CN105039221B - One plant of cold water fish probiotics Arthrobacter strain and application thereof - Google Patents
One plant of cold water fish probiotics Arthrobacter strain and application thereof Download PDFInfo
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Abstract
The invention discloses one plant of cold water fish probiotics Arthrobacter strains and application thereof.The present invention is separated from the intestinal mucosa of the rainbow trout parr in the natural mountain stream in Liaoning Province Benxi identifies one plant of bacterial strain to cold water fish with ntiviral characteristic and immunological regulation and enhancing immune function characteristic: HZC10.According to the observation of the morphological feature of bacterium colony and the measurement of 16SrDNA base sequence and homology analysis, bacterial strain qualification result are as follows: HZC10 belongs to arthrobacterium, deposit number are as follows: CGMCC No.10704.In vitro cell experiment proves: arthrobacterium HZC10 bacterial strain and EPC cell (fish epithelial cells) co-culture, and have external anti-IHNV virus in the characteristic of EPC cell Proliferation.Zoopery proves: arthrobacterium HZC10 bacterial strain has immunological regulation to cold water fish and enhances the characteristic of immune function, and arthrobacterium HZC10 bacterial strain individually feeds the characteristic also to cold water fish with anti-IHNV infection.It can be seen that: arthrobacterium HZC10 bacterial strain of the invention has certain application prospect in terms of cold water fish anti-virus aspect and immunological regulation and enhancing cold water fish immune function.
Description
Technical field
The present invention relates to probiotics bacterial strains and application thereof, in particular to one plant of prebiotic Arthrobacter of cold water fish (rainbow trout)
HZC10 bacterial strain and application thereof, the invention belongs to microorganisms technical fields.
Background technique
Probiotics is the normal flora of humans and animals enteron aisle, respiratory tract, genital tract and skin and mucosa system, it is many research at
Fruit discovery lactic acid bacteria and intestinal mucosa collectively constitute protective barrier, prevent the invasion of virus, bacterium etc., the mankind more and more clearly recognize
Know powerful effect of the probiotics to human and animal's life and health.A large amount of Immunology Today results of study have confirmed probiotics
(especially lactic acid bacteria) to the regulatory function of animal cell immunity, humoral immunity and intestinal mucosa local immunity, and to siberian crabapple
The essential and basic decisive action of system development and immune homeostasis；Research in recent years also found that probiotics has and inhibit tumour and resist
Cancer effect so that probiotic lactobacillus in recent years once become modern functions food, animal feed additive, mucosa-immune vaccine,
One of research hotspot of medical domain, or even the research hotspot as modern inherent immunity field.
The anti-infective and antiviral mechanism study of probiotics shows that probiotics not only can be by adjusting mucous membrane in recent years
Flora distribution promotes certain pathogen to agglomerate, and it is prevented to be adhered on mucous membrane, to prevent it from further invading；Or
Person probiotics is directly played a role by being adsorbed on mucous membrane surface (including intestinal mucosa and respiratory mucosa), is adsorbed on mucous membrane surface
Probiotics can degrade viscous protein and using this endogenic nutrition can keep substantially its quantity stablize, thus directly
Bacterium and virus is prevented to infect mucous membrane；Or probiotics is interacted by adherency with mucosal immune system, effectively
Ground stimulation mucosal immune system plays so that mucosal immune system be made to be in suitably stable state of activation (immune homeostasis)
Immunoregulation effect is very beneficial for resisting the invasion of adventitious viruses or bacterium.Therefore pass through the reasonable presence of lactic acid bacteria and fit
Work as stimulation, the defense function of mucosal immune system can be improved, to effectively prevent the absorption and intrusion of virus.
Numerous research reports also demonstrate, are present in the benefit of gastrointestinal tract mucosa, oral cavity respiratory mucosa and genital tract surface
Raw bacterium has dual protective effect to intestinal mucosa and respiratory mucosa as a kind of resident thallus of work, and on the one hand it can be with
Adhere to field planting in mucous membrane surface, safeguards gastrointestinal tract and respiratory mucosa microbial flora balance；Another aspect probiotics can be straight
The mucosal immune system for being connected to the immune system especially host of host occurs interaction and induces mucosa-immune；And it can stimulate
The development of the immune maincenter organ such as spleen, thymus gland and the bursa of farbricius can also promote macrophage vigor or play adjuvant effect；It is logical
The synthesis such as influence mucosal system cell factor and secretion are crossed, to enhance T, B cell to the reactivity worth of antigenic stimulus, is effectively sent out
Wave specific immunity humidification；Mucous membrane surface, which resides probiotics, can activate the associated lymphoid tissue in mucous membrane, increase sIgA
Biosynthesis improves alimentary canal mucous membrane and respiratory mucosa immune function, is generated by induction of lymphocyte and macrophage thin
Intracellular cytokine plays immunoregulation effect, to enhance body mucosa-immune function.
The immune system of fish is more rudimentary, and the inherent immunity system of fish is the anti-infectious principal immune system of fish,
Fish antibody forms, antibody titer longer than the time required to mammal and increases relatively slowly, and cold water fish antibody titer increases then more slowly
A bit, cryogenic conditions will limit the release of antibody, while the immunological memory ability of fish is weaker than mammal very much.Identical immunogene
Route of entry is different, and the immunological effect of generation is different.In fish primary response, the fish antibody duration is longer.Fish tool
There is immunological memory respond, needs if it is desired to obtaining stronger secondary immunoresponse by longer time, while answering again
Answering the antibody titer of generation, to generate antibody titer again than mammal much lower, therefore the effect of the beneficial bacteria of intestinal tract of fish is just
Seem most important.But aquatic products field of probiotic bacteria than terrestrial animal probiotics research and application relatively a little later, but
Research and use development of the probiotics in aquatic products in recent years is rapider, and the use of probiotics, which has become, promotes aquatic livestock raw
Long and control disease important means.
Due to being influenced by regional climate, cold water salmon fishes (Atlantic salmon, rainbow trout etc.) are mostly rich in albumen, unsaturation
Fatty acid, low cholesterol, without perverse between flesh, easy processing, the characteristics of can only surviving in chilly, free of contamination flowing water environment, certainly
Its organic, green, and pollution-free commodity nature is determined, has become the healthy food of the developed countries such as America and Europe high praise, be international mainstream
Cultivation and trade aquatic products kind, but epidemic disease takes place frequently in cold water fish rainbow trout breeding process in recent years, brings to cold water fish culture
Massive losses organize relevant departments expert research cooperation through country, determine that its Major Diseases is infectious hematopoietic organ necrosis
Sick (infectious hematopoitic necrosis, IHN) is that World Organization for Animal Health (OIE) regulation must report
Disease, be China as defined in two class epidemic diseases.Fish body itself probiotics how is screened, is delayed by the anti-infectious function of probiotics
Solution IHN give China's cold water fish aquaculture bring heavy losses, provides safeguard for industry sound development, becomes cold water fish probiotics
The highest priority of research.
Heilongjiang Province has advantageous salmonidae fish germ plasm resource and climatic environment, is China's salmon trout fish-egg and seed
Major production base, therefore, Heilongjiang Province just have the germ plasm resource treasure-house of advantageous salmon fishes cold water fish probiotics,
The use of probiotics has become the important means for promoting aquatic livestock growth and control disease.In recent years, the antiviral work of probiotics
Research has become hot spot, and Antiviral Mechanism also becomes the emphasis of scientists concern.
Arthrobacterium (Arthrobacter sp.) HZC10 bacterial strain that the present invention obtains be arthrobacterium Pseudomonas important member it
One, In vitro cell model has been referred in the research of the interaction of probiotics virus host, and zoopery also demonstrates
Arthrobacterium HZC10 bacterial strain is to the ntiviral characteristic of cold water fish and immunological regulation and enhancing immune function characteristic.
Summary of the invention
There is ntiviral characteristic and immunological regulation and enhancing to exempt from cold water fish the object of the present invention is to provide one plant
The prebiotic Arthrobacter of the cold water fish of epidemic disease functional characteristic (Arthrobacter sp.) HZC10 bacterial strain.
The prebiotic Arthrobacter of cold water fish of the invention (Arthrobacter sp.) HZC10 strain isolation is from Liaoning Province Benxi
The intestinal mucosa of rainbow trout parr in natural mountain stream, the well-grown on GM17 culture medium are screened and are crossed pure through temperature gradient
The one plant of bacterial strain obtained is separated, is named as HZC10, classification naming is arthrobacterium (Arthrobacter sp.), in April, 2015
It is preserved within 9th China Committee for Culture Collection of Microorganisms's common micro-organisms center, microbial preservation number are as follows: CGMCC
No.10704, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.
Arthrobacterium HZC10 bacterial strain has the property that
Microorganism is characterized in: aerobic or facultative anaerobic bacteria, the well-grown on solid and liquid GM17 culture medium；With thermophilic
Cold strain characteristic, the well-grown between 14 DEG C -20 DEG C are hardly grown more than 20 DEG C, and bacterium colony is on GM17 solid medium
Yellow-white, neat in edge, surface is smooth wet, slightly swells, opaque medium sized bacterium colony, has a kind of peculiar Yoghourt fragrant
The taste of sample；Gram's staining typical positive, thallus are median size, the bacillus of form rule；It, can be in pH with acid-resistant property
It is grown on 4.0 GM17 fluid nutrient medium, well-grown on the GM17 fluid nutrient medium between pH 5.0-6.0；With resistance to gallbladder
Salt characteristic can be grown on the GM17 fluid nutrient medium for being 0.3~0.5% containing high cholic acid salinity；.
The 16SrDNA sequence phase of the 16SrDNA sequence of arthrobacterium HZC10 bacterial strain of the present invention and the arthrobacterium delivered at present
It like property up to 98% or more, is analyzed according to 16SrDNA sequence, arthrobacterium HZC10 bacterial strain is accredited as arthrobacterium (Arthrobacter
Sp.), 16SrDNA sequence is as shown in SEQ ID NO:1 in nucleotide sequence.
In vitro cell experiment proves: the arthrobacterium HZC10 bacterial strain and EPC cell (fish epithelial cells) that the present invention screens are altogether
Culture, the characteristic being proliferated on EPC cell with external infectivity resistant Hematopoietic Necrosis disease viral (IHNV).Zoopery card
Bright: the arthrobacterium HZC10 bacterial strain that the present invention screens has immunological regulation to cold water fish and enhances the characteristic of immune function；And this
Inventing the arthrobacterium HZC10 bacterial strain screened and individually feeding to cold water fish also has infectivity resistant Hematopoietic Necrosis disease virus
(IHNV) characteristic infected.
Therefore, further, the invention also provides the cold water fish probiotics Arthrobacter strains to prepare cold water fish
Application in feed addictive.And
The cold water fish probiotics Arthrobacter strain is preparing the viral epidemic disease of cold water fish infectivity resistant Hematopoietic Necrosis's disease
Application in seedling preparation.And
The cold water fish probiotics Arthrobacter strain is in preparing cold water fish vaccine immunity regulator or immunopotentiator
Application.And
The cold water fish probiotics Arthrobacter strain is preparing the application in cold water fish live vector vaccine.
In the present invention, it is preferred to, the cold water fish probiotics Arthrobacter strain be used alone or with cold water fish forage,
Cold water fish vaccine is used in combination.
In the present invention, it is preferred to, the cold water fish vaccine immunity regulator or immunopotentiator are cold by improving
Water fish complement protein C3 is horizontal and then achievees the purpose that adjusting or enhancing cold water fish immune function.
In the present invention, it is preferred to, what the cold water fish probiotics Arthrobacter strain was expressed as heterologous protein secretion
Live vector host strain.
To sum up, the isolated prebiotic Arthrobacter HZC10 bacterial strain of one plant of cold water fish of the present invention has cold water fish disease-resistant
Malicious characteristic and immunological regulation and enhancing immune function characteristic in cold water fish anti-virus aspect and immunological regulation and and enhance cold
There is certain application prospect in terms of water fish immune function.
Detailed description of the invention
Fig. 1 is the morphological feature figure of arthrobacterium HZC10 bacterial strain of the present invention, wherein A figure is the bacterium of arthrobacterium HZC10 bacterial strain
Characteristic pattern is fallen, B figure is the morphological feature figure of arthrobacterium HZC10 bacterial strain under G dyeing microscope；
Fig. 2 is growth curve figure of the arthrobacterium HZC10 bacterial strain of the present invention in the GM17 fluid nutrient medium of different pH value；
Fig. 3 is that proliferation of the arthrobacterium HZC10 bacterial strain of the present invention in the GM17 fluid nutrient medium of different cholic acid salinity is bent
Line chart；
Fig. 4 is that arthrobacterium HZC10 bacterial strain of the present invention and EPC cell (fish epithelial cells) co-culture the examination of extracorporeal antivirus effect characteristic
Test result figure, wherein *: P < 0.05；
Fig. 5 is attribute testing knot of the arthrobacterium HZC10 bacterial strain of the present invention to cold water fish immunological regulation and enhancing immune function
Fruit figure, wherein * * *: P < 0.001.
Specific embodiment
The present invention is further described with attached drawing combined with specific embodiments below, the advantages and features of the present invention will be with
Description and it is apparent.But examples are merely exemplary, and it is not intended to limit the scope of the present invention in any way.Art technology
Personnel should be understood that without departing from the spirit and scope of the invention can details and form to technical solution of the present invention
It modifies or replaces, but these modifications and replacement are fallen within the protection scope of the present invention.
Separation, purifying and the identification of the arthrobacterium HZC10 bacterial strain of the present invention of embodiment 1
GM17 solid culture based formulas (g/L):
Soya peptone 5.0, peptone 2.5, casein peptone 2.5, yeast extract 2.5, powdered beef 5.0, lactose 5.0, Vitamin C
Sour sodium 0.5, sodium β-glycerophosphate 19.0, magnesium sulfate 0.25, agar 15 sterilize 15 minutes for 110 DEG C after dissolution, adjust pH7.2,
Pour plate is spare.
GM17 Liquid Culture based formulas (g/L):
Soya peptone 5.0, peptone 2.5, casein peptone 2.5, yeast extract 2.5, powdered beef 5.0, lactose 5.0, Vitamin C
Sour sodium 0.5, sodium β-glycerophosphate 19.0, magnesium sulfate 0.25 sterilize 15 minutes for 110 DEG C after dissolution, adjust pH7.2, spare.
(1) bacterial strain isolates and purifies
Rainbow trout parr in the natural mountain stream in Liaoning Province Benxi, should reduce to the greatest extent rainbow trout stress, carry out body surface rapidly
Disinfection dissects juvenile fish fish body, with the cotton swab of sterilizing acquisition oral cavity, top, middle part, the lower part of the gill, anus and intestinal tube
The positions such as mucous membrane are wiped repeatedly smearing, with the cotton swab of acquisition respectively in (1) MRS culture medium group: MRS meat soup solid medium
Coated plate is carried out on plate；(2) coated plate GM17 culture medium group: is carried out in GM17 solid medium tablets；(3) LB culture medium group: LB
Coated plate is carried out in solid medium tablets.By three kinds of plates in duplicate distinguish stationary culture in 4 DEG C, 15 DEG C, 20 DEG C, 30 DEG C,
It is cultivated 72 hours in 37 DEG C of incubators.Picking carries out scribing line purifying training again according to the apparent bacterium colony of colonial morphology picking feature
It supports, the repeated multiple times scribing line purifying culture of the apparent bacterium colony progress of picking feature is consistent to separation colonial morphology, size again, chooses
It takes single colonie to carry out Gram's staining observation, will determine as cultivating in the corresponding fluid nutrient medium of single purebred access, obtain
The separation bacteria strain of purifying.In order to obtain low temperature resistant psychrophile strain, the most suitable growth training is selected to the bacterial strain isolated and purified
It supports base (it was found that arthrobacterium HZC10 bacterial strain optimum medium is GM17 culture medium) and carries out temperature gradient screening, selected temperature is
37 DEG C, 30 DEG C, 20 DEG C, 15 DEG C, 4 DEG C, discovery arthrobacterium HZC10 bacterial strain can grow at 4 DEG C, grow between 14 DEG C -18 DEG C
Well, it is not grown more than 20 DEG C.Its cultural characteristic is: aerobic or facultative anaerobic bacteria, raw on solid and liquid GM17 culture medium
Long good, bacterium colony is yellow-white on GM17 culture medium, and neat in edge, surface is smooth wet, slightly swells, and opaque is medium big
Small bacterium colony has a kind of taste of peculiar Yoghourt perfume (or spice) sample；Gram's staining typical positive, Gram's staining typical positive, bacterium
Body is median size, the bacillus of form rule, finally by the pure culture conservation of the GM17.Arthrobacterium HZC10 bacterial strain bacterium colony
Feature is as shown in Figure 1A, and it is as shown in Figure 1B that G dyes micro- sem observation.
(2) physiological and biochemical property of bacterial strain
Arthrobacterium HZC10 bacterial strain has psychrophile strain characteristic, and the well-grown between 14 DEG C -20 DEG C is several more than 20 DEG C
It does not grow；With acid-resistant property, can be grown on the GM17 fluid nutrient medium of pH 4.0, between pH 5.0-6.0
Well-grown on GM17 fluid nutrient medium；It, can be in the GM17 for being 0.3~0.5% containing high cholic acid salinity with bile tolerance characteristic
It is grown on fluid nutrient medium；The physiological and biochemical property of arthrobacterium HZC10 bacterial strain are as follows: glycerol (+)；Red tinea alcohol (-)；D- I
Uncle is sugared (+)；L-arabinose (+)；Ribose (+)；D- xylose (-)；L- xylose (-)；Adonite (-)；Galactolipin (-)；Portugal
Grape sugar (+)；Fructose (-)；Mannose (-)；Sorbose (-)；Rhamnose (-)；Melampyrin (-)；Inositol (-)；Mannitol
(+)；Sorbierite (-)；Alpha-Methyl-D-MANNOSE glycosides (-)；Alpha-Methyl-D-Glucose glycosides (-)；N- acetyl-aminoglucose (+)；
Amarogentin (+)；Arbutin (+)；Aesculin (+)；Salicin (-)；Cellobiose (-)；Maltose (-)；Lactose (-)；Honey
Disaccharides (-)；Sucrose (-)；Trehalose (-)；Synanthrin (-)；Pine three is sugared (-)；Gossypose (-)；Starch (-)；Glycogen
(-)；Xylitol (-)；It is sugared (-) to hold together ox；D- turanose (+)；D- lyxose (-)；D-Tag (-)；D- rock sugar
(-).(note: (+) indicates available；(-) expression cannot utilize).
(3) the 16srDNA sequence analysis of bacterial strain
Using 16srDNA universal primer, the PCR product of 16srDNA is obtained, PCR product after purification is sequenced, is tied
Fruit discovery: the 16SrDNA of the 16SrDNA sequence (shown in SEQ ID NO:1) of bacterial strain HZC10 and the arthrobacterium delivered at present, together
Source property reaches 98% or more.Comprehensive morphological feature and 16SrDNA Sequence Identification, bacterial strain HZC10 are arthrobacterium.
The above-mentioned Strain Designation screened is HZC10, and classification naming is arthrobacterium (Arthrobacter sp.), in
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, microbial preservation number on April 9th, 2015
Are as follows: CGMCC No.10704, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences's microbe research
Institute.
The thermophilic cold characteristic research of the arthrobacterium HZC10 bacterial strain of the present invention of embodiment 2
The unique thermophilic cold characteristic of arthrobacterium HZC10 bacterial strain:
(1) experimental method: preparing solid and liquid GM17 culture medium, arthrobacterium HZC10 bacterium solution crossed on culture medium,
Be respectively set 14 DEG C, 20 DEG C, 25 DEG C, 30 DEG C and 37 DEG C totally 5 cultivation temperatures cultivated, be observed continuously 4 days.
(2) experimental result: well-grown (being shown in Table 1) between 14 DEG C -18 DEG C, bacterium colony is milky white on GM17 culture medium
Color, neat in edge, surface is smooth wet, and protuberance, opaque relatively macrocolony is not grown more than 20 DEG C, without appointing on culture medium
What its bacterium colony occurs, and illustrates that the bacterial strain is typical psychrophile strain.
Table 1
| Temperature | 4℃ | 14℃ | 18℃ | 20℃ |
| Strain growth situation | Well-grown (√) | Well-grown (√) | (√) can be grown | It does not grow (×) |
The acid-resistant property of the arthrobacterium HZC10 bacterial strain of the present invention of embodiment 3 is studied
The unique acid-resistant property of arthrobacterium HZC10 bacterial strain:
(1) experimental method: arthrobacterium HZC10 bacterial strain in 15 DEG C of condition of culture, observe bacterial strain different pH (pH3.0,
PH4.0, pH5.0, pH6.0 and pH6.5) GM17 fluid nutrient medium in growing state.
(2) experimental result: arthrobacterium HZC10 bacterial strain has good adaptability in the culture medium of different pH, and pH is
It can be proliferated in acid GM17 culture medium when 4.0；Proliferation is very fast in the slant acidity GM17 culture medium that pH is 5.0；PH value is
It is proliferated rapidly in 6.5 weakly acidic pH GM17 culture medium.It is low in enteron aisle to show that arthrobacterium HZC10 has for the growth curve for seeing Fig. 2
Tolerance under pH environment, arthrobacterium HZC10 bacterial strain have the ability colonized in intestinal acidity environment.
The resistance to cholate characteristic research of the arthrobacterium HZC10 bacterial strain of the present invention of embodiment 4
The unique resistance to cholate characteristic of arthrobacterium HZC10 bacterial strain:
(1) experimental method: arthrobacterium HZC10 bacterial strain observes bacterial strain dense containing different cholates in 15 DEG C of condition of culture
Spend the growth feelings in the GM17 fluid nutrient medium of (cholic acid salinity 0.10%, 0.20%, 0.30%, 0.40% and 0.50%)
Condition.
(2) experimental result: can be with fast breeding in the case where salinity containing cholic acid is 0.0%~0.2% GM17 culture medium；?
The GM17 culture medium that salinity containing cholic acid is 0.3~0.5% still can be comparatively fast proliferated.The growth curve for seeing Fig. 3, shows arthrobacterium
HZC10 bacterial strain has the ability colonized in the high cholate of enteron aisle.
The extracorporeal antivirus effect characteristic research of the arthrobacterium HZC10 bacterial strain of the present invention of embodiment 5
Arthrobacterium HZC10 bacterial strain and EPC cell (fish epithelial cells) co-culture extracorporeal antivirus effect attribute testing:
(1) experimental method: EPC cell is tested first 2 days in testing the culture of recovery in first 7 days and passes on cell in addition again
In the M199 culture medium of 10% heat-inactivated fetal bovine serum, adjustment cell density is 1 × 107/ mL is laid in 96 orifice plates, every hole 100
μ L cell suspension, is incubated at 28 DEG C, 3%CO2Under the conditions of.Be observed continuously cell growth state, experiment cover in cell (1.8 ×
106A/hole) after start within second day.Experiment starts to carry out within first 3 days HZC10 arthrobacterium culture to be measured, takes out bacterium solution 1.5mL and surveys
OD600Value, it is 1 that adjustment strain to be tested, which reaches OD value, takes out 300 μ L bacterium solutions by infection multiplicity (MOI) for 1:40 and is centrifuged in sterilizing
Guan Zhong, 8000g are centrifuged 2 minutes, 100 μ L of gentle aspiration bacterium solution culture supernatant, remaining culture liq supernatant are discarded, by remaining thallus
And 100 μ L of bacterium solution culture supernatant is added in 200 μ L cell culture mediums, (cell culture medium contains heat inactivation tire ox blood for mixing
Clearly, dual anti-) keep phage-phage supernatant-cell culture medium mixed liquor final concentration of containing 2% heat-inactivated fetal bovine serum and 1 ‰ pairs
It is anti-.Culture solution in 96 orifice plates for covering with cell is sucked, pays attention to having to softly guaranteeing that cell quantity is consistent, 100 μ are added in every hole
(100 μ L PBS are added in control group to the Bacteria Culture supernatant and cell culture medium mixed liquor of L strain to be tested and cell culture medium is mixed
Close liquid), while IHNV virus liquid (potency 10 is added by 1:1000 in co-culture medium8.5TCID50).It co-cultures in containing
There are 15 DEG C, 3%CO2Under the conditions of co-culture for 24 hours, respectively draw test group and control group culture supernatant, measure TCID50Value, into
Row compares.
(2) the result shows that: arthrobacterium HZC10 bacterial strain group, virus titer 108.3TCID50, control group is
108.5TCID50, statistically analyze and show significant difference (P < 0.05) (see Fig. 4).Show arthrobacterium HZC10 and EPC cell (on fish
Chrotoplast) co-culture the characteristic with extracorporeal antivirus effect (IHNV).
The immunological regulation of the arthrobacterium HZC10 bacterial strain of the present invention of embodiment 6 and the characteristic research of enhancing immune function
Zoopery verifies arthrobacterium HZC10 bacterial strain to the characteristic of the immunological regulation of cold water fish and enhancing immune function.
6.1 experimental animals:
Selection rainbow trout average weight is 20g juvenile fish, tests previous moon grouping raising in 15 DEG C of constant temperature and supplies oxygen recirculated water
In race's case, 50 tails in each independent sink.
6.2 experimental methods:
Before on-tests, rejuvenation and secondary culture are carried out to thermophilic cold prebiotic candidate strain HZC10, tests first 2 days and carries out 1:
100 switching cultures, take out bacterium solution when bacterial strain reaches logarithmic phase and survey OD600Value.Rainbow trout mash feed and bacterium solution use small-sized
Coating pellet is made in grain fodder machine, and the coating feed prepared is slightly dried in 20 DEG C of air blast constant incubators.Every group
Fish feeding contains bacterium 2 × 107The pellet (the daily feeding 20g/50 of feed containing bacterium tail) of cfu/g, while parallel control group is set
1 group (daily feeding chow diet 20g/50 tail), every group is 50 tails.Every day entry feed intake and the state of mind etc., continuously
After a week, kidney and spleen (have 3 in the disposable EP pipe for being placed in sterilizing respectively in aseptic collection head-kidney and part in pipe for feeding
A sterilizing steel ball), tissue grinder is crushed with oscillator immediately, 10000g relative centrifugal force 2 minutes, take immediately immediately
The content of the clear ELISA kit measurement Complement C_3 with fish complement protein C3, and be compared.
6.3 experimental result
The result shows that: feeding group feed intake and the state of mind and control group indifference, feed intake are normal (at 2 minutes
Interior whole, which is robbed, has eaten), the state of mind is good；And the C3 content of feeding group kidney and spleen is extremely significant higher than control group, difference
Extremely significant (P < 0.001) (see Fig. 5).Show that HZC10 new strains have fabulous immunological regulation to cold water fish and enhance immune function
The characteristic of energy.
The arthrobacterium HZC10 bacterial strain of the present invention of embodiment 7 studies the ntiviral characteristic of cold water fish
Zoopery verifies arthrobacterium HZC10 new strains to the ntiviral characteristic of cold water fish.
7.1 experimental animals:
Selection rainbow trout average weight is 20g juvenile fish, tests previous moon grouping raising in 15 DEG C of constant temperature and supplies oxygen recirculated water
In race's case, 50 tails in each independent sink.
7.2 experimental methods:
Before on-test, thermophilic cold prebiotic candidate strain HZC10 carries out rejuvenation and secondary culture, tests first 2 days progress 1:100
Switching culture takes out bacterium solution when bacterial strain reaches logarithmic phase and surveys OD600Value.Taken bacteria suspension bacterium is set to reach 2 × 107CFU/mL。
Coating pellet is made using minitype fodder-pelleting machine in rainbow trout mash feed and bacterium solution, and the coating feed prepared is at 20 DEG C
It is slightly dried in air blast constant incubator.Every group of fish feeding contains bacterium 2 × 107The pellet of cfu/g (raise containing bacterium by daily feeding
Expect 20g/50 tail, daily matching while using), while 1 group of parallel control group (daily feeding chow diet 20g/50 tail) is set, every group
It is 50 tails.Every day entry feed intake and the state of mind etc., continuous feeding after a week, carry out attacking malicious (fish infectious hematopoietic organ
Necrotic virus IHNV), it attacks on the day of poison fasting one day, takes out 40 tail test fish at random in each test group, use MS-222 (anesthesia
Agent) test fish is impregnated to anesthesia in batches, dorsal fin injection, which is carried out, with the IHNV virus liquid of 8 LD50 dosage immediately attacks poison, 100 μ L/
Tail.23d is observed continuously after attacking poison, counts the accumulative death toll of each group, and calculates relative immunity protective rate (Relative
Percent Survival, RPS): RPS=(1-MiMc) × 100%, in formula: Mi is the immune group death rate；Mc is control group
The death rate.
7.3 experimental result
The result shows that: the experimental group of feeding arthrobacterium HZC10 new strains can make cold water fish rainbow trout obtain 22.5% to attack
Poison protection, and control group 100% is dead.It the results are shown in Table 2.
Table 2
Claims (8)
1. the one plant of prebiotic Arthrobacter of cold water fish (Arthrobacter sp.) bacterial strain, is named as HZC10, microbial preservation number is
CGMCC No.10704, the deposit date is on April 9th, 2015, and depositary institution is China Committee for Culture Collection of Microorganisms
Common micro-organisms center.
2. application of the cold water fish probiotics Arthrobacter strain described in claim 1 in preparation cold water fish forage additive.
3. cold water fish probiotics Arthrobacter strain described in claim 1 is preparing cold water fish infectivity resistant Hematopoietic Necrosis's disease
Application in virus vaccine formulations.
4. application according to claim 3, which is characterized in that the cold water fish probiotics Arthrobacter strain is used alone
Or it is used in combination with cold water fish forage, cold water fish vaccine.
5. cold water fish probiotics Arthrobacter strain described in claim 1 is preparing cold water fish vaccine immunity regulator or immune increasing
Application in strong agent.
6. application according to claim 5, which is characterized in that the cold water fish vaccine immunity regulator or Immune-enhancing effect
Agent is that cold water fish complement protein C3 is horizontal to achieve the purpose that adjusting or enhancing cold water fish immune function by improving.
7. cold water fish probiotics Arthrobacter strain described in claim 1 is preparing the application in cold water fish live vector vaccine.
8. application according to claim 7, which is characterized in that the cold water fish probiotics Arthrobacter strain is as external source
The live vector host strain of protein secretion expression.
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| CN1668328A (en) * | 2002-07-15 | 2005-09-14 | 诺瓦提斯公司 | Vaccine against salmonid rickettsial septicaemia based on arthrobacter cells |
| CN1668538A (en) * | 2001-04-20 | 2005-09-14 | 上海金迪生物技术工程有限公司 | Extinction type biological treatment machine for organic garbage |
| CN1751064A (en) * | 2003-02-14 | 2006-03-22 | 诺瓦提斯公司 | Hsp60 from arthrobacter |
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| CN1668538A (en) * | 2001-04-20 | 2005-09-14 | 上海金迪生物技术工程有限公司 | Extinction type biological treatment machine for organic garbage |
| CN1668328A (en) * | 2002-07-15 | 2005-09-14 | 诺瓦提斯公司 | Vaccine against salmonid rickettsial septicaemia based on arthrobacter cells |
| CN1751064A (en) * | 2003-02-14 | 2006-03-22 | 诺瓦提斯公司 | Hsp60 from arthrobacter |
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Address after: 150001, Ma 427 street, Nangang District, Heilongjiang, Harbin Patentee after: HARBIN VETERINARY Research Institute CHINESE ACADEMY OF AGRICULTURE SCIENCE Patentee after: Harbin national biological Polytron Technologies Inc. Patentee after: Heilongjiang River Fisheries Research Institute of fisheries, Chinese Academy of Fishery Sciences Address before: 150001, Ma 427 street, Nangang District, Heilongjiang, Harbin Patentee before: HARBIN VETERINARY Research Institute CHINESE ACADEMY OF AGRICULTURE SCIENCE Patentee before: HARBIN ANIMAL BIOLOGICAL PRODUCTS NATIONAL ENGINEERING RESEARCH CENTER CO.,LTD. Patentee before: Heilongjiang River Fisheries Research Institute of fisheries, Chinese Academy of Fishery Sciences |
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Granted publication date: 20190416 |