoR gene knockout strain has a good immune protection effect on large yellow croaker, and has a good preventive effect on large yellow croaker visceral white spot disease, which is specifically manifested in that the anti-PQLYC4 specific antibody IgM level in the serum of large yellow croaker is significantly higher than that of the control group, the expression of immune-related genes is significantly increased compared with the control group, and there is a higher relative immune protection rate after the virus attack, and the bacterial load of the target tissue is significantly lower than that of the control group. It also has technical environmental and product safety, and has practical commercial development and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : Growth curves of Pseudomonas proteus PQLYC4 and Pseudomonas proteus deoR gene knockout strain ΔdeoR.

[0014] Figure 2 :Effects of Pseudomonas proteus deoR knockout strain ΔdeoR on fish growth. A: injection immunization; B: immersion immunization.

[0015] Figure 3 :Survival curve of yellow croaker challenged with poison on day 42 after the first immunization. A:Injected with 5×10 4 CFU / mL immunization 42 days after infection survival curve; B: injection of 1×10 5 CFU / mL immunization 42 days after infection survival curve; C: injection of 5×10 5 CFU / mL immunization 42 days after infection survival curve; D: soaking 1×10 6CFU / mL immunization 42 days after infection survival curve; E: soaking 1×10 7 CFU / mL immunization 42 days after infection survival curve; F: soaking 1×10 8 CFU / mL survival curve of infection on the 42nd day after immunization.

[0016] Figure 4 : Changes in IgM antibodies (AB) and relative protection rates (RPS) (CD) for injection and immersion vaccinations from 14 to 150 days after primary immunization.

[0017] Figure 5 : The number of Pseudomonas plecoglossicida in the head kidney, spleen, liver and kidney of each immunization group on the 42nd day after the first immunization and the 5th day after the virus challenge. A: injection immunization; B: immersion immunization.

[0018] Figure 6 : Relative expression changes of immune-related genes in spleen and head kidney 24 hours after challenge on day 42 after primary immunization. A: Injection of 5×10 4 CFU immunization, challenge on day 42; B: injection of 1×10 5 CFU immunization, challenge on day 42; C: injection of 5×10 5 CFU immunization, challenge on day 42; D: soaking 1×10 6 CFU immunization 42 days after infection; E: soak 1×10 7 CFU immunization 42 days after infection; F: soak 1×10 8 CFU immunization was carried out on the 42nd day.

[0019] Figure 7 : PCR verification of the deoR knockout strain ΔdeoR of Pseudomonas aeruginosa. A: Primer pair Up-F / Down-R verifies double-crossover clones; B: Primer pair Check-F3 / R3 verifies double-crossover clones. DETAILED DESCRIPTION

[0020] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0021] The pRE112 suicide plasmid used in the following examples was purchased from Shanghai Huzhenye Co., Ltd., with the product number HZ0122.

[0022] The holder of Pseudomonas plecoglossicida PQLYC4 used in the following examples is the College of Oceanography of Fujian Agriculture and Forestry University. The biological material has been disclosed in non-patent literature. The source of the literature is: Wei Jinping, Li Yunfeng, Qiu Wei, et al. Expression, purification and preparation of polyclonal antibodies of Pseudomonas plecoglossicida ExsA protein [J]. Journal of Agricultural Biotechnology, 2023, 31(4): 883-888. The College of Oceanography of Fujian Agriculture and Forestry University has guaranteed that within 20 years from the application date of the invention patent "A Pseudomonas plecoglossicida attenuated vaccine with deoR gene knocked out" with Fujian Agriculture and Forestry University as the applicant, the biological material Pseudomonas plecoglossicida PQLYC4 will be distributed to the public.

[0023] The commercial seamless cloning kit used in the following examples was purchased from Beijing Quanshijin Biotechnology Co., Ltd., with the item number CU201.

[0024] The formula of the LB solid culture medium used in the following examples is: 10 g of peptone, 5 g of yeast powder, 20 g of agar and 10 g of NaCl, and distilled water is added to 1000 mL; the pH value is 7.0-7.2.

[0025] The formula of the LB liquid culture medium used in the following examples is: 10 g of peptone, 5 g of yeast powder and 10 g of NaCl, and distilled water is added to 1000 mL; the pH value is 7.0-7.2.

[0026] The formula composition of the TSB liquid culture medium used in the following examples is (by weight percentage): 17 g of trypticase peptone, 3 g of soy peptone, 2.5 g of glucose, 5 g of NaCl and 2.5 g of K2HPO4, and the balance is distilled water, which is added to 1000 mL; the pH value is 7.3-7.5.

[0027] The formula of the TSA solid culture medium used in the following examples is: 15 g of tryptone, 5 g of soy peptone, 5 g of NaCl and 15 g of agar, and distilled water is added to 1000 mL; the pH value is 7.3±0.2.

[0028] The nucleotide sequence of the deoR gene described in the following examples is shown in SEQ ID NO.1.

[0029] Embodiment 1: The construction of the deoR gene knockout strain of Pseudomonas aeruginosa is as follows: S1: Using suicide plasmid pRE112 as template, PCR technology was used to obtain the linearized fragment of pRE112 plasmid based on primer pair pRE112-F / R, and the gel was cut and recovered after 1.2% agarose electrophoresis detection. The recovered product was stored at -20℃ for later use. Among them, the PCR amplification system was: 2×Pfu PCR Mix 25μL, upstream primer 0.4μmol, downstream primer 0.4μmol, template 1ng, and H2O was added to 50μL. Among them, the PCR amplification program was: 95℃5min; 95℃30s, 55℃30s, 72℃2min, 35 cycles; 72℃10min, stored at 4℃.

[0030] pRE112-F:5'-TAATACGACTCACTATAGGG-3' pRE112-R:5'-TGCTAGTTATTGCTCAGCGG-3' S2: Using the genomic DNA of Pseudomonas proteus PQLYC4 as a template, the upstream homology arm fragment of the deoR gene (SEQ ID NO.2) was obtained by PCR technology based on the primer pair Up-F / R, and the downstream homology arm fragment of the deoR gene (SEQ ID NO.3) was obtained by PCR technology based on the primer pair Down-F / R. After 1.2% agarose electrophoresis detection, the gel was cut and recovered, and the recovered products were stored at -20°C for standby use. Among them, the PCR amplification system is: 2×Pfu PCR Mix 25μL, upstream primer 0.4μmol, downstream primer 0.4μmol, template 1ng, and H2O was added to 50μL. Among them, the PCR amplification program is: 95℃5min; 95℃30s, 55℃30s, 72℃1min, 35 cycles; 72℃10min, 4℃ storage.

[0031] Up-F: 5'-tgattacgaattcgagctcgGCTTGTCGGCGAACAGCTCGG-3' Up-R: 5'-TGGCAAGGCAGAATAACTCCtcatgtgtggaatcgttggt-3' Down-F: 5'-GGAGTTATTCTGCCTTGCCAtggcgcttaagtttcgattc-3' Down-R: 5'-taaaacgacggccagtgccaCCGATGGCCTGGTGGTCGCGAC-3' S3: According to the instructions of the commercial seamless cloning kit, the upstream homology arm fragment of the deoR gene obtained in step S2 and the downstream homology arm of the deoR gene were connected with the linearized fragment of the pRE112 plasmid obtained in step S1 to obtain the recombinant plasmid pRE112-ΔdeoR. The seamless cloning system is: 2×Assembly Basic Mix 5μL, pRE112 plasmid linearized fragment 0.1pmol, deoR gene upstream homology arm fragment 0.2pmol, deoR gene downstream homology arm fragment 0.22pmol, add water to make up to 10μL. The seamless cloning procedure is: 50℃15min.

[0032] S4: The recombinant plasmid pRE112-ΔdeoR was conventionally heat-shock transformed into Escherichia coli S17-1λpir competent cells, revived at 37°C and 220rpm for 1h, and then centrifuged at 4000rpm for 2min. 100μl of the supernatant was retained to resuspend the bacteria, and spread on LB solid culture medium plates containing 5μg / mL tetracycline. The cells were allowed to stand for a period of time until the bacterial solution was absorbed, and then inverted and cultured at 37°C for 16h. Then, colony PCR was used to verify the positive clones based on the primer pair Check-F1 / R1, that is, an electrophoresis band appeared at 2000bp, proving that the recombinant plasmid pRE112-ΔdeoR was successfully transferred into Escherichia coli S17-1λpir and used as the conjugation donor bacteria.

[0033] Check-F1:5'-GTCATAGCCGCGGTACTCAA-3' Check-R1: 5'-GCTGGTGCTTTATGGCGATG-3' S5: The conjugation donor bacteria were inoculated into LB liquid medium containing 5 μg / mL tetracycline, cultured at 37°C overnight, and then transferred to a new LB liquid medium containing 5 μg / mL tetracycline at an inoculum volume of 1% (v / v), and cultured at 37°C until OD 600 The value was 0.6, and 100 mL of cells were collected by centrifugation. The cells were washed three times with LB liquid medium to obtain the cells of the strain for conjugation and set aside. Pseudomonas proteus PQLYC4 was inoculated into TSB liquid medium and cultured at 25°C overnight. Then, it was transferred to a new TSB liquid medium at an inoculum volume of 1% (v / v) and cultured at 25°C until OD 600The value was 0.6, 100 mL of cells were collected by centrifugation, the cells were washed 3 times with LB liquid medium, and then resuspended with 1 ml LB liquid medium to obtain the recipient bacterial resuspension. The obtained conjugation strain cells and the recipient bacterial resuspension were fully mixed, centrifuged at 4000 rpm for 2 min, 50 μl of supernatant was retained to resuspend the cells, and spread on the non-antibiotic LB solid medium plate, let it stand for a while until the bacterial solution was absorbed, and inverted culture at 25 ° C for 24 h to grow bacterial lawns.

[0034] S6: Use TSB liquid culture medium to wash and recover the bacterial moss on the non-antibiotic LB solid culture medium plate, dilute to an appropriate concentration and then spread on a TSA solid culture medium plate containing 5 μg / mL tetracycline, and culture inverted at 25°C for 24 to 48 hours until a monoclonal colony grows on the plate. Use primer pair Up-F / Down-R and Pseudomonas aeruginosa-specific primer pair Check-F2 / R2 to perform colony PCR to detect single exchange clones. If a band of 500 bp in length is amplified using the primer pair Up-F / Down-R and a band of 500 bp in length is amplified using the primer pair Check-F2 / R2, it is confirmed to be a single exchange clone.

[0035] Up-F: 5'-tgattacgaattcgagctcgGCTTGTCGGCGAACAGCTCGG-3' Down-R: 5'-taaaacgacggccagtgccaCCGATGGCCTGGTGGTCGCGAC-3' Check-F2: 5'-ACGTTCAGGCGAGTTGGTCAT-3' Check-R2: 5'-TCGGAAGTCACCATTCGCAA-3' S7: The single exchange clone was inoculated into TSB liquid medium containing 15wt% sucrose, cultured at 25°C overnight, and then diluted to a suitable concentration and spread onto a TSA solid medium plate containing 15wt% sucrose. The plate was inverted and cultured at 25°C for 24h until a single clone colony grew on the plate. The double exchange clone was detected by colony PCR using primer pair Up-F / Down-R and knockout identification primer pair Check-F3 / R3. If a band of 1000bp in length was amplified using primer pair Up-F / Down-R and no band was amplified using primer pair Check-F3 / R3, it was confirmed to be a double exchange clone, i.e., the Pseudomonas aeruginosa deoR gene knockout strain (ΔdeoR) in which the deoR gene was knocked out ( Figure 7 ).

[0036] Check-F3: 5'-GTCATAGCCGCGGTACTCAA-3' Check-R3: 5'-TCGGAAGTCACCATTCGCAA-3' Embodiment 2: Take equal amounts (OD 600 =0.6) of Pseudomonas proteus PQLYC4 and ΔdeoR were inoculated into TSB liquid medium at an inoculum volume of 1% (v / v), and cultured in a 25°C incubator for 30 h with shaking. The OD was measured every 1 h. 600 There were 3 replicates in each group, and the average value was taken to make a growth curve.

[0037] like Figure 1 As shown, the growth rate of ΔdeoR was not significantly different from that of Pseudomonas aeruginosa PQLYC4 (P>0.05).

[0038] Embodiment 3: Before the experiment, the yellow croaker was temporarily kept in a 10T circulating water aquaculture pond of the Mindong Fisheries Research Institute in Ningde City, with 200 yellow croakers in each aquaculture pond. The yellow croaker was domesticated for 1 week and routinely fed at a ratio of 3% of the fish weight, once in the morning and once in the evening; the swimming state of the yellow croaker was observed, and daily sampling (necropsy and PCR detection methods) was performed to ensure that the experimental fish were negative for Pseudomonas proteus. The heavy water temperature of the circulating water system was controlled at 18-20°C, and 20% of filtered seawater was replaced every morning. The concentration of Pseudomonas proteus PQLYC4 and ΔdeoR bacterial solutions was diluted to 1×10 7 CFU / mL, 1×10 6 CFU / mL, 1×10 5 CFU / mL, 1×10 4 CFU / mL, 1×10 3 CFU / mL and 1×10 2 CFU / mL. 50 large yellow croakers were intraperitoneally injected with 200 μL of bacterial solution per tail, and the control group was injected with an equal volume of sterile 1× PBS. The infected water temperature was controlled at 18°C. The large yellow croakers were then cultured in a 500L circulating culture pond and observed for 14 consecutive days. The mortality and pathological symptoms of each group were recorded, and the LD of Pseudomonas proteus PQLYC4 and ΔdeoR were calculated. 50 .

[0039] As shown in Table 1 , the virulence of ΔdeoR was significantly lower than that of P. deformans PQLYC4.

[0040] Table 1 Comparison of virulence between wild type and vaccine strains Embodiment 4: The survival and growth of large yellow croaker inoculated with ΔdeoR and sterile 1×PBS were studied. Both intraperitoneal injection and immersion inoculation were used for immunization. The final concentration of injection immunization was 5×10 4 CFU / mL, 1×10 5 CFU / mL and 5×10 5 CFU / mL, and the injection dose was 1×10 4 CFU / tail, 5×10 4 CFU / tail, 1×10 5 CFU / tail, final concentration for immersion immunity is 1×10 6 CFU / mL, 1×10 7 CFU / mL and 1×10 8 CFU / mL, immersion time was 30min. During the 150-day experimental period, the growth and survival of large yellow croaker inoculated with ΔdeoR were recorded, and large yellow croakers were regularly sampled to observe whether the tissues (head kidney, spleen, liver, kidney) had symptoms. After the end of the experiment, the remaining surviving large yellow croakers were dissected again to observe the symptoms. Ten large yellow croakers were randomly selected from the ΔdeoR immune group and the 1×PBS control group at 0d, 28d, 56d, 84d, 112d, and 140d after the initial inoculation and weighed.

[0041] The results showed that during the experimental period, no large yellow croaker inoculated with ΔdeoR or sterile 1×PBS died of visceral white spot disease, no typical symptoms were found in autopsy, and the clinical manifestations were normal. Figure 2 As shown, there was no significant difference in body weight between large yellow croaker inoculated with ΔdeoR and that in the control group (P>0.05).

[0042] Embodiment 5: Immunization by injection: Large yellow croaker was randomly divided into 4 groups (group A with an immunization dose of 5×10 4 CFU / mL, immune dose for group B: 1×10 5 CFU / mL, immune dose for group C: 5×10 5 CFU / mL, blank group was 1×PBS negative control group). After the fish were anesthetized with an appropriate amount of eugenol, ΔdeoR was injected intraperitoneally. After each group was immunized, the large yellow croaker was quickly transferred to a breeding bucket. No feeding was performed before and after the injection and on the day of immunization. A second booster immunization was performed in the same way on the 14th day after the initial immunization.

[0043] Immersion vaccination: Large yellow croaker was randomly divided into 4 groups (the final immune concentration in group D was 1×10 6 CFU / mL, final concentration of group E was 1×10 7CFU / mL, final concentration of group F was 1×10 8 CFU / mL, sterile seawater negative control group), 200 fish in each group, 200 μL per fish, the immunized group was aerated and soaked for 60 min in the corresponding ΔdeoR bacterial solution concentration for immersion infection, and the control group was soaked in sterile seawater for 60 min. After soaking for 60 min, the yellow croaker was quickly transferred to the seawater used for fish farming and soaked for 5 min, and washed 3 times in total. The yellow croakers in the immunized group and the control group were returned to the original breeding barrels, and no feeding was performed before and after the soaking immunization and on the day of immunization. A second booster immunization was performed in the same way on the 14th day after the soaking immunization.

[0044] On the 42nd day after the initial immunization, 50 yellow croakers from each immunization group were immersed in Pseudomonas proteus PQLYC4 to challenge the yellow croakers in the immunization group and the control group to investigate the immune protection of the vaccine strain. 4 CFU / mL of Pseudomonas proteus PQLYC4 bacterial solution was soaked for 60 minutes, then washed three times with aquaculture seawater and placed in aquaculture buckets (the water flow must be gentle during the washing process), observed for 14 days, and the activity, morbidity and mortality of the fish were recorded, and the relative immune protection rate was calculated statistically. The incidence and mortality of large yellow croaker in the injection vaccination, immersion vaccination and control groups within 14 days after the virus attack were counted. The relative protection rate was calculated according to the following formula = [l-(mortality rate of large yellow croaker in the immunization group % / mortality rate of large yellow croaker in the control group %)] × 100%.

[0045] The results are as follows Figure 3 , Table 2, Table 3. As can be seen from the table, after inoculation with ΔdeoR, the tolerance level of large yellow croaker to wild strain infection was significantly improved, proving that ΔdeoR vaccination has a good immune protection effect on large yellow croaker.

[0046] Table 2 Immune protection after challenge on day 42 after injection immunization Table 3 Immune protection after challenge on day 42 after immersion immunization Embodiment 6: Serum sampling was performed on large yellow croaker. The serum sampling time points were 0d, 14d, 28d, 42d, 60d, 90d, 120d, and 150d after the initial immunization in Example 5. Fifteen large yellow croakers were randomly selected from the immunization group and the 1×PBS control group, and blood was collected from the tail vein. After standing at room temperature for 2h, the blood was centrifuged at 4°C and 3000rpm for 15min, and the supernatant was taken. The changes in serum antibody levels in large yellow croakers after immunization were determined by ELISA.

[0047] like Figure 4As shown, after injection and immersion inoculation of ΔdeoR, the adaptive humoral immune response in large yellow croaker was significantly activated on the 14th day, and a certain level of IgM antibodies could still be detected after 150 days.

[0048] Embodiment 7: The TaqMan fluorescence quantitative PCR method was used to establish a qPCR absolute standard curve to quantify the bacterial load after the virus attack in Example 5. The gyrB gene was amplified using the DNA of Pseudomonas proteus as a template, and the amplified product was cloned into the pMD-19T vector and transformed into Escherichia coli DH5α to screen positive clones. The recombinant pMD-19T-gyrB plasmid was purified using a plasmid extraction kit (TIANGEN, China), and the concentration was determined by an ultra-micro spectrophotometer. The constructed recombinant plasmid was used as a quantitative marker for Pseudomonas proteus, and it was diluted with sterile deionized water into a total of 9 concentration gradients, namely 1.0×10 8 Copy number / μL-1.0×10 1 The number of copies / μL was used as a template to perform qPCR using the Premix Ex Taq (Probe qPCR) kit to establish a standard curve between the number of recombinant plasmid copies and the ct value. On the 42nd day after immunization, the bacterial load in the head kidney, spleen, liver, and kidney of each group was measured. Figure 5 As shown in the figure, the bacterial load in each tissue and organ after ΔdeoR immunization was significantly reduced compared with the control group. The above results show that ΔdeoR injection and immersion immunization can effectively inhibit the proliferation of Pseudomonas aeruginosa in large yellow croaker and have a good protective effect on large yellow croaker.

[0049] Embodiment 8: 24 h after the challenge with the virus in Example 5, spleen and head kidney tissues of large yellow croaker were collected from each group, and the relative expression levels of humoral immunity-related genes (IgM, IgT, MHCIIα, CD4-1) and cellular immunity molecules (MHCI, CD8α, ZAP70) were detected by quantitative real-time PCR (qPCR). -ΔΔCt After normalization with β-actin, the relative expression of each gene in the inoculated group relative to the PBS control group was quantified. β-actin and 18S rRNA were used as internal references, and the ChamQ Universal SYBR qPCR Master Mix kit produced by Nanjing Novozyme Biotechnology Co., Ltd. was used for fluorescence quantitative PCR. For specific operations, see the instructions. The relative expression of each gene in the inoculated group relative to the PBS control group was quantified using 2 -ΔΔCt Method, ΔCt = Ct target gene -(Ct β-actin +Ct 18SrRNA ) / 2. See Figure 6 ΔdeoR injection immunization group A (5×10 4 CFU / mL), B(1×10 5 CFU / mL), C(5×10 5 CFU / mL; immersion immunization group D (1×10 6 CFU / mL), E(1×10 7 CFU / mL), F(1×10 8 CFU / mL) in the head kidney and spleen of large yellow croaker, the expression levels of immune genes IgM, IgT, CD4-1, CD8α, MHCI, MHCIIα and ZAP70 were significantly upregulated.

[0050] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A Pseudomonas aeruginosa dOR A method for constructing a gene knockout strain, characterized in that: The following steps are involved: S1: Linearize the pRE112 suicide plasmid using PCR to obtain the pRE112 plasmid linearized fragment; Using the genome of wild-type Pseudomonas proteus strain as template, PCR amplified dOR Gene upstream homology arm fragment, dOR The homology arm fragment downstream of the gene; Using a commercial seamless cloning kit dOR Gene upstream homology arm fragment, dOR The downstream homology arm fragment of the gene and the linearized fragment of the pRE112 plasmid were connected to obtain the recombinant plasmid pRE112- △deoR ; S2: Recombinant plasmid pRE112- △deoR Transformation into E. coli E. coli In S17-1λpir, conjugation donor bacteria were obtained; S3: Conjugation transfer between the conjugation donor bacteria and the wild-type strain of Pseudomonas proteus; S4: Through screening and identification of single-exchange clones and double-exchange clones, Pseudomonas proteus was obtained dOR Knockout strains.

2. The construction method according to claim 1, characterized in that: The nucleotide sequences of the primer pair used to linearize the pRE112 suicide plasmid were: pRE112-F: 5'-TAATACGACTCACTATAGGG-3', pRE112-R: 5'-TGCTAGTTATTGCTCAGCGG-3'; dOR The nucleotide sequences of the primer pair used for the upstream homology arm fragment of the gene were: Up-F: 5'-tgattacgaattcgagctcgGCTTGTCGGCGAACAGCTCGG-3', Up-R: 5'-TGGCAAGGCAGAATAACTCCtcatgtgtggaatcgttggt-3'; dOR The nucleotide sequences of the primer pair used for the downstream homology arm fragment of the gene were: Down-F: 5′-GGAGTTATTCTGCCTTGCCAtggcgcttaagtttcgattc-3′, Down-R: 5′-taaaacgacggccagtgccaCCGATGGCCTGGTGGTCGCGAC-3′.

3. The construction method according to claim 1, characterized in that: dOR The nucleotide sequence of the upstream homology arm fragment of the gene is shown in SEQ ID NO.

2. dOR The nucleotide sequence of the downstream homology arm fragment of the gene is shown in SEQ ID NO.

3.

4. The construction method according to claim 1, characterized in that: Screening of single-exchange clones specifically refers to screening by plates containing 5 μg / mL tetracycline, and identification of single-exchange clones specifically refers to PCR identification by primer pair Up-F / F and Check-F2 / R2; screening of double-exchange clones specifically refers to screening by plates containing 15 wt% sucrose, and identification of double-exchange clones specifically refers to PCR identification by primer pair Up-F / F and Check-F3 / R3; the nucleotide sequence of primer pair Up-F / F is: Up-F: 5'-tgattacgaattcgagctcgGCTTGTCGGCGAACAGCTCGG-3', Down-R: 5'-taaaa cgacggccagtgccaCCGATGGCCTGGTGGTCGCGAC-3'; the nucleotide sequence of the primer pair Check-F2 / R2 is: Check-F2: 5'-ACGTTCAGCGAGTTGGTCAT-3', Check-R2: 5'-TCGGAAGTCACCATTCGCAA-3'; the nucleotide sequence of the primer pair Check-F3 / R3 is: Check-F3: 5'-GTCATAGCCGCGGTACTCAA-3', Check-R3: 5'-TCGGAAGTCACCATTCGCAA-3'.

5. A Pseudomonas aeruginosa dOR A gene knockout strain, characterized in that: The method is constructed according to any one of claims 1 to 4.

6. The Pseudomonas proteus according to claim 5 dOR The application of gene knockout strains in the preparation of drugs for the prevention and treatment of diseases caused by Pseudomonas aeruginosa infection in aquatic animal breeding.

7. The use according to claim 6, characterized in that: The disease is white spot disease of large yellow croaker viscera, and the medicine is a vaccine.

8. An attenuated vaccine of Pseudomonas aeruginosa, characterized in that: The vaccine comprises the Pseudomonas aeruginosa according to claim 5 dOR Knockout strains.

9. The attenuated Pseudomonas aeruginosa vaccine according to claim 8, characterized in that: The dosage forms of the vaccine include injection vaccine and immersion vaccine.

10. Knockout dOR The application of the gene in reducing the pathogenicity of Pseudomonas aeruginosa and preparing an attenuated vaccine of Pseudomonas aeruginosa is characterized by: Said dOR The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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