CN103141425B - Producing method of sterile zebra fish - Google Patents
Producing method of sterile zebra fish Download PDFInfo
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- CN103141425B CN103141425B CN201310096246.9A CN201310096246A CN103141425B CN 103141425 B CN103141425 B CN 103141425B CN 201310096246 A CN201310096246 A CN 201310096246A CN 103141425 B CN103141425 B CN 103141425B
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
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Abstract
The invention discloses a producing method of a sterile zebra fish. The producing method comprises the following steps of: (1), putting a zebra fish oosperm to AB-GZM solution for cultivating for 3 hours to 8 hours at 25 DEG C to 29 DEG C; (2), rinsing the zebra fish oosperm by the AB-GZM solution; (3), soaking the rinsed zebra fish oosperms in 0.005g/L of povidone-iodine aqueous solution for one minute to two minutes; (4), rinsing the soaked zebra fish oosperm by sterile GZM solution; (5), bleaching the rinsed zebra fish oosperm by bleaching agent for 10 minutes to 20 minutes; (6), rinsing the bleached zebra fish oosperm by the sterile GZM solution, and putting the oosperm in the sterile for cultivating at 25 DEG C to 29 DEG C after bleaching, wherein the photoperiod is L10:D14 to L14:D10; and (7), changing half of the sterile GZM solution every day after the embryo is hatched, and obtaining the sterile zebra fish in the hatched zebra fish. According to the producing method of the sterile zebra fish disclosed by the invention, the zebra fish oosperm is obtained by natural propagation, and finally the sterile zebra fish is obtained. The producing method provides convenient materials for researching the interaction between the microorganism and the zebra fish as well as a plurality of biological processes; furthermore, the producing method has a very important reference meaning to further researching the internal mechanism of a relationship between the microorganism and a vertebrate host thereof.
Description
Technical field
The present invention relates to the production method of a kind of aseptic zebra fish.
Background technology
The bacterial disease theory being proposed by Pasteur and Ke He in 1870 has changed our generalities greatly, and the various relations of we and microorganism have been described.The achievement of bacterium theoretical research is having far-reaching progress aspect medical science and public health, and microorganism is had to generalities widely, for example a pathogene.In host and microbial interaction relation, that long-term viewpoint centered by pathogene can be ignored this fact by let us, be that the most of microbe that animal runs into does not cause obvious disease, and can be considered to the emigration people in symbiosis or mutualism relation with host.These microorganism residents comprise bacterium, and archeobacteria and eukaryotic microorganisms wherein have many can not cultivation in vitro.In the genome epoch, interactional research let us between nonpathogenic host and microorganism is benefited huge, and the composition of let us to these microbiologic populations and the cognition of ability are significantly increased.The large main body of this research is set up, and the non-pathogenic member of animal microorganism is having brought into play significant effect aspect the normal postnatal growth of animal and physiology, from immune stable state in body to metabolic aspect.In addition, micropopulation is also relevant with the morbidity of numerous disease, comprises allergy, inflammatory bowel disease, cancer and obesity.The raising of the understanding that molecule between host and its field planting microorganism is exchanged makes us be hopeful to find the new medical treatment strategy that promotes human and animal's health.
We known to now how to contribute to host's biology and pathology about microbiologic population be all to obtain from research germfree animal.Aseptic this word be derive from Greece's word " gnosis " (knowledge) and " bios " (life), and refer to a kind of experimental situation be microorganisms all in environment be all determine or do not have.The foundation of aseptic experiment is that basis is in the ability without any cultivated animals under microbiological condition, with specific microorganism fungus kind field planting or more complicated cooperation in its body.The concept of aseptic experiment can be traced back to Pasteur, and he is that animal do not having under the condition of microorganism in the hypothesis of 1885 propositions is impossible survival.This hypothesis has only just been overthrown after 11 years, and Nuttal in 1896 and Thierfelder produce first germfree animal---cavy with aseptic caesarean birth, and support 13 days.This reports at the beginning and just successfully produces in the near future aseptic chicken, goat and a group other animals, birds and amphibian.Although what these were early stage studies confirm that animal can survive under the condition that there is no microorganism, but the continuous production of aseptic vertebrate is to beginning to all not succeeding eventually, until Reyniers in 1940 and his colleague just can succeed in Lun De university at University of Notre Dame and Gustafsson and his colleague.The success of the continuous production of aseptic vertebrate is mainly owing to obtaining crucial progress in germfree animal alimentology.Although the feasibility of germfree animal has been overthrown the hypothesis of Pasteur, be right on the one hand, there is no can affect in microorganism situation many aspects of animal biology.
Although most aseptic vertebrate research all concentrates on mammal and birds, also someone attempts cultured fishes under aseptic condition.Baker and Ferguson report successfully to obtain aseptic fish for the first time, ovoviviparity platy, after absorption of yolk at its with sterilization forage feed several time-of-weeks of can surviving.After this discovery, successfully obtain again aseptic oviparity Tilapia mossambica, many oviparity salmon kind, many ovoviviparity flower Medaka section fish and many oviparity sheepshead.Although the report of these initiatives confirms aseptic fish and can survive within a certain period of time, but they do not comprise grown form and the physiological property of aseptic fish, the specific nutrition demand of aseptic fish is not described yet, do not assess the result in aseptic fish field planting with microorganism, can not obtain and grow into sexually matured aseptic fish yet.
In in the past 30 years, zebra fish has become the model animal of an important basis and biomedical research.Zebra fish-egg is fertilized in vitro, and this causes embryo before hatching becomes free living juvenile fish---and-after fertilization 3 days is to grow in its diaphragm.It is after fertilization 5 days that zebra fish juvenile fish starts feed, and young fish is in after fertilization beginning in 14 days to the development by metamorphosis of adult fish.Zebra fish has extra attractability feature and analyzes the interaction between host and microorganism, comprise abundant genetic background and genetic resources, in growth course, optical transparency characteristic allows us to observe in vivo host and microbial cell, and can control further genescreen (host and microorganism can) and Chemical Screening.These attributes, in conjunction with zebra fish and mammalian genes group, have autoploidy widely between anatomical level and physiological level, allow zebra fish as human biology and useful model animal of pathology.
Germfree animal pattern is convenient to a series of parameter of molecule, first, germfree animal can for microorganisms how field planting or microbial metabolic products how to affect host living beings process, comprise gene expression, grow physiology, immunity and life-span.This can make germfree animal be exposed to indivedual bacterial classifications at particular point in time, and the reaction that then specific microorganism or product analyze host completes.Host's function and the gene outcome of microorganism can detect by carrying out genetic manipulation at corresponding bacterial classification.The second, germfree animal is that an extraordinary platform goes interaction between microorganisms and microorganism in live body host's physiological environment.We can allow their field planting in aseptic host with the combination of definite microbe species or genotype, then just can be with cultivating or the competition of DNA sequence dna detection microbiologic population between in vivo.Interaction between these hosts and microorganism and microorganism and microorganism can be used as a function and is used for analyzing microbial gene type, community competition, host gene type, host's developmental stage, host's physiology, the history of field planting microorganism in host, anatomy location, diet and other environment and physiological parameter.
Share except these experimental techniques between aseptic zebra fish and aseptic mammal, zebra fish also has unique advantage, so zebra fish should come into one's own in gnotobiology field.One of them advantage is that zebra fish allows us to observe host's active somatic cell at the optical transparency of the early property of growing, microbial cell and molecular events.Although can only observe the live cell of zebra fish and the cell of microorganism in the bright visual field of optics, these researchs can strengthen by go to express report fluorescin by the genetic engineering modified specific adjusting sequence of host or microorganism.In addition the fluorescence probe that, the external world provides can in vitro or detect specific cellular type and biological processes in body.
Summary of the invention
The object of this invention is to provide the production method of a kind of aseptic zebra fish.
The production method of aseptic zebra fish provided by the present invention, specifically can comprise the steps:
(1) zebra fish fertilized egg is placed in to AB-GZM solution, in 25-29 ℃, and the temperature difference is less than under the condition of 0.5 ℃ and cultivates 3-8h;
(2) described fertilized egg after treatment step (1) is carried out to rinsing with described AB-GZM solution;
(3) the described fertilized egg after step (2) rinsing is placed in to the povidone iodine aqueous solution that concentration is 0.005g/L, soaks 1-2min(and note: can not exceed 2min, otherwise can increase rate of embryonic death);
(4) the described fertilized egg after step (3) immersion is carried out to rinsing with aseptic GZM solution;
(5) the described fertilized egg after step (4) rinsing is bleached to 10-20min with bleaching agent, can jiggle several times during this time, described fertilized egg is suspended in described bleaching agent, notice that solution does not have drum;
Described bleaching agent is that concentration is the aqueous sodium hypochlorite solution of 0.02g/L.
(6) the described fertilized egg after step (5) bleaching is carried out to rinsing with described aseptic GZM solution, after rinsing, described fertilized egg being placed in to described aseptic GZM solution cultivates, cultivation temperature is 25-29 ℃, and the photoperiod is L10:D14 to L14:D10, until described incubating oosperm obtains young fish;
(7) half amount of described young fish being carried out to described aseptic GZM solution every day is changed liquid and (by old broth out half volume, is supplemented the new described aseptic GZM solution of respective volume; Note egg membrane sucking-off to be gone in order to avoid affect water quality when changing liquid for the first time simultaneously) cultivate, until described young fish grows up to the zebra fish for object size, from the zebra fish of described object size, obtain the aseptic zebra fish of object.
In one embodiment of the invention, the zebra fish of described object size is specially after described incubating oosperm the zebra fish of 1-6 days.
The solvent of described GZM solution is water, and solute is sea salt; Described sea salt is in every liter of described GZM solution, to contain sea salt described in 60mg at the content of described GZM solution.
The solvent of described AB-GZM solution is water, and solute and concentration thereof are as follows: the streptomycin of the ammonia benzyl mycin of the amphotericin B of 250ng/mL, the kanamycin of 5 μ g/mL, 100 μ g/mL, the penicillin of 10U/ml, 10U/ml, the described sea salt of 59 μ g/mL.
In said method, in step (1), the temperature of described cultivation is specially 28.5 ℃; The time of described cultivation is specially 4.5h.
In said method, in step (3), the time of described immersion is specially 1min.
In said method, in step (5), the time of described bleaching is specially 15min.
In said method, in step (6), the temperature of described cultivation is specially 28.5 ℃; The photoperiod of described cultivation is specially L14:D10; Described fertilized egg is placed in to described aseptic GZM solution and cultivates, culture density is in aseptic GZM solution, to cultivate fertilized egg as described in 40-60 (as 50) described in every 100mL.
In said method, in step (7), the method that obtains the aseptic zebra fish of described object from the zebra fish of described object size specifically can comprise the steps: the described GZM solution for the zebra fish of cultivating described object size to carry out aseptic detection, and the zebra fish of the described object size of cultivating in aseptic described GZM solution is after testing considered as aseptic zebra fish.
In one embodiment of the invention, the method for described " the described GZM solution for the zebra fish of cultivating described object size is carried out to aseptic detection " is specially TSA plating method or 16sV3PCR method.
More concrete, described TSA plating method comprises the steps: culture fluid to be measured to coat on TSA flat board, cultivates one week in 25-29 ℃ (as 28.5 ℃) under aerobic conditions, observes on flat board whether have colony growth.If there is colony growth on flat board, the zebra fish of raising with institute's culture fluid of surveying is also non-sterile, then in further cultivation process, by its removal; If there is no colony growth on flat board, definite zebra fish with surveyed culture fluid raising is aseptic zebra fish.Described 16sV3PCR method comprises the steps: take culture fluid to be measured as template, carries out pcr amplification with the primer of one section of conserved sequence design in directed toward bacteria 16S rDNA V3 district; If pcr amplification product has size for the object band of 198bp, the zebra fish of raising with institute's culture fluid of surveying is also non-sterile, then in further cultivation process, by its removal; If pcr amplification product does not have the object band that size is 198bp, definite zebra fish with surveyed culture fluid raising is aseptic zebra fish.
In said method, the described rinsing in step (2), step (4) and step (6) all can rinsing 3 times.
In said method, also comprise the step of zebra fish being carried out in the 5th day feeding from after fertilization.Institute's feeding thing can be aseptic tetrahymena.In one embodiment of the invention, be specially aseptic tetrahymena thermophila.Its frequency of administration be two days once, each feeding amount is that GZM solution (raising GZM solution used in zebra fish process) adds 300 described aseptic tetrahymena thermophilas described in every 1mL.
A further object of the present invention is to provide a kind of AB-GZM solution.
AB-GZM solution provided by the present invention, solvent is water, and solute and concentration thereof are as follows: the ammonia benzyl mycin of the amphotericin B of 250ng/mL, the kanamycin of 5 μ g/mL, 100 μ g/mL, the penicillin of 10U/ml, the streptomycin of 10U/ml, the sea salt of 59 μ g/mL.
Described AB-GZM solution also belongs to protection scope of the present invention in the application of producing in aseptic fish.
In above-mentioned application, described aseptic fishing gear body can be aseptic zebra fish.
In the present invention, all described zebra fishs to be all specially TU be zebra fish.
Described sea salt is the salt of gained after evaporation of seawater.In the present invention, all described sea salt is red coral sea salt; Be specially red coral sea salt marketing center (Beijing) product, its catalog number is h-38.
The present invention obtains zebra fish fertilized egg by natural propagation, then by a series of antibiotic and chemical substance treatment, obtains aseptic embryo, is finally placed in aseptic environment and cultivates, and finally obtains aseptic zebra fish.The present invention, for the interaction between microorganisms and zebra fish and many biological processes provide material easily, also has very important reference significance for the inherent mechanism of relation between the further microorganisms of people and its vertebrate host.
Embodiment
The experimental technique using in following embodiment if no special instructions, is conventional method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
In following embodiment, photoperiod Lx:Dy represents light irradiation time x hour every day, dark duration y hour, x+y=24.
Zebra fish: be zebra fish for coming from the TU of Peking University, be documented in " Yang Hanshuo; Tang Minghai; Deng Hongxin etc. receive and rise Ultra Performance Liquid Chromatography-flight time mass spectrum and detect the Primary Study of zebrafish embryo small molecular polypeptide. Sichuan University's journal (medicine); the 06th phase in 2007 " in a literary composition, the public can obtain from Institute of Feeds,China Academy of Agriculture Sciences.
Tetrahymena thermophila (Tetrahymena thermophila): be documented in " Zhang Yujun; Wang Qinglu; hole is great. the different section water quality in Xiao Fu river, Zibo City is on the amphigenetic impact of tetrahymena. Anhui agronomy circular; 2010; 16(11): 58-59 " in a literary composition, the public can obtain from Institute of Feeds,China Academy of Agriculture Sciences.
Amphotericin B, kanamycin, ammonia benzyl mycin, povidone iodine (complex compound of polyvinylpyrrolidone and iodine) (polyvinyl pyrrolidone-iodine complex, PVP-I), penicillin are purchased, streptomycin is all purchased from Sigma company.
Bleaching agent: mother liquor: the aqueous sodium hypochlorite solution that concentration is 10g/L; Working solution: the aqueous sodium hypochlorite solution that concentration is 0.02g/L.
Sea salt: red coral sea salt is red coral sea salt marketing center (Beijing) product, and its catalog number is h-38.
GZM solution: 1L water is mixed to autoclaving after mixing with 1.5mL sea salt solution (the every L water of 40g sea salt).The content that adds up to sea salt in described GZM solution is 60mg/L.
Amphotericin B storage liquid (250 μ g/mL), kanamycin storage liquid (10mg/mL), ammonia benzyl mycin storage liquid (20mg/mL), penicillin-streptomycin storage liquid (content of penicillin and streptomycin is 1U/ μ L).The solvent of each storage liquid is water above.
AB-GZM solution: penicillin-streptomycin storage liquid of the ammonia benzyl mycin storage liquid of the kanamycin storage liquid of the amphotericin B storage liquid of the GZM solution of 49.6mL, 50 μ L, 25 μ L, 250 μ L and 500 μ L is mixed, after mixing, filter with 0.2 μ m filter, divide and install to 50-ml conical pipe ,-20 ℃ of preservations.The content that adds up to amphotericin B in described AB-GZM solution is 250ng/mL, and the content of kanamycin is 5 μ g/mL, and the content of ammonia benzyl mycin is 100 μ g/mL, and the content of penicillin and streptomycin is respectively 10U/mL, and the content of sea salt is 59 μ g/mL.
The production of embodiment 1, aseptic zebra fish and effect assessment
(1), is put into a pair of sexual maturity zebra fish in a clean zebra fish hatching cylinder latter two hour of feeding in the afternoon, and the middle lamina of septum pellucidum of placing separates female milter, and water is provided by aseptic filtration system.
(2) the next morning, 8:00 took dividing plate away, under nature, raun lays eggs, milter carries out in vitro fertilization, after 1h, fish is transferred to another one fish jar, collect fertilized egg with suction pipe immediately, be transferred in the wide-mouth bottle of the sterilizing that AB-GZM solution is housed, 28.5 ℃ cultivate 4.5h(prompting: 3-8h all can).
(3) remove the low quality fertilized egg with white point or whiting, choose high-quality fertilized egg transparent and that diaphragm is complete and carry out following operation, about 30min of used time;
(4) with the fertilized egg of choosing in AB-GZM solution rinse step (3), rinsing 3 times (about 10min of used time), then immerses fertilized egg in the PVP-I aqueous solution that concentration is 0.005g/L gently, soaks 1min.(noting: can not exceed 2min, otherwise can increase fertilized egg embryo's lethality)
(5) with aseptic GZM solution rinsing through step (4) fertilized egg after treatment, rinsing 3 times (about 10min of used time), then the bleaching agent working solution that is 0.02g/L by clorox concentration (with obtaining after distilled water diluting bleaching agent mother liquor) is filled it up with wide-mouth bottle, covers tightly.Bleaching 15min(prompting: 10-20min all can), the compartment time jiggles during this time, and fertilized egg is suspended in bleaching agent.The bubble of noting having tried not in solution.
(6) with aseptic GZM solution rinsing through step (5) fertilized egg after treatment, rinsing 3 times (about 10min of used time), then fertilized egg is transferred in blake bottle or in 12 orifice plates and is cultivated, culture density is in the GZM solution of every 100ml, to support 50 fertilized egg embryos, be positioned in super-clean bench or cell culture incubator, in 28.5 ℃ of temperature, under the condition of photoperiod L14:D10, cultivate.
(8) after 48-72h, fertilized egg embryo is hatched into young fish, carries out afterwards half amount every day and changes liquid, removes the old culture fluid of half volume, supplements isopyknic aseptic GZM solution simultaneously.
(9) hatch into after young fish from fertilized egg embryo, timing was observed zebra fish survival condition and was added up its survival rate on the one hand every day, timing is got 100 μ L culture fluids and is carried out that TSA is dull and stereotyped to be cultivated on the other hand, gets 10 μ L culture fluids and carry out 16sV3PCR and detect the aseptic of gained zebra fish.
Selected fertilized egg number × 100% in the zebra fish number/step (3) of wherein, surviving when survival rate=observation;
Zebra fish number × 100% of aseptic rate=aseptic zebra fish number/survival
TSA plating method detects the method for operating of zebra fish aseptic: culture fluid to be measured is coated on TSA flat board, after inversion, cultivated one week in 28.5 ℃ under aerobic conditions, observe on flat board whether have colony growth.Result determination methods: if there is colony growth on flat board, the zebra fish of raising with institute's culture fluid of surveying is also non-sterile, then in further cultivation process, by its removal; If there is no colony growth on flat board, definite zebra fish with surveyed culture fluid raising is aseptic zebra fish.Wherein, TSA flat board: Trypticase Soy Broth (OXOID, England), article No. is CM0129, adds after 20g agar autoclaving by 30g/L, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices, can use after cooling.
16sV3PCR method detects the principle of zebra fish aseptic: carry out pcr amplification according to bacterium at one section of conserved sequence design primer in 16S rDNA V3 district, amplified fragments is 198bp.Detection method: take culture fluid to be measured as template, carry out the existence of any bacterium in inspection instrument with 16s rRNA target gene.Reaction system is: 10 × PCR buffer2.5 μ l, dNTP mix (2.5mmol/L) 2 μ l, primer 338F (20 μ mol/L) 0.5 μ l, primer 519R (20 μ mol/L) 0.5 μ l, rTaq (2.5U/ μ L) 0.5 μ l, 10 μ l templates, 4 μ l water cumulative volume 20 μ l.PCR response procedures is: 94 ℃ of 5min; 94 ℃ of 30s, 53 ℃ of 30s, 72 ℃ of 30s, 30 circulations; 72 ℃ are extended 10min.Result determination methods: if pcr amplification product has size for the object band of 198bp, the zebra fish of raising with institute's culture fluid of surveying is also non-sterile, then in further cultivation process, by its removal; If pcr amplification product does not have the object band that size is 198bp, definite zebra fish with surveyed culture fluid raising is aseptic zebra fish.
338F：5’-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3’；
519R：5’-ATTACCGCGGCTGCTGG-3’。
(10) form latter the 5th day from fertilized egg, young zebra fish is carried out to feeding.Within every two days, feed once (warm tip: the frequency that please indicate feeding.As fed how many times every day, or every how long feed once).Each feeding carries out according to following operation: cultured aseptic tetrahymena B2086 is transferred in the EP pipe of sterilizing, the centrifugal 4min of 2000rpm, discard upper strata culture fluid, with aseptic GZM solution Eddy diffusion, the amount that adds approximately 300 tetrahymenas according to the GZM culture fluid of 1ml (raise zebra fish process in GZM solution used) afterwards adds to cultivate to be had in the vessel of zebra fish.
Wherein, aseptic tetrahymena thermophila's cultural method is as follows:
A) recovery of tetrahymena: get 100 μ l tetrahymena preservative fluids, receive in the SPP medium of 20ml, 30 ℃, cultivate in the shaking table of 135r/min one week.
B) growth: the tetrahymena having recovered is that 1mlSPP medium adds 10 μ l tetrahymenas by 1:100() inoculum concentration receive in SPP medium, 30 ℃, in the shaking table of 135r/min, cultivate 48h.The aseptic of TSA plating method checking tetrahymena, concrete grammar and result criterion are referring to associated description in step (9).Learn from else's experience and identify that aseptic tetrahymena is for subsequent use.
SPP medium: 2%(2g/100mL) peptone (OXOID, LP0042), 1%(1g/100mL) yeast extract (OXOID, LP0021), 2%(2g/100mL) glucose (traditional Chinese medicines, Beijing), 0.003%(0.003g/100mL) ironic citrate (traditional Chinese medicines, Beijing); PH7.0.
The present inventor, with reference to said method, has carried out three experiments (in each experimental procedure (3), selected fertilized egg number is 400 pieces) altogether, and result is got the mean value repeating three times.
After fertilized egg embryo is hatched into the young fish 1-6 days (1dpf-6dpf), the statistics that repeats the survival rate of experiment to zebra fish and aseptic rate for three times is as shown in table 1.As can be seen from the table, 1-6 days after fertilized egg embryo is hatched into young fish, the survival rate of zebra fish is up to more than 93%; During by the 6th day, the aseptic rate of zebra fish still can reach more than 75%.Visible, by this method, the aseptic zebra fish of acquisition that can be simple and quick, for providing the interaction between microorganisms and host that very important material is provided.The mechanism of curing the disease of for example pathogenic bacteria, the basic research such as the prebiotic effect of probio and biomedical research
Repeat experiment statistics result three times of the survival rate of table 1 zebra fish 1dpf-6dpf and aseptic rate
Claims (10)
1. the production method of aseptic zebra fish, comprises the steps:
(1) in vitro zebra fish fertilized egg is placed in to AB-GZM solution, cultivates 3-8h in 25-29 ℃;
(2) described fertilized egg after treatment step (1) is carried out to rinsing with described AB-GZM solution;
(3) the described fertilized egg after step (2) rinsing is placed in to the povidone iodine aqueous solution that concentration is 0.005g/L, soaks 1-2min;
(4) the described fertilized egg after step (3) immersion is carried out to rinsing with aseptic GZM solution;
(5) the described fertilized egg after step (4) rinsing is bleached to 10-20min with bleaching agent;
(6) the described fertilized egg after step (5) bleaching is carried out to rinsing with described aseptic GZM solution, after rinsing, described fertilized egg being placed in to described aseptic GZM solution cultivates, cultivation temperature is 25-29 ℃, and the photoperiod is L10:D14 to L14:D10, until described incubating oosperm obtains young fish;
(7) half amount of described young fish being carried out to described aseptic GZM solution every day is changed liquid and is cultivated, until described young fish grows up to the zebra fish for object size, obtains the aseptic zebra fish of object from the zebra fish of described object size;
The solvent of described aseptic GZM solution is water, and solute is sea salt; Described sea salt is in every liter of described aseptic GZM solution, to contain sea salt described in 60mg at the content of described aseptic GZM solution;
The solvent of described AB-GZM solution is water, and solute and concentration thereof are as follows: the ammonia benzyl mycin of the amphotericin B of 250ng/mL, the kanamycin of 5 μ g/mL, 100 μ g/mL, the penicillin of 10U/ml, the streptomycin of 10U/ml, the sea salt of 59 μ g/mL.
2. method according to claim 1, is characterized in that: in step (1), the temperature of described cultivation is 28.5 ℃; Or
The time of described cultivation is 4.5h.
3. method according to claim 2, is characterized in that: in step (3), the time of described immersion is 1min.
4. method according to claim 3, is characterized in that: in step (5), the time of described bleaching is 15min.
5. method according to claim 4, is characterized in that: in step (6), the temperature of described cultivation is 28.5 ℃; Or
The photoperiod of described cultivation is L14:D10.
6. method according to claim 5, is characterized in that: in step (6), described fertilized egg is placed in to described aseptic GZM solution and cultivates, culture density is in aseptic GZM solution, to cultivate 40-60 described fertilized egg described in every 100mL.
7. method according to claim 6, it is characterized in that: in step (7), the method that obtains the aseptic zebra fish of described object from the zebra fish of described object size comprises the steps: the described aseptic GZM solution for the zebra fish of cultivating described object size to carry out aseptic detection, and the zebra fish of the described object size of cultivating in aseptic described aseptic GZM solution is after testing the aseptic zebra fish of object.
8. according to arbitrary described method in claim 1-7, it is characterized in that: in described method, also comprise the step of hatching gained zebra fish being carried out in the 5th day feeding after described fertilized egg; Institute's feeding thing is aseptic tetrahymena.
9.AB-GZM solution, it is characterized in that: the solvent of described AB-GZM solution is water, and solute and concentration thereof are as follows: the ammonia benzyl mycin of the amphotericin B of 250ng/mL, the kanamycin of 5 μ g/mL, 100 μ g/mL, the penicillin of 10U/ml, the streptomycin of 10U/ml, the sea salt of 59 μ g/mL.
10. described in claim 9, AB-GZM solution is in the application of producing in aseptic fish, and described aseptic fish is aseptic zebra fish.
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