WO2019095566A1 - 一种防控水产养殖动物气单胞菌出血病的减毒活疫苗 - Google Patents

一种防控水产养殖动物气单胞菌出血病的减毒活疫苗 Download PDF

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WO2019095566A1
WO2019095566A1 PCT/CN2018/075580 CN2018075580W WO2019095566A1 WO 2019095566 A1 WO2019095566 A1 WO 2019095566A1 CN 2018075580 W CN2018075580 W CN 2018075580W WO 2019095566 A1 WO2019095566 A1 WO 2019095566A1
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aeromonas
vaccine
aquatic
veronii
infection
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French (fr)
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周志刚
杨雅麟
冉超
高辰辰
张震
解明旭
何夙旭
张进雄
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中国农业科学院饲料研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/0208—Specific bacteria not otherwise provided for
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine

Definitions

  • the invention relates to a live attenuated vaccine for preventing and controlling hemorrhagic disease of aquaculture animals.
  • inactivated vaccines are the main prevention and treatment methods for hemorrhagic diseases caused by Aeromonas, but the destruction of antigen by inactivated vaccines leads to unstable immune response and lack of immunogenicity for a long time. Therefore, the development of a pathogen vaccine will be one of the important non-anti-control methods for the prevention and treatment of Aeromonas hemorrhagic disease.
  • the invention provides a live attenuated vaccine for preventing and controlling hemorrhagic disease of aquaculture animals.
  • the inventor of the present invention cooperated with the Pearl River Fisheries Research Institute of the Chinese Academy of Fishery Sciences to refer to the laboratory analysis of the aerobics of the aquaculture fish in the southern part of China in the period of 2009-2014, and found Aeromonas victoria.
  • A. veronii infection accounted for the largest proportion (50%)
  • Aeromonas hydrophila infection only accounted for 20.8%
  • the mixed infection of the two accounted for 16.7% of the total infection.
  • the researchers measured multiple isolates of these two species in a sterile zebrafish system by bathing and found that the virulence of Aeromonas vivax was generally higher than that of Aeromonas hydrophila.
  • Aeromonas hydrophila can only establish infections in the case of mixed infection with Aeromonas vivax and stress-induced barrier damage. This provides a new idea for the prevention and control of diseases of cultured fish aeruginosa sepsis, and it is expected to change the situation in which the existing measures are unclear.
  • the present invention first provides a recombinant strain obtained by knocking out the gas lysin gene in Aeromonas vivax.
  • the aerolysin gene is a gene encoding a gas lysin protein.
  • the aerolysin protein is as follows (1) or (2):
  • the gas lysin gene is as follows (a1) or (a2) or (a3) or (a4):
  • (a3) a DNA molecule which hybridizes under stringent conditions to a DNA sequence defined by (a1) or (a2) and which encodes a protein having the same function;
  • the knockout is to knock out the entire open reading frame or knock out part of the gene segment.
  • the knockout may be performed by knocking out sequence 5 of the sequence listing in Aeromonas urticae genomic DNA from positions 91 to 1381 of the 5' end.
  • the knockout is achieved by homologous recombination.
  • the homologous recombination is carried out by introducing a specific DNA fragment into Aeromonas aeruginosa; the specific DNA fragment includes an upstream homologous arm and a downstream homology arm of the aerolysin gene; and the upstream homologous arm is as a sequence listing Sequence 3 is shown at nucleotides 3092-4276 from the 5' end; the downstream homologous arm is shown in sequence 3 of the sequence listing from nucleotides 4277-5408 at the 5' end.
  • the specific DNA fragment is shown, for example, in Sequence 3 of the Sequence Listing from nucleotides 3092 to 5408 at the 5' end.
  • the homologous recombination is carried out by introducing a recombinant vector containing the specific DNA fragment into Aeromonas vivata; the recombinant vector is obtained by ligating a linearized plasmid vector, a fragment A and a fragment B; As shown in Sequence 1 of the Sequence Listing; the fragment B is as shown in Sequence 2 of the Sequence Listing.
  • the connection is achieved by seamless cloning.
  • the plasmid vector is plasmid pRE112.
  • the recombinant vector is specifically shown in SEQ ID NO: 3 of the Sequence Listing.
  • the present invention also protects a recombinant strain, which is a recombinant strain having the specific DNA fragment obtained by introducing a specific DNA fragment into Aeromonas vivax for homologous recombination; the specific DNA fragment includes an upstream of a gas lysin gene a homology arm and a downstream homology arm; the upstream homology arm such as sequence 3 of the sequence listing is shown at nucleotides 3092-4276 from the 5' end; the downstream homology arm is as sequence 3 of the sequence listing from 5 'The nucleotides at positions 4277-5408 are shown.
  • the homologous recombination is carried out by introducing a recombinant vector containing the specific DNA fragment into Aeromonas vivata; the recombinant vector is obtained by ligating a linearized plasmid vector, a fragment A and a fragment B; As shown in Sequence 1 of the Sequence Listing; the fragment B is as shown in Sequence 2 of the Sequence Listing.
  • the connection is achieved by seamless cloning.
  • the plasmid vector is plasmid pRE112.
  • the recombinant vector is specifically shown in SEQ ID NO: 3 of the Sequence Listing.
  • Aeromonas vivax may be A. veronii Hm091.
  • Any of the above recombinant bacteria may specifically be Aeromonas veronii Hm091 ⁇ aer.
  • Aeromonas veronii Hm091 ⁇ aer deposited on October 09, 2017 at the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC; Address: No. 1 Beichen West Road, Chaoyang District, Beijing) No. 3, Institute of Microbiology, Chinese Academy of Sciences; Zip Code: 100101), with the accession number CGMCC No. 14776.
  • the present invention also protects the use of any of the above-described recombinant bacteria for preparing a vaccine;
  • the vaccine is any one of the following (b1) to (b4):
  • the present invention also protects a method of preparing a vaccine comprising the steps of: packaging any of the above-described recombinant bacteria as an active ingredient of a vaccine; the vaccine is any one of the following (b1) to (b4):
  • the present invention also protects the use of any of the above-described recombinant bacteria in the preparation of a product; the use of the product is at least one of the following (c1) to (c5):
  • the present invention also protects an aquatic animal, Aeromonas vivax vaccine, which has an active ingredient of any of the above recombinant bacteria.
  • the present invention also protects an aquatic animal Aeromonas hydrophila vaccine, the active ingredient of which is any of the above recombinant bacteria.
  • the present invention also protects the aquatic animal Aeromonas vaccine, the active ingredient of which is any of the above recombinant bacteria.
  • the invention also protects the aquatic animal hemorrhagic disease vaccine, wherein the active ingredient is any of the above recombinant bacteria.
  • the present invention also protects a product having an active ingredient of any of the above-described recombinant bacteria; the use of the product is at least one of the following (d1) to (d5):
  • the present invention also protects the use of any of the above-described recombinant bacteria as at least one of the following (f1) to (f5):
  • the manner of use of any of the above vaccines may specifically be oral or dip bath.
  • Any of the above products may specifically be an aquatic animal feed additive.
  • Aeromonas may be Aeromonas victorii or Aeromonas hydrophila.
  • Aeromonas vivax may be A. veronii Hm091.
  • Aeromonas hydrophila may specifically be Aeromonas hydrophila AJ-1.
  • any of the above-mentioned aquatic animals may specifically be zebrafish, grass carp, carp, carp, carp, carp, carp, carp, carp, tilapia, cockroach, white pheasant, silver carp, cockroach, yellowtail owl, cockroach, channel catfish, Goldfish, scutellaria, gums and other aquatic animals such as frogs, frogs, shrimps, crabs, crickets, turtles, etc.
  • Figure 1 is a schematic diagram showing the construction process of the knockout plasmid of Example 1.
  • Example 2 is a result of identification of knockout bacteria of Example 1.
  • Figure 3 is a graph showing the results of fluorescence localization in Example 2.
  • Figure 5 is a statistical result of the immunoprotective effect of Example 4.
  • the genomic DNA reference sequence of the Aeromonas vilii gas lysin gene is shown in SEQ ID NO: 5 of the Sequence Listing.
  • the DNA shown in SEQ ID NO: 5 shows the protein shown in SEQ ID NO: 6.
  • Plasmid pRE112 BioVector Plasmid Vector Culture Cell Genetics Collection, Cat. No. 3573410.
  • Aeromonas aeruginosa A. veronii Hm091 References: Zhang Defeng, Liu Lihui, Li Ningqiu, et al. The epidemiological characteristics of different species of Aeromonas fishes in southern China[J]. Fisheries Science, 2015, 34(11) :673-682.; The public can obtain it from the Feed Research Institute of the Chinese Academy of Agricultural Sciences.
  • A.hydrophila NJ-1 References: Li J, Ni X D, Liu Y J, et al. Detection of three virulence genes alt, ahp and aerA in Aeromonas hydrophila and their relationship with actual virulence to Zebrafish [J]. Journal of Applied Microbiology, 2011, 110 (3): 823-30.; public can be obtained from the Feed Research Institute of the Chinese Academy of Agricultural Sciences.
  • E.coli MC1061 BioVector NTCC Typical Culture Collection, No. MC1061.
  • E. coli S17-1 ( ⁇ pair): BioVector NTCC Typical Culture Collection, number: s17-1.
  • E.coli MC1061 single colonies were inoculated into 50 ml LB liquid medium, cultured overnight at 37 ° C, 200 rpm, and inoculated into a 1 l flask containing 500 ml of LB liquid medium at a 5% inoculum, 37 ° C
  • the flask is iced for 15 to 30 minutes (shake the flask at a time to evenly cool the bacteria), and then transfer the bacteria solution to the pre-cooling of the ice bath.
  • centrifuge at 1000g, 4°C for 15min discard the supernatant, resuspend the cells with deionized water precooled in 500ml ice bath; centrifuge at 1000g for 20min at 4°C, remove the supernatant, collect the cells and resuspend in 250ml ice bath.
  • glycerol aqueous solution 1000g, centrifuged at 4 ° C for 20min, remove the supernatant, resuspend the cells with 1ml ice bath pre-cooled GYT liquid medium; the bacteria liquid is divided into ice bath pre-cooled In a 1.5 ml sterile EP tube, 50 ⁇ l per tube, rapidly cooled with liquid nitrogen, and stored in a -80 ° C refrigerator.
  • E. coli S17-1 ( ⁇ pair) single colonies were inoculated into 50 ml of LB liquid medium, cultured overnight at 37 ° C, 200 rpm, and inoculated with 500 ml of LB liquid medium at a dose of 5%.
  • a 1l flask incubate at 37 ° C and 250 rpm.
  • the flask is iced for 15 to 30 minutes (shake the flask for a uniform cooling) and then transfer the bacteria solution.
  • centrifuge tube To the pre-cooled pre-cooled centrifuge tube, centrifuge at 1000g, 4°C for 15min, discard the supernatant, resuspend the cells with 500ml ice bath pre-cooled deionized water; centrifuge at 1000g, centrifuge at 20°C for 20min, remove the supernatant, and collect the bacteria.
  • the body was resuspended in a 10% (mentioned percentage) glycerin aqueous solution pre-cooled in a 250 ml ice bath, centrifuged at 1000 g, 4 ° C for 20 min, the supernatant was removed, and the cells were resuspended in a 1 ml ice bath precooled GYT liquid medium;
  • the liquid was placed in a 1.5 ml sterile EP tube pre-cooled in an ice bath, 50 ⁇ l per tube, rapidly cooled with liquid nitrogen, and stored in a refrigerator at -80 °C.
  • Zebrafish Tu line zebrafish provided by Peking University zebrafish water laboratory.
  • the plasmid pRE112 was linearized by PCR method (reaction procedure: 98 ° C, 5 min, 32 ⁇ [98 ° C, 20 s; 58 ° C, 20 s; 72 ° C, 1 min], 72 ° C, 5 min) to obtain a linearized plasmid fragment. .
  • primers consisting of primers Aer-up-F and primer Aer-up-R were used for PCR amplification to obtain PCR amplification products (sequence table) Sequence 1, with an upstream homology arm).
  • Aer-up-F 5'-TGAATTCCCGGGAGAATGATCTCGGCGGTACCTGG-3';
  • Aer-up-R 5'-GATCCACACCGGTAAATCAGGGTAGACAGGTTCAG-3'.
  • primers consisting of primers Aer-down-F and primers Aer-down-R were used for PCR amplification to obtain PCR amplification products (sequence table) Sequence 2, with a downstream homology arm).
  • Aer-down-F 5'-CCTGTCTACCCTGATTTACCGGTGTGGATCTGGAC-3';
  • Aer-down-R 5'-GCTTCTTCTAGAGGTTGAGTGAAGGTGGAGCTGAG-3'.
  • step 4 use HiFi DNA Assembly Master Mix (NEB, Cat. No. E2621L)
  • the linearized plasmid obtained in step 1, the PCR amplification product obtained in step 2, and the PCR amplification product obtained in step 3 are homologously recombined (method reference kit instructions)
  • the knockout plasmid was obtained (the circular plasmid shown in SEQ ID NO: 3 of the Sequence Listing, which has been verified by sequencing).
  • the knockout plasmid obtained in the first step was transformed into MC1061 competent cells, and electroporated at 25 uF, 2.5 kV, and 200 ohm. Immediately after electroporation, add 1 ml of LB liquid medium, incubate at 37 ° C, shaker at 200 r / min for 1-1.5 h, centrifuge, remove 900 ⁇ l, leave 100 ⁇ l of resuspension, and then apply all to 30ug/ml chloramphenicol (Cm) Incubate on LB solid plates overnight at 37 °C. For the single colonies grown overnight, positive colonies were screened by colony PCR (using the primer Aer-up-F and the primer Aer-up-R for PCR detection to obtain a 2348 bp band).
  • step 2 Single colonies identified as positive in step 1 were inoculated into 30 ml LB liquid medium containing 30 ug/ml chloramphenicol (Cm), cultured at 37 ° C, shaker at 200 r / min for 12 h, and single colony extraction plasmid was picked.
  • the Hind III restriction endonuclease was subjected to restriction enzyme digestion to obtain a positive plasmid of a band of 8029 bp in size.
  • the correct positive plasmid identified in step 2 was transformed into S17 ( ⁇ ) competent cells, and electrotransformed at 25 uF, 2.5 kV, and 200 ohm. Immediately after electrotransfer, add 1 ml of LB liquid medium, incubate at 37 ° C, shaker at 200 r / min for 1 to 1.5 h, centrifuge, remove 900 ⁇ l, leave 100 ⁇ l of resuspension, and then apply all to 30 ug / ml chloramphenicol (Cm) Incubate on LB solid plates overnight at 37 °C. For the single colonies grown after overnight, positive colonies were screened by colony PCR (using the primer Aer-up-F and the primer Aer-up-R for PCR detection to obtain a 2348 bp band).
  • step 3 The single colonies identified as positive in step 3 were re-streaked to LB solid plates containing 30 ug/ml chloramphenicol (Cm) using an inoculating loop and incubated at 37 ° C for 24 h.
  • Cm chloramphenicol
  • A. veronii Hm091 was inoculated to LB solid plates containing 30 ug/ml chloramphenicol (Cm) and incubated at 37 ° C for 24 h.
  • step 4 The colonies obtained in step 4 were separately scraped and the colonies obtained in step 5 were combined (scraping step 4 to obtain colonies on the plate of step 5), and binding was carried out at 37 ° C for 8 h.
  • step 6 the colony was scraped off, diluted with LB liquid medium, and applied to LB solid plate containing 30 ⁇ g/ml chloramphenicol (Cm) + 100 ⁇ g/ml ampicillin (Amp), and cultured at 37 ° C. 24h.
  • step 7 single colonies were picked and inoculated onto LB solid plates containing 30 ⁇ g/ml chloramphenicol (Cm) + 100 ⁇ g/ml ampicillin (Amp) and cultured at 37 ° C for 24 h.
  • Cm chloramphenicol
  • Amp ampicillin
  • step 8 single colonies were picked and inoculated on LB solid plates without antibiotics for 24 h at 37 °C.
  • step 9 an appropriate amount of colony was scraped with an inoculating loop and streaked onto an LB solid plate containing 15% (mass%) sucrose, and cultured at 15-25 ° C for 3 days at low temperature to select a plasmid-loss strain.
  • step 10 After completing step 10, 50-80 monoclonal clones were picked and streaked onto LB solid plates containing 30 ⁇ g/ml chloramphenicol (Cm) + 100 ⁇ g/ml ampicillin (Amp), and cultured at 37 ° C for 24 h.
  • Cm chloramphenicol
  • Amp ampicillin
  • step 12 pick a single colony and screen out the knockout mutant strain by colony PCR detection.
  • Aeromonas aeruginosa A. veronii Hm091 was used as a control.
  • Primer pairs consisting of primers Aer-up-F and primers Aer-down-R, primers Aer-confirm-F and primers Aer-confirm-R were identified.
  • the primer sequences and expected product fragment lengths are shown in Table 1.
  • the primer Aer-confirm-F and the primer Aer-confirm-R are primers designed for the Aer gene.
  • the wild type (WT) amplified the target fragment by 1722 bp, and the knockout strain (Hm091 ⁇ aer) did not amplify the target fragment; primer Aer -up-F and the primer Aer-down-R are primers designed for the upstream and downstream sequences of the Aer gene.
  • the sequence of the amplified fragment of the wild type (WT) is about 4000 bp, and the amplified fragment of the knockout (Hm091 ⁇ aer) The sequence is about 2300 bp, which is about 1700 bp smaller than the size of the Aer gene fragment.
  • Aeromonas veronii Hm091 ⁇ aer Aeromonas veronii Hm091 ⁇ aer
  • Aeromonas veronii Hm091 ⁇ aer Aeromonas veronii Hm091 ⁇ aer
  • Aeromonas victoria Aeromonas victoria
  • Aeromonas veronii Hm091 ⁇ aer deposited on October 09, 2017 at the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC; Address: No. 1 Beichen West Road, Chaoyang District, Beijing) No. 3, Institute of Microbiology, Chinese Academy of Sciences; Zip Code: 100101), with the accession number CGMCC No. 14776.
  • Aeromonas aeruginosa A. veronii Hm091 into 30 ml LB liquid medium, incubate at 37 ° C, shaker 200 r / min for 18 h; take 2 ml of bacterial solution, centrifuge at 5000 r / mim for 10 min, remove the supernatant, with PBS Wash twice, centrifuge to remove the supernatant, and resuspend the cells by adding 1 ml of 0.1 M sodium bicarbonate buffer to obtain A. veronii Hm091 sodium bicarbonate buffer.
  • hydrophila NJ-1 into 30 ml LB liquid medium, incubate at 37 ° C, shaker at 200 r / min for 18 h; take 2 ml of bacterial solution, centrifuge at 5000 r / mim for 10 min, remove the supernatant, Wash twice with PBS, remove the supernatant by centrifugation, and resuspend the cells by adding 1 ml of 0.1 M sodium bicarbonate buffer to obtain A. hydrophila NJ-1 sodium bicarbonate buffer.
  • Aeromonas aeruginosa A. veronii Hm091 ⁇ aer into 30 ml LB liquid medium, incubate at 37 ° C, shaker 200 r / min for 18 h; take 2 ml of bacterial solution, centrifuge at 5000 r / mim for 10 min, remove the supernatant, Wash twice with PBS, remove the supernatant by centrifugation, and resuspend the cells by adding 1 ml of 0.1 M sodium bicarbonate buffer to obtain A. veronii Hm091 ⁇ aer sodium bicarbonate buffer.
  • Red fluorescent dye (Texas) -X) (Thermo Fisher Scientific T7471) was dissolved in DMSO at a concentration of 10 mg/ml to obtain a red fluorescent dye solution.
  • a blue fluorescent dye (Pacific BlueTM) (Invitrogen P10163) was dissolved in DMSO at a concentration of 10 mg/ml to obtain a blue fluorescent dye solution.
  • red fluorescent dye solution obtained in step 4 50 ⁇ l was added to the A.hydrophila NJ-1 sodium bicarbonate buffer obtained in step 2, and incubated at room temperature for a period of 1.5 h, protected by light, and then centrifuged to remove the supernatant and washed with PBS. The supernatant was removed by centrifugation and finally resuspended in 1 ml of PBS to obtain a red fluorescent labeling solution of A. hydrophila NJ-1.
  • A. hydrophila NJ-1 into 30 ml LB liquid medium incubate at 37 ° C, shaker at 200 r / min for 18 h; take 2 ml of bacterial solution, centrifuge at 5000 r / mim for 10 min, remove the supernatant, Wash twice with PBS, remove the supernatant by centrifugation, and resuspend the cells with PBS to obtain A.hydrophila NJ-1 solution.
  • Aeromonas aeruginosa A. veronii Hm091 ⁇ aer into 30 ml LB liquid medium, incubate at 37 ° C, shaker 200 r / min for 18 h; take 2 ml of bacterial solution, centrifuge at 5000 r / mim for 10 min, remove the supernatant, Wash twice with PBS, remove the supernatant by centrifugation, and resuspend the cells with PBS to obtain A. veronii Hm091 ⁇ aer solution.
  • the zebrafish 2 days after hatching were randomly divided into the following 5 groups (each group of fish was divided into 6-well plates, 10 ml of water per well, 15 fish):
  • Group A (control group): zebrafish was infected with PBS;
  • Group B (A.hydrophila NJ-1 red fluorescently labeled infection group): The zebrafish was infected with A.hydrophila NJ-1 red fluorescent labeling solution. The concentration of A.hydrophila NJ-1 in the infected well was 4 ⁇ 10 7 CFU/ Ml;
  • Group C (A. veronii Hm091 red fluorescently labeled infection group): zebrafish was infected with A. veronii Hm091 red fluorescent labeling solution, and the concentration of A. veronii Hm091 in the infected well was 4 ⁇ 10 7 CFU/ml;
  • Group D A. hydrophila NJ-1 red fluorescent label + unlabeled A. veronii Hm091 infection group: A. hydrophila NJ-1 red fluorescent labeling solution and A. veronii Hm091 solution were used to infect zebrafish, A. hydrophila NJ-1 The concentration in the infected well was 4 ⁇ 10 7 CFU/ml, and the concentration of A. veronii Hm091 in the infected well was 4 ⁇ 10 7 CFU/ml;
  • Group E (A. hydrophila NJ-1 red fluorescent label + A. veronii Hm091 blue fluorescent label): Infected zebrafish with A. hydrophila NJ-1 red fluorescent labeling solution and A. veronii Hm091 blue fluorescent labeling solution, A.
  • the concentration of hydrophila NJ-1 in the infected well was 4 ⁇ 10 7 CFU/ml, and the concentration of A. veronii Hm091 in the infected well was 4 ⁇ 10 7 CFU/ml;
  • Group F (A. veronii Hm091 ⁇ aer red fluorescently labeled infection group): The zebrafish was infected with A. veronii Hm091 ⁇ aer red fluorescent labeling solution, and the concentration of A. veronii Hm091 ⁇ aer in the infected well was 4 ⁇ 10 7 CFU/ Ml;
  • Group G (A. hydrophila NJ-1 red fluorescent label + unlabeled A. veronii Hm091 ⁇ aer infection group): zebrafish was infected with A. hydrophila NJ-1 red fluorescent labeling solution and A. veronii Hm091 ⁇ aer solution, A. The concentration of hydrophila NJ-1 in the infected well was 4 ⁇ 10 7 CFU/ml, and the concentration of A. veronii Hm091 ⁇ aer in the infected well was 4 ⁇ 10 7 CFU/ml.
  • the small fish were fixed with 4% paraformaldehyde; the fixed small fish was photographed under a laser confocal microscope to observe the localization of bacteria in zebrafish.
  • veronii Hm091 infection group not only a strong fluorescent signal was observed in the intestinal tract of the zebrafish, but also around the intestinal tract and the head. Fluorescent signals (C and E) can be detected in the ministry; interestingly, the red fluorescently labeled Aeromonas hydrophila NJ-1+ is infected with the blue fluorescent marker A. veronii Hm091. In the group, the fluorescent signal of NJ-1 can also be detected around the zebrafish gut and in the head (D and E).
  • Aeromonas urticae can destroy the intestinal barrier of zebrafish, prompting itself and other Aeromonas, especially Aeromonas hydrophila, to enter the body of the fish, resulting in fish Hemolysis and death. Aeromonas vivax A. veronii Hm091 ⁇ aer does not have the ability to destroy the intestinal barrier of zebrafish.
  • Aeromonas aeruginosa A. veronii Hm091 onto LB solid medium, incubate at 37 ° C for 12 h, then pick up the monoclonal inoculation into 30 ml of LB liquid medium, incubate at 37 ° C, shaker 200 r / min 18h.
  • Aeromonas aeruginosa A. veronii Hm091 ⁇ aer onto LB solid medium, incubate at 37 °C for 12 h, then pick up the monoclonal inoculation into 30 ml of LB liquid medium, 37 ° C, shaker 200r / Min culture for 18h.
  • Fig. 4A is the statistical result of the survival time at a concentration of 3.3 ⁇ 10 7 CFU/ml
  • Fig. 4B is the statistical result of the survival time at a bath concentration of 2.5 ⁇ 10 7 CFU/ml
  • Fig. 4C is 1.67 ⁇ 10 Survival time statistics at 7 CFU/ml bath concentration.
  • the results showed that A. veronii Hm091 ⁇ aer had no zebrafish death at the bath concentration of 3.3 ⁇ 10 7 CFU/ml, 2.5 ⁇ 10 7 CFU/ml and 1.67 ⁇ 10 7 CFU/ml, respectively. However, after infecting wild plants, 100% mortality of zebrafish can be caused within 24 hours.
  • the results showed that knocking out Aer significantly reduced the virulence of Aeromonas vivax, and A. veronii Hm091 ⁇ aer was safe to bathe at an infection concentration of ⁇ 3.3 ⁇ 10 7 CFU/ml.
  • Control group Normally rearing zebrafish for 2 weeks, feeding the basal feed twice a day;
  • Group 2 feeding immunization group: Normally rearing zebrafish for 2 weeks, feeding the basal feed containing Aeromonas aeruginosa A. veronii Hm091 ⁇ aer (2 ⁇ 10 7 CFU/g) twice a day;
  • Group 3 (immersion immunization group): Normally rearing zebrafish for 2 weeks, once a week bath containing A. veronii Hm091 ⁇ aer water (2 ⁇ 10 7 CFU / ml, bath time 12h) Feed the basic feed twice a day.
  • each group of zebrafish was collected from the tail vein, and the zebrafish immunoglobulin index was tested according to the instruction of the fish IGM kit (purchased from Nanjing Institute of Bioengineering).
  • the average concentrations of the three groups of zebrafish immunoglobulins were determined as follows: control group: 7.304175 ng/ml; feeding immunization group: 13.646945 ng/ml; bath immunization group: 16.00466 ng/ml.
  • the results showed that the serum immunoglobulin content of zebrafish increased after vaccine immunization.
  • Control group Normally rearing zebrafish for 2 weeks, feeding the basal feed twice a day;
  • Group 2 feeding immunization group: Normally rearing zebrafish for 2 weeks, feeding the basal feed containing Aeromonas aeruginosa A. veronii Hm091 ⁇ aer (2 ⁇ 10 7 CFU/g) twice a day;
  • Group 3 Immersion immunization group: Normally rearing zebrafish for 2 weeks, bath once a week with A. veronii Hm091 ⁇ aer water (4 ⁇ 10 7 CFU / L, bath time 12h) Feed the basic feed twice a day.
  • each group of zebrafish was bled through the tail vein, and serum was taken for determination of serum antibody titer.
  • the 96-well hemagglutination method was used.
  • the A. veronii Hm091 culture solution was centrifuged at 12000 rpm, and then resuspended in an equal amount of physiological saline to obtain a bacterial suspension (concentration: 4 ⁇ 10 7 CFU/ml).
  • Add 80 ⁇ l of physiological saline to the first well with a micropipette add 50 ⁇ l to each well; add 20 ⁇ l of the test serum to the first well, mix by pipetting, and add 50 ⁇ l to the second well (1:2 dilution).
  • the experiment was repeated twice (first group and second group), and the results are shown in Table 2.
  • the A. veronii Hm091 ⁇ aer vaccine against A. veronii Hm091 ⁇ aer vaccine was used to immunize adult zebrafish by feeding and bathing.
  • the serum antibody agglutination titers of the control group, the feeding group and the bathing group were 2 3 ⁇ 2 4 , 2 4 ⁇ 2 5 and 2 4 , the serum antibody level in the feeding group was slightly higher than the serum antibody level in the control group.
  • Group 1 (control group, CK): Normally rearing zebrafish for 2 weeks, feeding the basal feed twice a day;
  • Group 2 feeding immunization group, T1: Normally rearing zebrafish for 2 weeks, feeding twice a day with a basic diet containing Aeromonas aeruginosa A. veronii Hm091 ⁇ aer (2 ⁇ 10 7 CFU/g) ;
  • Group 3 immersion immunization group, T2: Normally rearing zebrafish for 2 weeks, bath once a week with A. veronii Hm091 ⁇ aer water (3 ⁇ 10 7 CFU / ml, bath time 12h), feed the basic feed twice a day.
  • each group of zebrafish was randomly divided into the following three groups:
  • Group A (A.hydrophila NJ-1 + ammonium chloride): zebrafish (dipping bath) was infected with 200 mg/L ammonium chloride, and 2 ⁇ 10 7 CFU/ml of Aeromonas vivax A. veronii was added after 12 h. Hm091.
  • Group B Aeromonas vivax A. veronii Hm091 was infected with zebrafish (dipping bath) at a concentration of 2 x 10 7 CFU/ml.
  • Group C Aeromonas aeruginosa A. veronii Hm091 and Aeromonas hydrophila A.hydrophila NJ-1 at 2 ⁇ 10 7 CFU/ml for each strain The concentration of the infected zebrafish (dipping bath).
  • RPS immune protection rate
  • Fig. 5A is the statistical result of the survival rate of each group when NJ-1+ ammonium chloride is challenged
  • Fig. 5B is the statistical result of the survival rate of each group when HM091 is challenged
  • Fig. 5C is the HM091+NJ-1 challenge.
  • the survival rate of each group was statistically obtained.
  • the results in Figure 5 show that the death time of the zebrafish in the immunization group was prolonged, but there was no obvious protective effect.
  • the feeding immunization method has obvious immunoprotective effect on NJ-1+ ammonium chloride, Hm091, Hm091+NJ-1 bathing dysfunction, and the survival rate of zebrafish is obviously improved.
  • the results in Table 3 show that the immune protection rate of the immunization group is low and does not play a significant protective role.
  • the immune protection rate of NJ-1+HM091 in the feeding immunization group reached 33%, which had a certain protective effect.
  • the immune protection rate of HM091 reached 80%, which had strong immunoprotective effect.
  • the present invention is based on the gas lysin of Aeromonas vivax causing damage to the zebrafish infection site and further promoting the entry of the relevant bacteria into the fish body is the main cause of the outbreak of the bleeding disease, from the infection of the Vickis air cell
  • the development of a vaccine for the prevention and treatment of hemorrhagic diseases has prevented the development of a key bacterial vaccine that has long been a cause of bacterial infection due to the unclear pathogenesis of Aeromonas hemorrhagic disease, which has led to the late infection of bacteria, leading to long-term bacterial sepsis.
  • the invention adopts the principle of homologous recombination, and the specific gene fragment is definitely deleted to cause gene inactivation, so that the obtained gene-deficient strain has good genetic stability, and avoids the use of unstable genetic engineering methods such as transposon to construct an attenuated strain. The risk of genetic instability.
  • the live attenuated vaccine of the present invention does not carry any antibiotic marker, and there is no potential harm caused by releasing the resistant strain or the antibiotic resistance gene to the environment;
  • a gene knockout method is used to knock out the key virulence factors of Aerolysin to obtain an attenuated strain, and the virulence is significantly reduced compared with the parent strain, and it is better to avoid inactivation of the inactivated vaccine.
  • the live attenuated vaccine of the present invention has a certain cross-protection against different sources of Aeromonas, and has a broad spectrum;
  • the live attenuated vaccine of the present invention has certain immunoprotective power in zebrafish, indicating that the live attenuated vaccine of the present invention can effectively activate the immune response of zebrafish.
  • the vaccine provided by the present invention can significantly enhance the immunity of aquatic animals and improve the disease resistance phenotype.
  • the invention adopts an oral or bath attenuated live vaccine constructed by the technique of knocking out the key virulence factor without antibiotic labeling to reduce the pathogenicity of the pathogenic bacteria while retaining the immunogenicity, and the immune protection effect is remarkable, and the natural immune system reaction and cells are enhanced. Immunization enhances the ability to resist infection by other pathogens while greatly reducing the amount of immune work.

Abstract

提供了一种防控水产养殖动物气单胞菌出血病的减毒活疫苗。提供了一种重组菌，是将维氏气单胞菌中的气溶素基因敲除得到的。所述重组菌可以用于制备水产动物维氏气单胞菌疫苗和/或嗜水气单胞菌疫苗和/或气单胞菌疫苗和/或出血病疫苗。

Description

一种防控水产养殖动物气单胞菌出血病的减毒活疫苗 技术领域
本发明涉及一种防控水产养殖动物气单胞菌出血病的减毒活疫苗。
背景技术
中国是世界上最大的水产养殖生产国家，占世界水产养殖产量的60％以上。随着水产养殖在生产实践过程中的日益集约化和商业化，疾病暴发已经成为鱼类养殖业的主要问题。特别是气单胞菌引起的鱼类出血病已成为水产养殖业发展过程中的一个非常突出的问题。据估计，鱼类疾病爆发每年在全球水产养殖业中造成大约数十亿美元的经济。例如，由气单胞菌(Aeromonas spp.)引起的运动性气单胞菌败血症(MAS)的爆发，通常导致全球水产养殖中鱼类的高死亡率并造成严重的经济损失。在过去几十年中，抗生素是作为水产养殖中鱼类疾病的治理与控制以及促进鱼类生长的传统策略。然而，病原菌的耐药性问题以及动物产品中抗生素的残留问题已经成为全球人们所关注的问题，抗生素在水产养殖业中的禁用已势在必行。目前除使用抗生素外，灭活疫苗是气单胞菌所引起的出血病的主要防治方法，但灭活疫苗对抗原的破坏对导致免疫反应不稳定、缺少免疫原性作用时间不持久。因此开发病原菌疫苗将是防治气单胞菌出血病的重要无抗防治方法之一。
目前对细菌性败血症的疫苗防治由于前期对致病机理不明，导致主要是集中在肠道/鳃屏障破坏后趁虚而入的感染细菌嗜水气单胞菌疫苗方面的研制上，而关于破坏宿主物理屏障的关键细菌维氏气单胞菌疫苗的开发报道很少，主要包括传统的高毒力菌株的灭活疫苗，此外还报道了维氏气单胞菌菌蜕以及引入特异性适体肽的维氏气单胞菌疫苗，且多数是以注射的方式进行免疫，存在以下缺陷：1)灭活过程导致部分或完全丧失保护性抗原，只能引起不稳定或者水平低下的免疫应答反应，甚至不能激发正确免疫反应及引发副作用等；2)维氏气单胞菌菌蜕存在生产产量低，裂解基因存在毒性的缺点；3)特异性适体肽的维氏气单胞菌疫苗无法避免自身毒力基因表达致病的缺点，4)鱼类注射免疫存在工作量大的缺点。
发明公开
本发明提供了一种防控水产养殖动物气单胞菌出血病的减毒活疫苗。
本发明的发明人与中国水产科学研究院珠江水产研究所合作，参考该所2009-2014年对我国南方地区鲤科养殖鱼类的气单胞菌病流调分析，发现维氏气单胞菌A.veronii感染所占的比例最大(50％)，嗜水气单胞菌感染只占20.8％，另外，二者的混合感染占总感染情况的16.7％。研究人员在无菌斑马鱼体系中通过浸浴攻毒的方式测定了这两个种的多个分离株，发现维氏气单胞菌的毒力普遍高于嗜水气单胞菌。进一步通过转座子敲除筛选明确了维氏气单胞菌II型分泌系统分泌的气溶素(Aerolysin)是关键致病因子。嗜水气单胞菌只有在与维氏气单胞菌混合感染及应激引起屏障损伤情况下才可建立感染。这为养殖鱼类气单胞菌败血症的病害防控提供了新的思路，有望可以改变现有手段对该病效果不明朗的局面。
本发明首先提供了一种重组菌，是将维氏气单胞菌中的气溶素基因敲除得到的。
所述气溶素基因为编码气溶素蛋白的基因。
所述气溶素蛋白如下(1)或(2)：
(1)由序列表中序列6所示的氨基酸序列组成的蛋白质；
(2)将序列表中序列6所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由序列6衍生的蛋白质。
所述气溶素基因为如下(a1)或(a2)或(a3)或(a4)：
(a1)序列表的序列5所示的DNA分子；
(a2)编码区如序列表的序列5所示的DNA分子；
(a3)在严格条件下与(a1)或(a2)限定的DNA序列杂交且编码具有相同功能的蛋白质的DNA分子；
(a4)与(a1)或(a2)或(a3)限定的DNA序列具有90％以上同源性且编码具有相同功能的蛋白质的DNA分子。
所述敲除为敲除整个开放阅读框或敲除部分基因区段。
所述敲除具体可为将维氏气单胞菌基因组DNA中序列表的序列5自5’端第91至1381位敲除。
所述敲除是通过同源重组实现的。
所述同源重组是将特异DNA片段导入维氏气单胞菌实现的；所述特异DNA片段包括气溶素基因的上游同源臂和下游同源臂；所述上游同源臂如 序列表的序列3自5’端第3092-4276位核苷酸所示；所述下游同源臂如序列表的序列3自5’端第4277-5408位核苷酸所示。所述特异DNA片段如序列表的序列3自5’端第3092-5408位核苷酸所示。
所述同源重组是将含有所述特异DNA片段的重组载体导入维氏气单胞菌实现的；所述重组载体是将线性化质粒载体、片段甲和片段乙连接得到的；所述片段甲如序列表的序列1所示；所述片段乙如序列表的序列2所示。所述连接是通过无缝克隆实现的。所述质粒载体为质粒pRE112。所述重组载体具体为序列表的序列3所示。
本发明还保护一种重组菌，是将特异DNA片段导入维氏气单胞菌进行同源重组后得到的具有所述特异DNA片段的重组菌；所述特异DNA片段包括气溶素基因的上游同源臂和下游同源臂；所述上游同源臂如序列表的序列3自5’端第3092-4276位核苷酸所示；所述下游同源臂如序列表的序列3自5’端第4277-5408位核苷酸所示。
所述同源重组是将含有所述特异DNA片段的重组载体导入维氏气单胞菌实现的；所述重组载体是将线性化质粒载体、片段甲和片段乙连接得到的；所述片段甲如序列表的序列1所示；所述片段乙如序列表的序列2所示。所述连接是通过无缝克隆实现的。所述质粒载体为质粒pRE112。所述重组载体具体为序列表的序列3所示。
以上任一所述维氏气单胞菌具体可为维氏气单胞菌A.veronii Hm091。
以上任一所述重组菌具体可为维氏气单胞菌(Aeromonas veronii)Hm091△aer。
维氏气单胞菌(Aeromonas veronii)Hm091△aer，已于2017年10月09日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC；地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所；邮编：100101)，保藏编号为CGMCC No.14776。
本发明还保护以上任一所述重组菌在制备疫苗中的应用；所述疫苗为如下(b1)-(b4)中的任一种：
(b1)水产动物维氏气单胞菌疫苗；
(b2)水产动物嗜水气单胞菌疫苗；
(b3)水产动物气单胞菌疫苗；
(b4)水产动物出血病疫苗。
本发明还保护一种制备疫苗的方法，包括如下步骤：将以上任一所述重组菌作为疫苗的活性成分进行包装；所述疫苗为如下(b1)-(b4)中的任一种：
(b1)水产动物维氏气单胞菌疫苗；
(b2)水产动物嗜水气单胞菌疫苗；
(b3)水产动物气单胞菌疫苗；
(b4)水产动物出血病疫苗。
本发明还保护以上任一所述重组菌在制备产品中的应用；所述产品的用途为如下(c1)-(c5)中的至少一种：
(c1)预防和/或治疗水产动物维氏气单胞菌感染；
(c2)预防和/或治疗水产动物嗜水气单胞菌感染；
(c3)预防和/或治疗水产动物气单胞菌感染；
(c4)预防和/或治疗水产动物出血病；
(c5)增加水产动物的免疫力。
本发明还保护水产动物维氏气单胞菌疫苗，其活性成分为以上任一所述重组菌。本发明还保护水产动物嗜水气单胞菌疫苗，其活性成分为以上任一所述重组菌。
本发明还保护水产动物气单胞菌疫苗，其活性成分为以上任一所述重组菌。
本发明还保护水产动物出血病疫苗，其活性成分为以上任一所述重组菌。
本发明还保护一种产品，活性成分为以上任一所述重组菌；所述产品的用途为如下(d1)-(d5)中的至少一种：
(d1)预防和/或治疗水产动物维氏气单胞菌感染；
(d2)预防和/或治疗水产动物嗜水气单胞菌感染；
(d3)预防和/或治疗水产动物气单胞菌感染；
(d4)预防和/或治疗水产动物出血病；
(d5)增加水产动物的免疫力。
本发明还保护以上任一所述重组菌的应用，为如下(f1)-(f5)中的至少一种：
(f1)预防和/或治疗水产动物维氏气单胞菌感染；
(f2)预防和/或治疗水产动物嗜水气单胞菌感染；
(f3)预防和/或治疗水产动物气单胞菌感染；
(f4)预防和/或治疗水产动物出血病；
(f5)增加水产动物的免疫力。
以上任一所述疫苗的使用方式具体可为口服或浸浴。
以上任一所述产品具体可为水产动物饲料添加剂。
以上任一所述气单胞菌具体可为维氏气单胞菌或嗜水气单胞菌。
以上任一所述维氏气单胞菌具体可为维氏气单胞菌A.veronii Hm091。
以上任一所述嗜水气单胞菌具体可为嗜水气单胞菌A.hydrophila NJ-1。
以上任一所述水产动物具体可为斑马鱼、草鱼、鲫、鳙、鲢、鳜、鲤、青鱼、罗非鱼、鳗鲡、白鲫、银鲫、鲟、黄尾鲴、鳊、斑点叉尾鮰、金鱼、黄鳝、牙鲆等鱼类或蛙、大鲵、虾、蟹、鳖、龟等水生动物。
附图说明
图1为实施例1敲除质粒的构建流程示意图。
图2为实施例1敲除菌鉴定结果。
图3为实施例2荧光定位结果。
图4为实施例3安全性检测结果。
图5为实施例4免疫保护效果统计结果。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
维氏气单胞菌气溶素基因的基因组DNA参考序列如序列表的序列5所示。序列5所示的DNA编码序列6所示的蛋白质。
质粒pRE112：BioVector质粒载体菌种细胞基因保藏中心，货号：3573410。
维氏气单胞菌A.veronii Hm091：参考文献：张德锋,刘礼辉,李宁求,等.我国南方地区鱼源气单胞菌不同种类的流行特征[J].水产科学,2015,34(11):673-682.；公众可以从中国农业科学院饲料研究所获得。
嗜水气单胞菌A.hydrophila NJ-1：参考文献：Li J,Ni X D,Liu Y J,et al.Detection of three virulence genes alt,ahp and aerA in Aeromonas hydrophila and their relationship with actual virulence to zebrafish[J].Journal of Applied Microbiology,2011,110(3):823-30.；公众可以从中国农业科学院饲料研究所获得。
E.coli MC1061：BioVector NTCC典型培养物保藏中心，编号：MC1061。
E.coli S17-1(λpair)：BioVector NTCC典型培养物保藏中心，编号：s17-1。
MC1061感受态细胞：将E.coli MC1061单菌落接种于50ml LB液体培养基中，37℃、200rpm培养过夜，按5％的接种量接种到含有500ml LB液体培养基的1l三角瓶中，37℃、250rpm培养，当OD 600nm值为0.35～0.4时，将三角瓶冰浴15～30min(期间摇晃一下三角瓶，使菌液均匀的冷却)，然后将菌液转移至提前冰浴预冷的离心管中，1000g、4℃离心15min，弃上清，用500ml冰浴预冷的去离子水重悬菌体；1000g、4℃离心20min，去掉上清，收集菌体并重悬于250ml冰浴预冷的10％(提及百分比)甘油水溶液中，1000g、4℃离心20min，去掉上清，用1ml冰浴预冷的GYT液体培养基重悬菌体；将菌液分装到冰浴预冷的1.5ml无菌EP管中，每管50μl，用液氮迅速冷却后，放-80℃冰箱保存。
S17(λ)感受态细胞：将E.coli S17-1(λpair)单菌落接种于50ml LB液体培养基中，37℃、200rpm培养过夜，按5％的接种量接种到含有500ml LB液体培养基的1l三角瓶中，37℃、250rpm培养，当OD 600nm值为0.35～0.4时，将三角瓶冰浴15～30min(期间摇晃一下三角瓶，使菌液均匀的冷却)，然后将菌液转移至提前冰浴预冷的离心管中，1000g、4℃离心15min，弃上清，用500ml冰浴预冷的去离子水重悬菌体；1000g、4℃离心20min，去掉上清，收集菌体并重悬于250ml冰浴预冷的10％(提及百分比)甘油 水溶液中，1000g、4℃离心20min，去掉上清，用1ml冰浴预冷的GYT液体培养基重悬菌体；将菌液分装到冰浴预冷的1.5ml无菌EP管中，每管50μl，用液氮迅速冷却后，放-80℃冰箱保存。
斑马鱼：北京大学斑马鱼水体实验室提供的Tu品系斑马鱼。
实施例1、维氏气单胞菌敲除菌的构建
一、敲除质粒的构建
敲除质粒的构建流程示意图见图1，具体步骤如下：
1、用PCR的方法(反应程序：98℃，5min，32×[98℃，20s；58℃，20s；72℃，1min]，72℃，5min)将质粒pRE112线性化，得到线性化质粒片段。
2、以维氏气单胞菌A.veronii Hm091的基因组DNA为模板，采用引物Aer-up-F和引物Aer-up-R组成的引物对进行PCR扩增，得到PCR扩增产物(序列表的序列1，具有上游同源臂)。
Aer-up-F：5'-TGAATTCCCGGGAGAATGATCTCGGCGGTACCTGG-3'；
Aer-up-R：5'-GATCCACACCGGTAAATCAGGGTAGACAGGTTCAG-3'。
3、以维氏气单胞菌A.veronii Hm091的基因组DNA为模板，采用引物Aer-down-F和引物Aer-down-R组成的引物对进行PCR扩增，得到PCR扩增产物(序列表的序列2，具有下游同源臂)。
Aer-down-F：5'-CCTGTCTACCCTGATTTACCGGTGTGGATCTGGAC-3'；
Aer-down-R：5'-GCTTCTTCTAGAGGTTGAGTGAAGGTGGAGCTGAG-3'。
4、使用
Figure PCTCN2018075580-appb-000001
HiFi DNA Assembly Master Mix(NEB，货号：E2621L)将步骤1得到的线性化质粒、步骤2得到的PCR扩增产物和步骤3得到的PCR扩增产物进行同源重组连接(方法参照试剂盒说明书)，得到敲除质粒(序列表的序列3所示的环状质粒，已测序验证)。
二、敲除菌的构建和鉴定
1、将步骤一得到的敲除质粒转化MC1061感受态细胞，25uF、2.5kV、200ohm进行电转。电转后，立即加1ml LB液体培养基，37℃、摇床200r/min培养1-1.5h后，离心，去掉900μl，留100μl重悬，然后全部涂到含30ug/ml氯霉素(Cm)的LB固体平板上，37℃培养过夜。对过夜 后长出的单菌落通过菌落PCR检测筛选出阳性菌落(采用引物Aer-up-F和引物Aer-up-R进行PCR检测得到2348bp大小的条带)。
2、将步骤1鉴定为阳性的单菌落接种至含30ug/ml氯霉素(Cm)的30ml LB液体培养基中，37℃、摇床200r/min培养12h，挑取单菌落提取质粒，采用Hind III限制性内切酶进行酶切鉴定，得到8029bp大小的条带的为阳性质粒。
3、将步骤2鉴定正确的阳性质粒转化S17(λ)感受态细胞，25uF、2.5kV、200ohm进行电转。电转后，立即加1ml LB液体培养基，37℃、摇床200r/min培养1～1.5h后，离心，去掉900μl，留100μl重悬，然后全部涂到含30ug/ml氯霉素(Cm)的LB固体平板上，37℃培养过夜。对过夜后长出的单菌落通过菌落PCR检测筛选出阳性菌落(采用引物Aer-up-F和引物Aer-up-R进行PCR检测得到2348bp大小的条带)。
4、将步骤3鉴定为阳性的单菌落用接种环重新划线到含30ug/ml氯霉素(Cm)的LB固体平板上，37℃培养24h。
5、将维氏气单胞菌A.veronii Hm091接种划线到含30ug/ml氯霉素(Cm)的LB固体平板上，37℃培养24h。
6、分别刮取步骤4得到的菌落和步骤5得到的菌落进行结合(刮取步骤4获得菌落在步骤5的平板上划线)，37℃结合8h。
7、完成步骤6后，将菌落刮下，用LB液体培养基稀释后涂布到含30μg/ml氯霉素(Cm)+100μg/ml氨苄青霉素(Amp)的LB固体平板上，37℃培养24h。
8、完成步骤7后，挑取单菌落，接种到含30μg/ml氯霉素(Cm)+100μg/ml氨苄青霉素(Amp)的LB固体平板上，37℃培养24h。
9、完成步骤8后，挑取单菌落，接种到不含有抗生素的LB固体平板上37℃培养24h。
10、完成步骤9后，用接种环刮取适量菌落划线到含15％(质量百分比)蔗糖的LB固体平板上，15-25℃低温培养3天，筛选质粒丢失菌株。
11、完成步骤10后，挑取50-80个单克隆分别对应划线到含30μg/ml氯霉素(Cm)+100μg/ml氨苄青霉素(Amp)的LB固体平板上，37℃培养24h。
12、完成步骤11后，挑取单菌落通过菌落PCR检测筛选出敲除突变菌株。
采用维氏气单胞菌A.veronii Hm091(WT)作为对照。分别采用引物Aer-up-F和引物Aer-down-R组成的引物对、引物Aer-confirm-F和引物Aer-confirm-R组成的引物对进行鉴定。引物序列和预期产物片段长度见表1。
表1 引物信息和预期结果
Figure PCTCN2018075580-appb-000002
结果如图2所示。引物Aer-confirm-F和引物Aer-confirm-R是针对Aer基因设计的引物，野生菌(WT)扩增到目的片段1722bp，敲除菌(Hm091△aer)未扩增到目的片段；引物Aer-up-F和引物Aer-down-R是针对Aer基因上游序列和下游序列设计的引物，野生菌(WT)扩增片段的序列大约是4000bp，敲除菌(Hm091△aer)扩增片段的序列约为2300bp，正好比野生菌少Aer基因片段大小的约1700bp。
对野生菌和敲除菌进行测序，结果表明，敲除菌是将序列表中序列4所示的DNA片段取代了野生菌基因组气溶素基因中的相应片段得到的。
将其中一株通过上述方法得到的所有敲除菌命名为维氏气单胞菌(Aeromonas veronii)Hm091△aer，维氏气单胞菌(Aeromonas veronii)Hm091△aer简称为维氏气单胞菌A.veronii Hm091△aer，将其中一株进行保藏。
三、维氏气单胞菌(Aeromonas veronii)Hm091△aer的保藏
维氏气单胞菌(Aeromonas veronii)Hm091△aer，已于2017年10月09日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC；地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研 究所；邮编：100101)，保藏编号为CGMCC No.14776。
实施例2、维氏气单胞菌A.veronii Hm091△aer在斑马鱼中的定位
1、将维氏气单胞菌A.veronii Hm091接种至30ml LB液体培养基中，37℃、摇床200r/min培养18h；取2ml菌液，5000r/mim离心10min，去掉上清，用PBS洗两次，离心去掉上清，加1ml 0.1M的碳酸氢钠缓冲液重悬菌体，得到A.veronii Hm091碳酸氢钠缓冲液。
2、将嗜水气单胞菌A.hydrophila NJ-1接种至30ml LB液体培养基中，37℃、摇床200r/min培养18h；取2ml菌液，5000r/mim离心10min，去掉上清，用PBS洗两次，离心去掉上清，加1ml 0.1M的碳酸氢钠缓冲液重悬菌体，得到A.hydrophila NJ-1碳酸氢钠缓冲液。
3、将维氏气单胞菌A.veronii Hm091△aer接种至30ml LB液体培养基中，37℃、摇床200r/min培养18h；取2ml菌液，5000r/mim离心10min，去掉上清，用PBS洗两次，离心去掉上清，加1ml 0.1M的碳酸氢钠缓冲液重悬菌体，得到A.veronii Hm091△aer碳酸氢钠缓冲液。
4、将红色荧光染料(Texas
Figure PCTCN2018075580-appb-000003
-X)(赛默飞世尔科技T7471)按10mg/ml的浓度添加DMSO进行溶解，得到红色荧光染料溶液。将蓝色荧光染料(Pacific BlueTM)(Invitrogen P10163)按10mg/ml的浓度添加DMSO进行溶解，得到蓝色荧光染料溶液。
5、取50μl步骤4得到的红色荧光染料溶液加至步骤2得到的A.hydrophila NJ-1碳酸氢钠缓冲液中，室温避光轻微摇晃孵育1.5h，然后离心取上清，用PBS洗2次，离心去掉上清，最后用1ml PBS重悬，得到A.hydrophila NJ-1红色荧光标记溶液。
6、将50μl步骤4得到的红色荧光染料溶液加至步骤1得到的A.veronii Hm091碳酸氢钠缓冲液中，室温避光轻微摇晃孵育1.5h，然后离心取上清，用PBS洗2次，离心去掉上清，最后用1ml PBS重悬，得到A.veronii Hm091红色荧光标记溶液。
7、将50μl步骤4得到的蓝色荧光染料溶液加至步骤1得到的A.veronii Hm091碳酸氢钠缓冲液中，室温避光轻微摇晃孵育1.5h，然后离心取上清，用PBS洗2次，离心去掉上清，最后用1ml PBS重悬，得 到A.veronii Hm091蓝色荧光标记溶液。
8、将50μl步骤4得到的红色荧光染料溶液加至步骤3得到的A.veronii Hm091△aer碳酸氢钠缓冲液中，室温避光轻微摇晃孵育1.5h，然后离心取上清，用PBS洗2次，离心去掉上清，最后用1ml PBS重悬，得到A.veronii Hm091△aer红色荧光标记溶液。
9、将嗜水气单胞菌A.hydrophila NJ-1接种至30ml LB液体培养基中，37℃、摇床200r/min培养18h；取2ml菌液，5000r/mim离心10min，去掉上清，用PBS洗两次，离心去掉上清，加PBS重悬菌体，得到A.hydrophila NJ-1溶液。
10、将维氏气单胞菌A.veronii Hm091△aer接种至30ml LB液体培养基中，37℃、摇床200r/min培养18h；取2ml菌液，5000r/mim离心10min，去掉上清，用PBS洗两次，离心去掉上清，加PBS重悬菌体，得到A.veronii Hm091△aer溶液。
11、将孵化后2天的斑马鱼随机分为如下5组(将每组鱼分装到6孔板中，每孔10ml水，15尾鱼)：
组A(对照组)：采用PBS感染斑马鱼；
组B(A.hydrophila NJ-1红色荧光标记感染组)：采用A.hydrophila NJ-1红色荧光标记溶液感染斑马鱼，A.hydrophila NJ-1在感染孔中的浓度为4×10 7CFU/ml；
组C(A.veronii Hm091红色荧光标记感染组)：采用A.veronii Hm091红色荧光标记溶液感染斑马鱼，A.veronii Hm091在感染孔中的浓度为4×10 7CFU/ml；
组D(A.hydrophila NJ-1红色荧光标记+无标记A.veronii Hm091感染组)：采用A.hydrophila NJ-1红色荧光标记溶液和A.veronii Hm091溶液感染斑马鱼，A.hydrophila NJ-1在感染孔中的浓度为4×10 7CFU/ml，A.veronii Hm091在感染孔中的浓度为4×10 7CFU/ml；
组E(A.hydrophila NJ-1红色荧光标记+A.veronii Hm091蓝色荧光标记)：采用A.hydrophila NJ-1红色荧光标记溶液和A.veronii Hm091蓝色荧光标记液感染斑马鱼，A.hydrophila NJ-1在感染孔中的浓度为4×10 7CFU/ml，A.veronii Hm091在感染孔中的浓度为4×10 7CFU/ml；
组F(A.veronii Hm091△aer红色荧光标记感染组)：采用A.veronii Hm091△aer红色荧光标记溶液感染斑马鱼，A.veronii Hm091△aer在感染孔中的浓度为4×10 7CFU/ml；
组G(A.hydrophila NJ-1红色荧光标记+无标记A.veronii Hm091△aer感染组)：采用A.hydrophila NJ-1红色荧光标记溶液和A.veronii Hm091△aer溶液感染斑马鱼，A.hydrophila NJ-1在感染孔中的浓度为4×10 7CFU/ml，A.veronii Hm091△aer在感染孔中的浓度为4×10 7CFU/ml。
上述各组感染4h后用4％的多聚甲醛分别将小鱼进行固定；固定好的小鱼在激光共聚焦显微镜下拍照观察细菌在斑马鱼中的定位。
结果如图3所示。在对照组斑马鱼小鱼中，没有观察到任何的荧光信号(A)；在红色荧光标记的嗜水气单胞菌A.hydrophila NJ-1感染组(B)、红色荧光标记的维氏气单胞菌A.veronii Hm091△aer感染组(F)、红色荧光标记的嗜水气单胞菌A.hydrophila NJ-1+维氏气单胞菌A.veronii Hm091△aer感染组中(G)，在斑马鱼小鱼的肠道中可以观察到红色荧光信号，但仅仅局限于肠腔内。然而，在红色荧光标记或蓝色荧光标记的维氏气单胞菌A.veronii Hm091感染组中，在斑马鱼的肠道中不仅可以观察到很强的荧光信号，而且在肠道的周围以及头部都可以检测到荧光信号(C和E)；有趣的是，在红色荧光标记的嗜水气单胞菌NJ-1+有无蓝色荧光标记的维氏气单胞菌A.veronii Hm091感染组中，NJ-1的荧光信号同样可以在斑马鱼肠道的周围以及头部能检测到(D和E)。这些数据表明由维氏气单胞菌分泌的气溶素可以破坏斑马鱼的肠道屏障，促使它本身以及其他气单胞菌特别是嗜水气单胞菌进入到鱼的体内，导致鱼的溶血及死亡。而维氏气单胞菌A.veronii Hm091△aer则不具备破坏斑马鱼的肠道屏障的能力。
实施例3、维氏气单胞菌A.veronii Hm091△aer疫苗的安全性
1、将维氏气单胞菌A.veronii Hm091接种至LB固体培养基上，37℃培养12h，然后挑取单克隆接种至30ml的LB液体培养基中，37℃，摇床200r/min培养18h。
2、将维氏气单胞菌A.veronii Hm091△aer接种至LB固体培养基上， 37℃培养12h，然后挑取单克隆接种至30ml的LB液体培养基中，37℃，摇床200r/min培养18h。
3、分别将步骤1和步骤2培养的维氏气单胞菌A.veronii Hm091和维氏气单胞菌A.veronii Hm091△aer以不同的浓度(2.5×10 8CFU/ml、1.0×10 8CFU/ml、5.0×10 7CFU/ml、3.33×10 7CFU/ml、2.5×10 7CFU/ml和1.67×10 7CFU/ml)感染孵化后5天的斑马鱼(浸浴)，统计96h内斑马鱼的死亡情况。
结果如图4所示。图4中，图4A为3.3×10 7CFU/ml浸浴浓度下的存活时间统计结果，图4B为2.5×10 7CFU/ml浸浴浓度下的存活时间统计结果，图4C为1.67×10 7CFU/ml浸浴浓度下的存活时间统计结果。结果表明，维氏气单胞菌A.veronii Hm091△aer分别在3.3×10 7CFU/ml、2.5×10 7CFU/ml、1.67×10 7CFU/ml的浸浴浓度下没有斑马鱼出现死亡，而感染野生株后，在24小时内可造成斑马鱼100％的死亡率。结果表明敲除Aer明显降低维氏气单胞菌的毒力，维氏气单胞菌A.veronii Hm091△aer在感染浓度≤3.3×10 7CFU/ml下浸浴是安全的。
实施例4、维氏气单胞菌A.veronii Hm091△aer疫苗保护性研究
一、维氏气单胞菌A.veronii Hm091△aer疫苗对斑马鱼血清免疫球蛋白含量的影响
将3月龄的斑马鱼随机分为如下三组：
组1(对照组)：正常饲养斑马鱼2周，每天两次饱食投喂基础饲料；
组2(饲喂免疫组)：正常饲养斑马鱼2周，每天两次饱食投喂含维氏气单胞菌A.veronii Hm091△aer(2×10 7CFU/g)的基础饲料；
组3(浸浴免疫组)：正常饲养斑马鱼2周，每周浸浴含维氏气单胞菌A.veronii Hm091△aer的水一次(2×10 7CFU/ml，浸浴时间12h)，每天两次饱食投喂基础饲料。
经过各组处理后，每组斑马鱼经尾静脉采血，按鱼IGM试剂盒(购自南京建成生物工程研究所)说明书对进行斑马鱼免疫球蛋白指标检测。
根据试剂盒方案测定Logistic曲线，得出免疫球蛋白方程y＝(A-D)/[1+(x/C) B]+D(其中：A＝5.61378；B＝0.36217；C＝0.14147；D＝-0.12176； r 2＝0.99394)。经测定，三组斑马鱼免疫球蛋白浓度平均值如下：对照组:7.304175ng/ml；饲喂免疫组:13.646945ng/ml；浸浴免疫组:16.00466ng/ml。结果表明，经疫苗免疫后斑马鱼血清免疫球蛋白含量均有一定提升。
二、维氏气单胞菌A.veronii Hm091△aer疫苗斑马鱼的血清抗体凝集效价
将3月龄的斑马鱼随机分为如下三组：
组1(对照组)：正常饲养斑马鱼2周，每天两次饱食投喂基础饲料；
组2(饲喂免疫组)：正常饲养斑马鱼2周，每天两次饱食投喂含维氏气单胞菌A.veronii Hm091△aer(2×10 7CFU/g)的基础饲料；
组3(浸浴免疫组)：正常饲养斑马鱼2周，每周浸浴含维氏气单胞菌A.veronii Hm091△aer的水一次(4×10 7CFU/L，浸浴时间12h)，每天两次饱食投喂基础饲料。
经过各组处理后，每组斑马鱼经尾静脉采血，取血清用于测定血清抗体效价。采用96孔血凝板法进行。将维氏气单胞菌A.veronii Hm091培养液12000rpm离心后用等量生理盐水重悬，得到菌悬液(浓度为4×10 7CFU/ml)。用微量移液器于第l孔中加入80μl生理盐水，其余各孔加50μl；向第1个孔中加入20μl的待测血清，吹吸混匀后取50μl加入第2孔(1:2稀释)，再混匀后从第2孔取50μl加入第3孔(1:4稀释)，依此类推至第9孔(1：256稀释)，弃去50μl，第10孔作为对照；向每个孔中加入50μl维氏气单胞菌悬液，吹吸混匀，置于37℃培养箱中孵育1h，放置在4℃条件下过夜检测。
实验重复两次(第一组和第二组)，结果如表2所示。维氏气单胞菌A.veronii Hm091△aer疫苗通过饲喂和浸浴两种方式对成年斑马鱼进行免疫，对照组、饲喂组及浸浴组的血清抗体凝集效价分别为2 3～2 4、2 4～2 5及2 4，饲喂组血清抗体水平略高于对照组血清抗体水平。
表2 抗体效价检测结果
Figure PCTCN2018075580-appb-000004
Figure PCTCN2018075580-appb-000005
三、浸浴和口服维氏气单胞菌A.veronii Hm091△aer疫苗对斑马鱼的相对免疫保护率
组1(对照组，CK)：正常饲养斑马鱼2周，每天两次饱食投喂基础饲料；
组2(饲喂免疫组，T1)：正常饲养斑马鱼2周，每天两次饱食投喂含维氏气单胞菌A.veronii Hm091△aer(2×10 7CFU/g)的基础饲料；
组3(浸浴免疫组，T2)：正常饲养斑马鱼2周，每周浸浴含维氏气单胞菌A.veronii Hm091△aer的水一次(3×10 7CFU/ml，浸浴时间12h)，每天两次饱食投喂基础饲料。
经过各组处理后，每组斑马鱼随机分为如下三组：
组A(A.hydrophila NJ-1+氯化铵)：采用200mg/L氯化铵感染斑马鱼(浸浴)，12h后加入2×10 7CFU/ml的维氏气单胞菌A.veronii Hm091。
组B(A.veronii Hm091)：维氏气单胞菌A.veronii Hm091以2×10 7CFU/ml的浓度感染斑马鱼(浸浴)。
组C(A.veronii Hm091+A.hydrophila NJ-1)：维氏气单胞菌A.veronii Hm091和嗜水气单胞菌A.hydrophila NJ-1以每种菌2×10 7CFU/ml的浓度感染斑马鱼(浸浴)。
统计96h内斑马鱼的死亡情况，并根据公式计算免疫保护率(RPS)，RPS＝(1-免疫组死亡数/对照死亡数)×100％
结果如图5和表3所示。
表3 疫苗保护率统计结果
Figure PCTCN2018075580-appb-000006
Figure PCTCN2018075580-appb-000007
图5中，图5A为NJ-1+氯化铵攻毒时各组的存活率统计结果，图5B为HM091攻毒时各组的存活率统计结果，图5C为HM091+NJ-1攻毒时各组的存活率统计结果。图5结果表明，浸浴免疫组斑马鱼死亡时间延长，但没有明显的保护效果。而饲喂免疫方式对NJ-1+氯化铵、Hm091、Hm091+NJ-1浸浴功毒都有明显的免疫保护作用，斑马鱼存活率有明显提升。
表3结果表明，浸浴免疫组免疫保护率较低，没有起到明显的保护作用。而饲喂免疫组对NJ-1+HM091免疫保护率达到33％，有一定保护作用；对NJ-1+氯化铵组，HM091免疫保护率达到80％，有很强的免疫保护作用。
工业应用
本发明具有以下优点：
1)本发明基于维氏气单胞菌的气溶素导致斑马鱼感染部位损伤进一步促进相关细菌进入鱼体内是引起出血病爆发的主因这一致病机制，从引发感染的维氏气单胞菌出发研制防治出血病的疫苗，避免了长期以来因气单胞菌出血病致病机理不明导致重视引起后期感染细菌而忽视了引发损伤的关键细菌疫苗的开发，导致长期以来细菌性败血症的疫苗防治效果差这一现状；
2)本发明采用的是同源重组原理定点缺失特定基因片段导致基因失活，使获得的基因缺失菌株具有较好的遗传稳定性，避免使用转座子等不稳定基因工程方法构建减毒菌株带来的遗传不稳定的风险。本发明的减毒活疫苗不带有任何抗生素标记，不存在向环境释放抗性菌株或抗生素抗性基因的带来的潜在危害；
3)本发明中使用基因敲除的方法敲除气溶素(Aerolysin)关键毒力因子从而获得减毒株，毒力与亲本菌株相比显著降低，同时较好避免灭活疫苗灭活不彻底带了的安全性问题和灭活试剂带来的副作用；
4)本发明中的减毒活疫苗对不同来源的气单胞菌具有一定的交叉保护力，具有广谱性；
5)本发明中的减毒活疫苗在斑马鱼中具有一定的免疫保护力，说明本发明的减毒活疫苗能有效激活斑马鱼的免疫反应。
6)使用本发明提供的疫苗，可显著增强水产动物的免疫力，提高抗病 表型。
本发明采用无抗生素标记敲除关键毒力因子技术构建的口服或浸浴减毒活疫苗可以在保留免疫抗原性的同时降低病原菌的致病性，免疫保护效果显著，增强天然免疫系统反应和细胞免疫，增强抵抗其它病原菌的感染能力，同时大大减少免疫工作量。

Claims (17)

  1. 重组菌，是将维氏气单胞菌中的气溶素基因敲除得到的。
  2. 如权利要求1所述的重组菌，其特征在于：所述敲除为敲除整个开放阅读框或敲除部分基因区段。
  3. 重组菌，是将特异DNA片段导入维氏气单胞菌进行同源重组后得到的具有所述特异DNA片段的重组菌；所述特异DNA片段包括气溶素基因的上游同源臂和下游同源臂；所述上游同源臂如序列表的序列3自5’端第3092-4276位核苷酸所示；所述下游同源臂如序列表的序列3自5’端第4277-5408位核苷酸所示。
  4. 维氏气单胞菌(Aeromonas veronii)Hm091△aer，保藏编号为CGMCC No.14776。
  5. 权利要求1-3任一所述的重组菌在制备疫苗中的应用；所述疫苗为如下(b1)-(b4)中的任一种：
    (b1)水产动物维氏气单胞菌疫苗；
    (b2)水产动物嗜水气单胞菌疫苗；
    (b3)水产动物气单胞菌疫苗；
    (b4)水产动物出血病疫苗。
  6. 权利要求4所述的维氏(A.veronii)Hm091△aer在制备疫苗中的应用；所述疫苗为如下(b1)-(b4)中的任一种：
    (b1)水产动物维氏气单胞菌疫苗；
    (b2)水产动物嗜水气单胞菌疫苗；
    (b3)水产动物气单胞菌疫苗；
    (b4)水产动物出血病疫苗。
  7. 一种制备疫苗的方法，包括如下步骤：将权利要求1-3任一所述的重组菌作为疫苗的活性成分进行包装；所述疫苗为如下(b1)-(b4)中的任一种：
    (b1)水产动物维氏气单胞菌疫苗；
    (b2)水产动物嗜水气单胞菌疫苗；
    (b3)水产动物气单胞菌疫苗；
    (b4)水产动物出血病疫苗。
  8. 一种制备疫苗的方法，包括如下步骤：将权利要求4所述的维氏气单胞菌(Aeromonas veronii)Hm091△aer作为疫苗的活性成分进行包装；所述疫苗为如下(b1)-(b4)中的任一种：
    (b1)水产动物维氏气单胞菌疫苗；
    (b2)水产动物嗜水气单胞菌疫苗；
    (b3)水产动物气单胞菌疫苗；
    (b4)水产动物出血病疫苗。
  9. 权利要求1-3任一所述的重组菌在制备产品中的应用；所述产品的用途为如下(c1)-(c5)中的至少一种：
    (c1)预防和/或治疗水产动物维氏气单胞菌感染；
    (c2)预防和/或治疗水产动物嗜水气单胞菌感染；
    (c3)预防和/或治疗水产动物气单胞菌感染；
    (c4)预防和/或治疗水产动物出血病；
    (c5)增加水产动物的免疫力。
  10. 权利要求4所述的维氏气单胞菌(Aeromonas veronii)Hm091△aer在制备产品中的应用；所述产品的用途为如下(c1)-(c5)中的至少一种：
    (c1)预防和/或治疗水产动物维氏气单胞菌感染；
    (c2)预防和/或治疗水产动物嗜水气单胞菌感染；
    (c3)预防和/或治疗水产动物气单胞菌感染；
    (c4)预防和/或治疗水产动物出血病；
    (c5)增加水产动物的免疫力。
  11. 水产动物维氏气单胞菌疫苗，其活性成分为权利要求1-3任一所述的重组菌，或，权利要求4所述的维氏气单胞菌(Aeromonas veronii)Hm091△aer。
  12. 水产动物嗜水气单胞菌疫苗，其活性成分为权利要求1-3任一所述的重组菌，或，权利要求4所述的维氏气单胞菌(Aeromonas veronii)Hm091△aer。
  13. 水产动物气单胞菌疫苗，其活性成分为权利要求1-3任一所述的 重组菌，或，权利要求4所述的维氏气单胞菌(Aeromonas veronii)Hm091△aer。
  14. 水产动物出血病疫苗，其活性成分为权利要求1-3任一所述的重组菌，或，权利要求4所述的维氏气单胞菌(Aeromonas veronii)Hm091△aer。
  15. 一种产品，活性成分为权利要求1-3任一所述的重组菌，或，权利要求4所述的维氏气单胞菌(Aeromonas veronii)Hm091△aer；所述产品的用途为如下(d1)-(d5)中的至少一种：
    (d1)预防和/或治疗水产动物维氏气单胞菌感染；
    (d2)预防和/或治疗水产动物嗜水气单胞菌感染；
    (d3)预防和/或治疗水产动物气单胞菌感染；
    (d4)预防和/或治疗水产动物出血病；
    (d5)增加水产动物的免疫力。
  16. 权利要求1-3任一所述的重组菌的应用，为如下(f1)-(f5)中的至少一种：
    (f1)预防和/或治疗水产动物维氏气单胞菌感染；
    (f2)预防和/或治疗水产动物嗜水气单胞菌感染；
    (f3)预防和/或治疗水产动物气单胞菌感染；
    (f4)预防和/或治疗水产动物出血病；
    (f5)增加水产动物的免疫力。
  17. 权利要求4所述的维氏气单胞菌(Aeromonas veronii)Hm091△aer的应用，为如下(f1)-(f5)中的至少一种：
    (f1)预防和/或治疗水产动物维氏气单胞菌感染；
    (f2)预防和/或治疗水产动物嗜水气单胞菌感染；
    (f3)预防和/或治疗水产动物气单胞菌感染；
    (f4)预防和/或治疗水产动物出血病；
    (f5)增加水产动物的免疫力。
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