CN105886429B - A kind of Aeromonas hydrophila attenuation bacterium of antibiotic-free label and application - Google Patents

A kind of Aeromonas hydrophila attenuation bacterium of antibiotic-free label and application Download PDF

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CN105886429B
CN105886429B CN201610241140.7A CN201610241140A CN105886429B CN 105886429 B CN105886429 B CN 105886429B CN 201610241140 A CN201610241140 A CN 201610241140A CN 105886429 B CN105886429 B CN 105886429B
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aeromonas hydrophila
ahfgds
bacterial strain
zyah72
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CN105886429A (en
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周洋
李锦铨
林蠡
王小红
魏顺
李智
喻蔚
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Huazhong Agricultural University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • 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

Abstract

The invention discloses a kind of Aeromonas hydrophila attenuation bacterium of antibiotic-free label and applications.Based on strong virus force bacterial strain ZYAH72, lacked in the bacterial strain by gene engineering method and unmarked screening strategyaerA,alt,ahp,astWithhlyFive virulence factors, obtain five gene deletion strains AHFGDS, and the deposit number of the bacterial strain is CCTCC NO:M2015798.It is found by the applicant that the bacterial strain is compared with wild mushroom, it is pathogenic to be obviously reduced, but still kickback can occur with positive serum.The experiment proved that the infection of wild Aeromonas hydrophila can be resisted using the bacterial strain as a kind of attenuated live vaccine with the susceptible aquatic product animal of effective protection.

Description

A kind of Aeromonas hydrophila attenuation bacterium of antibiotic-free label and application
Technical field
The present invention relates to fresh water aquaculture fish attenuated live vaccines, and in particular to a kind of thermophilic aqueous vapor of antibiotic-free label Monad is attenuated bacterium and application.
Background technique
The bacterial disease frequency of disease development broken out in fish is high, and Epidemic Scope is wide, becomes and restricts the aquaculture of China's fresh water The important bottleneck of industry.
Aeromonas hydrophila is a kind of important fish-pathogenic bacteria, seriously endangers China by its bacterial septicemia caused Or even world aquaculture industry, wherein it is very serious with freshwater fish culturing industry, it is to cause China's freshwater aquaculture industry tremendous economic A kind of primary acute infectious disease of loss.
Caused lesion speed is exceedingly fast after infected by Aeromonas hydrophila fish, and existing research shows that the bacterium has a variety of cause a disease The factor participates in pathogenic course.Through overtesting comparative studies, this seminar has determined the major virulent factor packet for participating in pathogenic course It includes gas lysin (aerA), serine protease (ahp), hemolysin (hly), Cytotonic enterotoxin (alt), metalloproteinases (ast) etc..
The means that mainly prevent of the bacterial septicemia caused at present for the pathogen are antibiotic and thermophilic aqueous vapor unit cell Bacterium inactivated vaccine.But the abuse of antibiotic can cause flora imbalance and internal residual antibiotic etc. in animal body, if the mankind The animal of edible residual antibiotic or product, then may bring a series of food-safety problems, nor meet environmental-friendly With the policy of the strategy of sustainable development.Two vaccines have it is with strong points, the disease-resistant period is long, semelincident immunization, significant effect etc. Feature, wherein attenuated live vaccine, which even more has, prepares simple, immunization route conveniently, and immunizing potency is high, at low cost to wait remarkable advantages.
It is mainly inactivated vaccine that China, which prevents and treats the disease vaccine as caused by Aeromonas hydrophila, but inactivated vaccine would generally Have following defect: inactivation process causes partially or completely to lose protective antigens, can only cause unstable or low SI Immune response, or even correct immune response cannot be excited and cause side effect etc..And attenuated live vaccine passes through gene knockout Mode can reduce the pathogenic of pathogen, while immunogenicity can also be retained so that its immune protective effect is more significant, Also more persistently.Construct attenuated live vaccine by unmarked knockout technology, have the advantages that following significant: immunizing dose is low immune Number is few, and only need to be inoculated with a small amount of live vaccine can cause body to generate strong immunity；It does not need to cause using adjuvant The chance of quick reaction is smaller；Cross protection, live vaccine stimulate body to enhance natural immune system reaction and cellular immunity, Ke Yitong Shi Zengqiang resists the infection ability of other pathogens.
Summary of the invention
The purpose of the present invention is to provide a kind of antibiotic-free label Aeromonas hydrophila be attenuated bacterium, the bacterial strain in On December 29th, 2015 is sent to China typical culture collection center preservation, classification naming: Aeromonas hydrophila (Aeromonas Hydr ophila five-gene deletion strain) AHFGDS, address: Wuhan, China Wuhan University, deposit number: CCT CC NO:M2015798。
The purpose of the present invention is to provide the applications of the Aeromonas hydrophila AHFGDS of antibiotic-free label a kind of.Utilize this Aeromonas hydrophila (the Aeromonas hydrophila five-gene deletion strain) AHFGDS provided is provided It can be prepared into the live vaccine of infected by Aeromonas hydrophila.
In order to achieve the above object, the present invention takes following technical measures:
A kind of acquisition of the Aeromonas hydrophila attenuation bacterium of antibiotic-free label: Aeromonas hydrophila provided by the invention (Aerom onas hydrophila five-gene deletion strain) AHFGDS is strong virus force Aeromonas hydrophila Five gene deletion mutants of the aer A, alt, ahp, ast and hly of ZYAH72, and do not contain any antibiotic resistant gene Or other marker gene.
The bacterial strain is sent on December 29th, 2015 to China typical culture collection center preservation, classification naming: thermophilic water Aeromonas (Aeromonas hydrophila five-gene deletion strain) AHFGDS, address: Wuhan, China Wuhan University, deposit number: CCTCC NO:M2015798.
The five gene-deleted strains bacterial strain is compared with wild strain ZYAH72, and hemolytic activity significantly drops on blood agar plate It is low, not gassiness lysin (aerA), serine protease (ahp), hemolysin (hly), enterotoxin (alt), metalloproteinases (ast) Five virulence factors, in biochemical test in oxidizing ferment, arabinose, aesculin, arginine dihydrolase, glucose produce gas, Salicin is positive reaction, and 6%NaCl peptone water is negative reaction.It is grown in general T SB solid medium or fluid nutrient medium Well, preference temperature needed for growing is 28-30 DEG C.
The purpose of the present invention is to provide a kind of applications of the Aeromonas hydrophila attenuation bacterium of antibiotic-free label, including benefit With Aeromonas hydrophila provided by the invention (Aeromonas hydrophila five-gene deletion strain) AHFGDS is prepared into infected by Aeromonas hydrophila live vaccine or inactivated vaccine, especially suitable for Aeromonas hydrophila in aquatic livestock The prevention and treatment of infection.
The aquatic livestock includes but is not limited to: grass carp, crucian carp, flathead, silver carp, mandarin fish, carp, black carp, Tilapia mossambica, common eel, white crucian carp, The fish such as pond crucian carp, sturgeon, yellow tail silver xenocypris, bream, channel catfish, goldfish, swamp eel, lefteye flounder；It further include the frog, giant salamander, shrimp, crab, soft-shelled turtle, tortoise etc. Aquatic animal.
Aeromonas hydrophila attenuation bacterium of the present invention can also be used in the infected by Aeromonas hydrophila for preparing mammal Vaccine.Compared with prior art, the invention has the following advantages that
1. attenuated live vaccine of the invention is marked without any antibiotic, it is not present to Environment release resistant strain or resists The bring potential hazard of raw element resistant gene；
2. the present invention causes gene to inactivate using homologous recombination principle fixed point deletion specific gene segment, make acquisition Gene deletion strains have preferable genetic stability, avoid constructing attenuation bacterium using the labile genes engineering method such as transposons The risk of strain bring Genomic instability.
3. knocking out five virulence factors using the method for gene knockout in the present invention to obtain attenuated strain, virulence and parent Bacterial strain preferably avoids inactivated vaccine inactivation from being not thorough the safety issue of band and inactivate reagent strip compared to significant decrease Side effect；
4. the attenuated live vaccine in the present invention has certain cross-protection, tool to the Aeromonas hydrophila of separate sources There is broad spectrum activity；
5. the attenuated live vaccine in the present invention all has certain immune protective efficiency in different animal models, illustrate this The attenuated live vaccine of invention can effectively activate the immune response of a variety of hosts.
6. using vaccine provided by the invention, the immunity of aquatic livestock can be significantly increased, disease-resistant phenotype is improved.
Detailed description of the invention
Fig. 1: AHFGDS five gene delection verifies schematic diagram.
Wherein: M:DNA marker；The inside and outside 1-2AHFGDS aerA gene；The inside and outside 3-4ZYAH72aerA gene；5- The inside and outside 6AHFGDS alt gene；The inside and outside 7-8ZYAH72alt gene；The inside and outside 9-10AHFGDS ahp gene；11- The inside and outside 12ZYAH72ahp gene；The inside and outside 13-14AHFGDS ast gene；The inside and outside 15-16ZYAH72ast gene；17- The inside and outside 18AHFGDS alt gene；The inside and outside 19-20ZYAH72alt gene.
The growth curve schematic diagram of two bacterial strains of Fig. 2 ZYAH72 and AHFGDS.
Fig. 3 Aeromonas hydrophila ZYAH72 and attenuated strain AHFGDS the haemolysis schematic diagram on blood plate.
Fig. 4 Aeromonas hydrophila ZYAH72 and attenuated strain AHFGDS Quantitative Haemolytic schematic diagram.
Fig. 5 Aeromonas hydrophila ZYAH72 and attenuated strain AHFGDS tests schematic diagram to the pathogenicity of mouse.
Fig. 6 Aeromonas hydrophila ZYAH72 and attenuated strain AHFGDS tests schematic diagram to the pathogenicity of zebra fish.
Specific embodiment
Technical solution of the present invention is if not otherwise specified the ordinary skill in the art.The reagent or material, If not otherwise specified, both from commercial channel.
Embodiment 1:
The acquisition of the Aeromonas hydrophila attenuation bacterium AHFGDS of antibiotic-free label:
By gene engineering method, by aerA, alt, ahp, ast and the hly five of strong virus force Aeromonas hydrophila ZYAH72 A gene is knocked out, and after screening, is finally obtained five gene-deleted strain AHFGDS of a Hygrophilous monad (hereinafter referred to as For AHFGDS), the mutant strain of acquisition is one plant of attenuated strain without five genes (Fig. 1).The bacterial strain is in 2015 December 29 was sent to China typical culture collection center preservation, classification naming: Aeromonas hydrophila (Aero monas Hydrophila five-gene deletion strain) AHFGDS, address: Wuhan, China Wuhan University.Deposit number: CCTCC NO:M2015798。
Strong virus force Aeromonas hydrophila ZYAH72 (hereinafter referred to as ZYAH72) of the invention is applicant from Hubei province Morbidity crucian body in separation and one plant of strong virus force wild strain screening, the Chinese Typical Representative culture for being stored in Wuhan protects Hiding center, classification naming: Aeromonas hydrophila (Aeromonas hydrophila) ZYAH72, deposit number: CCTCC NO: M2015797, address: Wuhan, China Wuhan University.It has proven convenient that the strain contains aerA (the Genbank number of logging in AHA_0438), Alt (the Genbank number of logging in AHML_00550), ahp (the Genbank number of logging in AHA_2687), ast (the Ge nbank number of logging in AHA_0804) and five kinds of virulence genes of hly (the Genbank number of logging in AHML_02265), the missing of this five virulence genes not shadow The growth rate of the bacterial strain and the requirement to nutrition are rung, shows that the bacterial strain has good genetic stability by passage assays, Use general T SB culture medium culture.
It is demonstrated experimentally that the growth rate of AHFGDS and the demand to nutrition are not significantly different (Fig. 2) with parent strain.It chooses The AHFGDS single colonie in TSB solid medium tablets is taken, is inoculated in fresh TSB fluid nutrient medium, at 28 DEG C, 140rpm is shaken Bed is incubated overnight.Transferred fresh TSB fluid nutrient medium with the ratio of 1:100 (volume ratio) within second day, cultivated under similarity condition to OD595nm is 1.20 or so, and cell concentration is 2.0*10 at this time9CFU/mL.It is washed with PBS and the bacterium for being diluted to various concentration is outstanding Liquid (106-108CFU/mL), as AHFGDS live vaccine is used for following embodiment.
The same AHFGDS of strain culturing mode of ZYAH72, in following embodiment, if not otherwise specified, ZYAH72's used Bacterial strain or AHFGDS bacterial strain are viable bacteria.
Embodiment 2:
The experiment of qualitative and Quantitative Haemolytic shows the reduction of AHFGDS cytotoxicity:
In qualitative test, the single colonie of ZYAH72 and AHFGDS are picked them separately, is put onto sheep blood plate, discovery The haemolysis circle that AHFGDS is formed on blood plate is less than ZYAH72 (Fig. 3).
Fresh sheep erythrocyte is diluted to 1% using physiological saline.
Monoclonal on plate is selected, is cultivated 24 hours into TSB culture medium, 12000rpm is centrifuged 15min, takes supernatant.Make Fresh sheep erythrocyte is diluted to 1% with physiological saline, respectively takes 100 μ L and 100 μ L sheep red blood cell (SRBC)s (1% content) mixed It closes, 37 DEG C of incubation 2h measure its OD540nm light absorption value afterwards.It is observed that AHFGDS is aobvious compared with wild strain ZYAH72 Write the toxicity (Fig. 4) reduced to cell.This is because AHFGDS has been lacked caused by hemolysin gene.
Embodiment 3:
4 week old female Balb/c mouse are that experimental subjects proves AHFGDS virulence attenuation of:
It tests mouse used and defends control centre purchased from Wuhan City, Hubei Province disease, in laboratory rearing change without exception in 3 days Change is used as experiment mice.
Mouse infection experiment: the bacterial concentration of ZYAH72 and AHFGDS are separately adjusted to angularly 1*106CFU/mL, every mouse 200 μ L bacterium solutions are injected intraperitoneally, it is primary every 3h observation, dead mouse quantity is recorded, observing time continues one week, injects wild strain After ZYAH72, mouse is dead 8 interior for 24 hours, and the mouse for injecting AHFGDS shows AH FGDS to the poison of mouse without death Property is obviously reduced (Fig. 5).
Embodiment 4:
Using zebra fish as the infection experiment of object and median lethal dose LD50Measurement:
The zebra fish Wuhan City, Hubei Province Chinese Academy of Sciences is aquatic used in experiment is bought, the long 3-4CM of body, in laboratory rearing Variation without exception in 3 days is used as Experimental fish.
Zebra fish infection experiment: the bacterial concentration of ZYAH72 and AHFGDS are separately adjusted to angularly 2*109CFU/mL, every 3h Observation is primary, records dead zebra fish quantity, and observing time continues one week, as a result sees Fig. 4.Zebra fish is in injection wild strain After ZYAH72,12h is to have 60% zebra fish dead, all dead after 36h, and injects the zebra fish of AHFGDS in test Without death, show that AHFGDS is obviously reduced (Fig. 6) to the toxicity of zebra fish.
Median lethal dose LD50Measurement: the bacterial concentration of ZYAH72 and AHFGDS are separately adjusted to angularly 2*109CFU/mL is dilute Different gradient concentrations (see Table 1 for details for injection dosage) are interpreted into, respectively the healthy zebra fish of intraperitoneal injection, every group 10, every fish belly chamber Inject 20 μ L bacterium solutions, zebra fish death toll in observation one week, the Virulence Difference of more different Aeromonas hydrophilas, LD50It is worth basis Karber method calculates, and the results are shown in Table 1.The virulence of attenuated strain AHFGDS is relative to wild strain it can be seen from testing above ZYAH72 is decreased obviously.Meanwhile it can be observed that there is apparent bleeding in lesion zebra fish abdomen, after dissecting sick fish belly chamber It can be seen that spleen is obviously great, enteron aisle festers.And then without apparent symptom in the abdominal cavity of normal fish body.
LD of the table 1 Aeromonas hydrophila ZYAH72 and attenuated strain AHFGDS to zebra fish50Value
Embodiment 5:
The biological safety of Aeromonas hydrophila AHFGDS:
Sterile disposable sealed bag is taken, wherein the sterile PBS of 1mL is added, 2 zebra fish is placed into, gently helps and rub fish Body 3min or so is to wash lower mucus.It regathers liquid in sealed bag and collects supernatant, supernatant in 12000rpm, 4 DEG C of centrifugation 10min Use the sterilised membrane filter filtration sterilization of 0.22 μ L.It takes 100 μ L mucus that 100 μ L bacterium solutions are added, obtains containing about 1.5*105CFU/mL Initial reaction system, in 28 DEG C of placement 1h after mixing, gradient dilution applies plate count later, calculates the concentration of different strains.
Experimental result is shown in Table 2, show mucus to wild strain ZYAH72 almost without bactericidal effect, and AHFGDS bacterial strain Survival rate is 7.74% or so after handling by mucus, illustrates that attenuated strain is easy to be known by the Fungicidal substance in mucous membrane in the present invention It not and kills, it is difficult to break through the mucous membrane of zebra fish to enter in vivo, the attenuated live vaccine is safe in release in water body.
Table 2: zebra fish mucus sterilization experiment
Embodiment 6:
Aeromonas hydrophila AHFGDS is in the application being prepared into Aeromonas hydrophila live vaccine:
The present embodiment is illustrated for being prepared as live vaccine.
Safety experiment: the long 3-4CM zebra fish of 30 bodies carries out safety detection after raising 4 days in the lab, wherein 3 × 10 are used respectively10CFU/mL AHFGDS impregnates and 4 × 106CFU AHFGDS is injected intraperitoneally fish zebra fish and handles 15 zebras Fish, the results showed that zebra fish used all goes well, and without any death, splanchnotomy is not shown any abnormalities yet.
Immunization experiment:
It selects the long 3-4CM health zebra fish of body at random to test for Immunization, experimental group and processing mode are following (every 15 zebra fish of group):
Immersion immunity group: it is 1*10 that zebra fish, which is immersed in AHFGDS concentration,8In bacterium solution in CFU/mL, soaking time is It 8 minutes, is put into clear water impregnates 3min later, transfer in big water tank and continuously cultivate；
It impregnates control group: zebra fish being immersed in PBS 8 minutes, is put into clear water impregnates 3min later, transfer to big Water tank in continuously cultivate；
Intraperitoneal injection immune group: every zebra fish belly chamber injects 20 μ L and contains 3.30*104The work AHFGDS thallus of CFU Live vaccine；
Control group is injected intraperitoneally: every zebra fish belly chamber injects 20 μ LPBS；
All groups are raised under the same conditions.2 weeks after immune for the first time, carry out in the same manner immune for the second time.The After secondary immunity 2 weeks, attack poison to the above each group with Aeromonas hydrophila wild strain ZYAH72, (attacking toxic dose is 1 LD100).Then two weeks each group situations are observed, death toll is recorded.
The results show that impregnating control group and intraperitoneal injection control group abdomen bleeding after attacking poison with wild strain ZYAH72, occurring The symptom of septicemia, no zebra fish survival；15 zebra fish of immersion immunity group dead 7,15 zebras of immune group are injected intraperitoneally Fish dead 8, there are not any clinical symptoms in the zebra fish of survival, also without observing any lesion after abdominal cavity dissection Phenomenon.
Further, applicant utilizes traditional bacterial strain J-1 (CGMCC NO.3220, CN201010158115 cause of this field Characteristic of disease Aeromonas hydrophila detection kit) Immunization experiment has been carried out to also above-mentioned each group, except attack toxic bacterial strain it is different in addition to, Other conditions with it is above-mentioned identical.The results show that after attacking poison with J-1 bacterial strain, impregnate control group and intraperitoneal injection control group without Zebra fish survival, 15 zebra fish of immersion immunity group dead 7,15 zebra fish of intraperitoneal injection group dead 8.
Embodiment 7:
Aeromonas hydrophila AHFGDS is in the application being prepared into Aeromonas hydrophila live vaccine:
Safety experiment: the healthy grass carp of 100g-150g 30 carries out vaccine safety after raising in the lab 2 weeks Detection, wherein respectively using immersion (soaking concentration 3*1010The live vaccine of CFU/mL AHFGDS) and intraperitoneal injection (abdominal cavity note Penetrating 200 μ L concentration is 3*1010CFU/mL AHFGDS live vaccine, i.e., 6 × 109Every grass carp of CFU) 15 grass carps of processing, as a result table Bright grass carp used all goes well, and without any death, splanchnotomy is not shown any abnormalities yet.
Immunization experiment:
It selects healthy grass carp (100g-150g) at random to test for Immunization, experimental group and processing mode are following (every 15 grass carps of group):
Immersion immunity group: it is 3*10 that grass carp, which is immersed in AHFGDS concentration,8In bacterium solution in CFU/mL, soaking time 8 Minute, it is put into clear water impregnates 3min later, transfer in big water tank and continuously cultivate；
It impregnates control group: grass carp being immersed in PBS 8 minutes, is put into clear water impregnates 3min later, is transferred to big It is continuously cultivated in water tank；
Intraperitoneal injection immune group: every grass carp is injected intraperitoneally 200 μ L and contains 2*107The work epidemic disease of the work AHFGDS thallus of CFU Seedling；
Control group is injected intraperitoneally: 200 μ LPBS are injected intraperitoneally in every grass carp；
All groups are raised under the same conditions.2 weeks after immune for the first time, by same way to above-mentioned each group progress the Secondary immunity.After second 2 weeks immune, the above each group is carried out attacking poison with Aeromonas hydrophila wild strain ZYAH72 and (attacks toxic agent Amount is 1 LD100).Then two weeks each group situations are observed, death toll is recorded.
The results show that impregnating control group and intraperitoneal injection control group abdomen bleeding after attacking poison with wild strain ZYAH72, occurring The symptom of septicemia, no grass carp survival；Dead 4,15 grass carps of immersion immunity group, 15 grass carps of immune group dead 2 are injected intraperitoneally There are not any clinical symptoms in item, the grass carp of survival, also without observing any lesion phenomenon after abdominal cavity dissection.
Further, applicant has carried out Immunization to also above-mentioned each grass carp group using traditional bacterial strain J-1 of this field Experiment, except attack toxic bacterial strain it is different in addition to, other conditions with it is above-mentioned identical.The results show that being impregnated after attacking poison with J-1 bacterial strain Control group and intraperitoneal injection control group are survived without grass carp, and dead 2,15 grass carps of immersion immunity group, 15 grass carps of intraperitoneal injection group Dead 4.
Embodiment 8:
Live vaccine can effectively activating immune system and enhancing sterilizing ability:
Grass carp is immunized in the way of immersion immunity group and intraperitoneal injection immune group in embodiment 7, does not attack poison； Two exempt from therefrom to take within 7 days and 14 days 3 tails, syringe tail vein puncture blood collecting at random afterwards, and blood is divided into two parts collection, and liver is not added in portion Element is used to prepare antiserum；Anticoagulant heparin is added in another, for detecting the sterilizing ability of whole blood.
Experimental result, which is shown, detects anti serum titre by agglutination test and ELISA, detects it at wavelength 450nm Spectrophotometric shows that apparent antigen-antibody reaction has occurred in positive hole, it was demonstrated that the live vaccine can enhance immune system.Two groups Immune group grass carp, the leukocytes phagocytic activity in blood are apparently higher than control group.
Embodiment 9:
Aeromonas hydrophila AHFGDS is in the application being prepared into Aeromonas hydrophila live vaccine:
Safety experiment: 5 week old female Balb/c mouse carry out live vaccine safety detection after raising 3 days in the lab, 10 mouse peritoneals inject 200 μ L and contain 2*108The live vaccine of CFU, the results showed that 10 experiment mices are all gone well, without any Death, splanchnotomy are not shown any abnormalities yet.
Immunization experiment:
3 week old female Balb/c mouse are grouped at random after raising 3 days in the lab.Experimental group and processing mode are such as Under (every group 10):
Subcutaneous inoculation group: every 100 μ L of mouse subcutaneous injection contains 2*106The AHFGDS live vaccine of CFU；
Subcutaneous control group: 100 μ L PBS of subcutaneous injection；
Intragastric group: every 100 μ L of intragastric administration on mice contains 2*107The live vaccine of the AHFGDS of CFU；
Stomach-filling control group: 100 μ L PBS of stomach-filling.
All groups are raised under the same conditions.2 weeks after immune for the first time, by same way to above-mentioned each group progress the Secondary immunity.After second 2 weeks immune, the above each group is carried out attacking poison with Aeromonas hydrophila wild strain ZYAH72 and (attacks toxic agent Amount is 1 LD100).Then each group situation in two weeks is observed, death toll is recorded.
It is complete after subcutaneous control group and stomach-filling control group injection ZYAH72 the results show that after attacking poison with wild strain ZYAH72 Portion is dead；Subcutaneous inoculation group mouse 10 is only without death, and stomach-filling group mouse 10 dead 5.
Meanwhile applicant has investigated the mouse survival situation after attacking poison of J-1, J-1 challenge viral dosage except attack toxic bacterial strain it is different in addition to, Remaining with it is above-mentioned identical.The results show that the dead mouse 5 of subcutaneous inoculation group 10,10 dead mouses 5 of intragastric group Only.
Meanwhile the mouse that applicant exempts from each group for (not attacking poison) after two weeks to two has carried out antibody titer, separates liver, spleen Dirty and small intestine carries out pathology detection.Experimental result, which is shown, detects anti serum titre by agglutination test and ELISA, in wave Its spectrophotometric is detected at long 450nm, shows that apparent antigen-antibody reaction has occurred in positive hole, it was demonstrated that the vaccine has good Immunogenicity.Pathological tissue observation is carried out to each internal organs of mouse simultaneously, as a result two groups of immune group mouse and control group mice Equally, any clinical symptoms are not occurred.

Claims (2)

1. a kind of Aeromonas hydrophila attenuation bacterium of antibiotic-free label is in the live vaccine for preparing infected by Aeromonas hydrophila It is Aeromonas hydrophila AHFGDS using, the described Aeromonas hydrophila attenuation bacterium, deposit number are as follows: CCTCC NO: M2015798；The infected by Aeromonas hydrophila is that Aeromonas hydrophila ZYAH72 infects or Aeromonas hydrophila J-1 infects.
2. application according to claim 1, it is characterised in that: in the work epidemic disease of preparation mammal infected by Aeromonas hydrophila Application in seedling.
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