CN103289913A - Apostichopus japonicus Pseudomonas elyacovii HS1 strain and its use - Google Patents
Apostichopus japonicus Pseudomonas elyacovii HS1 strain and its use Download PDFInfo
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Abstract
The invention discloses an Apostichopus japonicus Pseudomonas elyacovii HS1 strain (CCTCCM2012512), which belongs to Pseudomonas. With a strain code of HS1, the strain has a Latin scientific name of Pseudomonas elyacovii, and is preserved in China Center for Type Culture Collection (CCTCC) on December 6, 2012, with a preservation number of CCTCCM2012512. The strain can be used as a feed additive for Apostichopus japonicus. Being able to secrete protease, amylase and other digestive enzymes, the strain has the ability to promote digestive absorption of Apostichopus japonicas and can improve the growth performance of Apostichopus japonicas. The strain can survive in water without killing alga. Being able to proliferate substantially, the strain can compete with pathogenic bacteria in water for nutrition and decompose organic matters in water so as to play a water purifying role. Being a colonizable dominant strain in a real sense, the Apostichopus japonicus Pseudomonas elyacovii HS1 strain can replace chemical drugs to undergo disease prevention and control without destroying the ecological environment.
Description
Technical field
The present invention relates to a kind of probiotic bacterium that from stichopus japonicus enteron aisle dominant microflora, separates, especially a kind of stichopus japonicus pseudomonas HS1 bacterial strain and purposes that can be used as the stichopus japonicus fodder additives.
Technical background
Stichopus japonicus (
Apostichopus japonicus) be one of eight sea treasure, have the title of " marine genseng ", have tonifying kidney and benefiting sperm, myogenic and nourish blood, delay body aging, keep many effects such as skin is smooth, since ancient times, just by the excellent tonic product of people as a kind of integration of drinking and medicinal herbs.Along with the surge of the market requirement, stichopus japonicus has become one of important breed variety in China marine site in recent years, has created huge economic benefit and social benefit, only 2006, whole nation apostichopus japonicus culture area just reaches 84200 hectares, and output breaks through 70000 tons, and direct economic benefit surpasses 10,000,000,000 yuan.Yet along with the continuous intensification of culturing intensive degree, culture environment of aquatic products goes from bad to worse and reason such as stichopus japonicus germ plasm resource degeneration, and the disease of culturing each stage of stichopus japonicus takes place frequently and seriously restricted the healthy and sustainable development of this industry.
Traditional disease control means mainly are to use microbiotic and chemicals, not only can destroy the normal colony balance of animal body, cause the animal body lower immune function, and can cause the increase of Resistant strain in the animal body, further strengthen the difficulty of disease control.For a long time, the abuse of microbiotic and chemicals has constituted very big threat to ecotope and human health, and the food-safety problem that drug residue brings also emerges in an endless stream.Culture cry more and more higher today ensuring food safety, pursue green ecological, probiotic bacterium receives increasing concern as a kind of disease control means of highly effective and safe.Probiotic bacterium is suppressing pathogenic bacteria, is regulating the intestinal microflora balance, is helping digest and significantly effect is being brought into play in aspects such as enhancing body immunizing power, improvement water quality.Compare with chemicals with microbiotic, probiotic bacterium has drawn from the animal body or its breeding environment, has highly effective and safe, natural advantage such as pollution-free.
The probiotic bacterium of using in the present aquaculture at home mainly contains photosynthetic bacterium, the antagonism bacterium, and nutrition is with product digestive ferment micropopulation (milk-acid bacteria, yeast etc.) and improve water quality flora (nitrobacteria, denitrifying bacteria etc.).These bacteriums are derived from terrestrial animal mostly or use for reference the development of terrestrial animal thinking, are applied to exist in the aquatic products be difficult to survive in breeding environment reach the deficiency that is difficult to field planting in animal body, and its effect is difficult to performance.The screening principle of probiotic bacterium is widely accepted in the world: must be harmless to the host; Can in host, field planting also breed; Can arrive the position that needs its performance function; It is consistent that actual effect is in vitro and in vivo wanted; Do not contain Disease-causing gene and drug resistant gene.Endogenous and the external source of probiotic bacterium kind is the important factor that influences probiotic effect, the screening probiotic bacterium is preferably simultaneously also to be valid approach in organism or the natural surroundings of its existence, and Gate thinks that normal dominant bacteria in the healthy animal or inferior dominant bacteria can be used as the source of probiotic bacterium.Dominant microflora has can field planting and to host and environmentally friendly advantage, but has the potentiality that are developed as efficient field planting probiotic bacterium.And for a long time, the screening study of aquatic animal specialty probiotic bacterium focuses mostly in the screening of intestinal microflora evaluation and some known common probiotic bacteriums (milk-acid bacteria, genus bacillus etc.), has ignored the effect of enteron aisle dominant microflora.
Summary of the invention
The present invention is in order to solve the above-mentioned technical problem of existing in prior technology, a kind of stichopus japonicus pseudomonas HS1 bacterial strain and purposes that can be used as the stichopus japonicus fodder additives to be provided.
Technical solution of the present invention is: a kind of stichopus japonicus pseudomonas HS1 bacterial strain (CCTCC M 2012512) belongs to pseudomonas, bacterial strain code HS1, Latin formal name used at school
Pseudomonas elyacovii, being preserved in Chinese typical culture collection center (CCTCC) on December 6th, 2012, its preserving number is CCTCC M 2012512.
Described stichopus japonicus pseudomonas HS1 bacterial strain is characterized in that: the gene fragment order of the 16S rDNA of described stichopus japonicus pseudomonas HS1 bacterial strain is as follows:
CAGGGGCGGGCGGGCCTAACACATGCAAGTCGAGCGGTAACAGAAAGTAGCTTGCTACTTTGCTGACGAGCGGCGGACGGGTGAGTAATGCTTGGGAACATGCCTTTAGGTGGGGGACAACAGTTGGAAACGACTGCTAATACCGCATAATGTCTACGGACCAAAGGGGGCTTCGGCTCTCGCCTAAAGATTGGCCCAAGTGGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCAACGATCCCTAGCTGGTTTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGTCAGGAGGAAAGGTTAGTAGTTAATACCTGCTAGCTGTGACGTTACTGACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTACGCAGGCGGTTTGTTAAGCGAGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATTTCGAACTGGCAAACTAGAGTGTGATAGAGGGTGGTAGAATTTCAGGTGTAGCGGTGAAATGCGTAGAGATCTGAAGGAATACCGATGGCGAAGGCAGCCACCTGGGTCAACACTGACGCTCATGTACGAAAGCGTGGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAACGATGTCTACTAGAAGCTCGGCTCTTCGGAGTTGTTTTTCAAAGCTAACGCATTAAGTAGACCCGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACACTTGACATACAGAGAACTTTCTAGAGATAGTTTGGTGCCTTCGGGAACTCTGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCCTTAGTTGCTAGCAGGTAATGCTGAGAACTCTAAGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGTGTAGGGCTACACACGTGCTACAATGGCGCATACAGAGTGCTGCGAACTCGCGAGAGTAAGCGAATCACTTAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGTATCAGAATGACGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTAGATAGTCTAACCTTAGGGAGGACGTTACCACGGGAGGGTTCCCTGA
A kind of above-mentioned stichopus japonicus pseudomonas HS1 bacterial strain is as the purposes of animal feedstuff additive.
Compared with prior art, pseudomonas HS1 bacterial strain of the present invention has the following advantages:
1) digestive ferments such as energy extracellular proteinase, amylase have the ability that promotes that stichopus japonicus digests and assimilates, and can improve the growth performance of stichopus japonicus;
2) in water body, can survive and algae is not had killing action; Propagation can and have been decomposed the organic matter in the water body with pathogenic bacteria competition nutrition in the water body in large quantities, plays the effect that purifies water;
3) but be truly field planting dominant bacteria, can regulate the normal microecological balance of enteron aisle, improve immunity of organisms, in enteron aisle, can stick the site with the pathogenic bacteria competition, can carry out diseases prevention and treatment and broken ring ecotope by the instead of chemical medicine.
Description of drawings
Fig. 1 is the phyletic evolution tree graph according to the stichopus japonicus pseudomonas HS1 bacterial strain of 16S rDNA sequence construct.
The culture presevation date: on December 6th, 2012
Depositary institution: Chinese typical culture collection center (CCTCC)
Preserving number: CCTCC M 2012512.
Embodiment
1. stichopus japonicus pseudomonas HS1 bacterial strain (CCTCC M 2012512) belongs to pseudomonas, bacterial strain code HS1, Latin formal name used at school
Pseudomonas elyacovii, being preserved in Chinese typical culture collection center (CCTCC) on December 6th, 2012, its preserving number is CCTCC M 2012512.
2. the preparation method of stichopus japonicus pseudomonas HS1 bacterial strain (CCTCC M 2012512):
1) stichopus japonicus source
Pick up from the marine site, Dalian.
2) isolation and purification of bacterial strain
With 75 % alcohol flushing stichopus japonicus body surface.Under aseptic condition, cut off the stichopus japonicus body cavity with scissors, take out the stichopus japonicus digestive tube, with 0.9 % normal saline flushing enteron aisle outer wall, extrude intestinal contents, the intestines wall is placed sterile test tube, after weighing the intestines wall is placed mortar, fully grinding with 5 mL stroke-physiological saline solution evenly is sample.With stroke-physiological saline solution sample carry out doubling dilution to 10
-6, each extent of dilution sample is all with revolving the concussion of nest vibrator evenly.Choose suitable gradient, get 100 μ L coating 2216E flat board respectively, each dull and stereotyped 3 repetition is cultivated 5 d for 28 ℃.The separation and purification of repeatedly ruling on the 2216E substratum of the bacterial strain of picking different shape is till single bacterium colony.With the purebred bacterial strain that obtains be stored in-80 ℃ standby.
The component of described 2216E substratum and proportioning are: peptone 5 g, and yeast extract paste 1 g, high ferric phosphate 0.01 g, seawater 1000 mL, pH 7.2 ~ 7.4,121 ℃ of sterilization 20 min.
The evaluation of stichopus japonicus pseudomonas HS1 bacterial strain:
1) morphologic feature
Stichopus japonicus pseudomonas HS1 bacterial strain is reddish-brown, smooth surface, well-grown in the growth of 2216E substratum.Cultural characteristic sees Table 1.
The cultural characteristic of table 1 stichopus japonicus pseudomonas HS1 bacterial strain
| Numbering | Shape | Color | Slickness | Degree of convexity | The edge | Transparency | Size | The thalline feature | Gramstaining |
| HS1 | The circle shape | Reddish-brown | Smooth moistening | High rising | Full edge | Opaque | 6 mm | Shaft-like single | Negative |
2), the molecular biology identification of stichopus japonicus pseudomonas HS1 bacterial strain.
(1) gene fragment order of stichopus japonicus pseudomonas HS1 bacterial strain 16S rDNA is measured
Stichopus japonicus pseudomonas HS1 bacterial strain pure growth is inoculated in ordinary broth, and 28 ℃ of shaking tables are cultivated 48 h, and centrifugal collection thalline adopts centrifugal column type bacterial genomes DNA extraction test kit to extract bacteria total DNA.The gene of amplification stichopus japonicus pseudomonas HS1 bacterial strain 16S rDNA adopts universal primer, and forward primer is:
5 ＇-CAGGCCTAACACATGCAAGTC-3 ＇ (corresponding to
E.coli16S rDNA 5 ＇ 43-63f), reverse primer is: 3 ＇-GGGCGGWGTGTACAAGGC-5 ＇ (corresponding to
E.coli16S rDNA 3 ＇ 1405-1387r), synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.PCR reaction system (50 μ L): Mix 25 μ L, ddH
2O 19 μ L, forward primer 1 μ L, reverse primer 1 μ L, DNA 4 μ L.PCR reaction conditions: at 95 ℃ of pre-sex change 2 min, enter the cyclic amplification stage: 95 ℃ of sex change 1 min → 55 ℃ of renaturation 30 s → 72 ℃ of extension 1 min, circulate 30 times, last 72 ℃ are extended 10 min.After finishing reaction, the PCR product detects with the agarose gel electrophoresis of 1 %, and reclaims test kit with gel and reclaim, Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's order-checking.The gene fragment order length of the 16S rDNA of pseudomonas HS1 bacterial strain is 1433 bp.
16S rDNA sequence information:
CAGGGGCGGGCGGGCCTAACACATGCAAGTCGAGCGGTAACAGAAAGTAGCTTGCTACTTTGCTGACGAGCGGCGGACGGGTGAGTAATGCTTGGGAACATGCCTTTAGGTGGGGGACAACAGTTGGAAACGACTGCTAATACCGCATAATGTCTACGGACCAAAGGGGGCTTCGGCTCTCGCCTAAAGATTGGCCCAAGTGGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCAACGATCCCTAGCTGGTTTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGTCAGGAGGAAAGGTTAGTAGTTAATACCTGCTAGCTGTGACGTTACTGACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTACGCAGGCGGTTTGTTAAGCGAGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATTTCGAACTGGCAAACTAGAGTGTGATAGAGGGTGGTAGAATTTCAGGTGTAGCGGTGAAATGCGTAGAGATCTGAAGGAATACCGATGGCGAAGGCAGCCACCTGGGTCAACACTGACGCTCATGTACGAAAGCGTGGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAACGATGTCTACTAGAAGCTCGGCTCTTCGGAGTTGTTTTTCAAAGCTAACGCATTAAGTAGACCCGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACACTTGACATACAGAGAACTTTCTAGAGATAGTTTGGTGCCTTCGGGAACTCTGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCCTTAGTTGCTAGCAGGTAATGCTGAGAACTCTAAGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGTGTAGGGCTACACACGTGCTACAATGGCGCATACAGAGTGCTGCGAACTCGCGAGAGTAAGCGAATCACTTAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGTATCAGAATGACGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTAGATAGTCTAACCTTAGGGAGGACGTTACCACGGGAGGGTTCCCTGA
(2) structure of stichopus japonicus pseudomonas HS1 bacterial strain sequential analysis and systematic evolution tree
With the stichopus japonicus pseudomonas HS1 bacterial strain 16S rDNA sequence of measuring, compare with all the prokaryotic organism 16S rDNA that measured in the GenBank database, according to the homologous sequence Search Results, download the number strain with the 16S rDNA sequence of the highest bacterial strain of representative strain similarity, together mate arrangement (Alignment) according to Clustal W method with representative strain, obtain phylogenetic tree with the Kimura2-Parameter Distance model in the MEGA5.0 software and neighbour-joining analytical method structure, and carry out 1000 Bootstraps checks.The phylogenetic tree of stichopus japonicus pseudomonas HS1 bacterial strain is seen Fig. 1.
Test example 1: stichopus japonicus pseudomonas HS1 strain enzyme-producing capability analysis
Select substratum, amylase to select substratum and lipase to select on the substratum in proteolytic enzyme the dibbling of stichopus japonicus pseudomonas HS1 bacterial strain, cultivate 48 h for 28 ℃.If periphery of bacterial colonies produces transparent ring, illustrate that bacterial strain has corresponding product enzyme activity, measure and the record transparent ring of periphery of bacterial colonies and colony diameter ratio.Relevant culture medium prescription is as follows:
Proteolytic enzyme is selected substratum: casein 10 g, yeast extract paste 1 g, agar 16 g, artificial seawater 1000 mL; Transfer pH to 7.4.121.5 ℃, 20 min sterilize.
Amylase is selected substratum: Zulkovsky starch 10 g, peptone 5 g, yeast extract paste 1 g, agar 16 g, artificial seawater 1000 mL; Transfer pH to 7.4.121.5 ℃, 20 min sterilize.
Lipase is selected substratum: peptone 10 g, tween-80 10 mL, CaCl
27H
20 0.1 g, agar 9 g, artificial seawater 1000 mL; Transfer pH to 7.4.121.5 ℃, 20 min sterilize.
Experimental result sees Table 2.
Table 2 stichopus japonicus pseudomonas HS1 strain enzyme-producing capability analysis
As shown in Table 2, stichopus japonicus pseudomonas HS1 bacterial strain energy extracellular proteinase, amylase and lipase have the ability that promoting digestion absorbs, and can improve the utilization ratio of feed in the apostichopus japonicus culture process, promote the healthy growth of stichopus japonicus.
Test example 2: the safety testing of stichopus japonicus pseudomonas HS1 bacterial strain
(1) hemolytic test: Sanguis Naemorhedi flat board rewarming to 25 that will be aseptic ℃, the pseudomonas HS1 inoculation that will activate again is on goat blood agar substratum, after cultivating 48 h in 25 ℃ of constant incubators, observe and find that zone of hemolysis does not appear in periphery of bacterial colonies, show that this bacterium does not have haematolysis ability.
(2) dipping bath test: choose the healthy young ginseng of small dimension and support temporarily in 6 glass jars that fill 10 L nature seawaters, every cylinder is put 20 young ginsengs.Experiment is divided into two groups of test group and control groups, every group of three repetitions, and the bacteria suspension that test group adds pseudomonas HS1 bacterial strain makes concentration reach 10
7Cfu/mL; Control group is aseptic seawater.Constant and the lasting aeration of duration of test water temperature does not change not bait throwing in of water, continues 7 days, and death all appears in test group and control group stichopus japonicus, and dissecting test group does not have pathological phenomenon.
Illustrate that stichopus japonicus pseudomonas HS1 bacterial strain does not have toxicity to stichopus japonicus, can use safely.
Test example 3: stichopus japonicus pseudomonas HS1 bacterial strain is to stichopus japonicus growth test and production application effect
In stichopus japonicus pseudomonas HS1 inoculation Yu Haiyang 2216E solid medium, place 28 ℃ of constant incubators to cultivate, wash scraper plate with physiological saline, with the gained bacteria suspension, with standby after the blood counting chamber mensuration concentration.Experiment is divided into two groups, and one group is that test group is namely by 10
9Cfu/g feed ratio adds bacterium throws something and feeds, and another group is only used feedstuff feeding for control group, and every group arranges three repetitions.Choose the relatively stichopus japonicus of uniformity of specification size, its initial weight is 495/jin, drops in the pond of 62 tons of capacity, carries out 60 days growth experiment by a definite date.The initial gross weight of each pond sea cucumber is 1397 g, test the feeds utilized existing feed of company that is, experiment condition is: extracting seawater used seawater of experiment after the sand filter from the ocean, water temperature is 18.5 ℃ ~ 20.8 ℃ in the breeding process, salinity 31 ~ 34 ‰, pH 7.5 ~ 7.9, and dissolved oxygen is not less than 5 mg/L; Every afternoon, the 16:00 point was fed, and daily ration, feeding quantity is 3 ~ 5 % of body weight; Next day, 12:00 point flowing water changed water 1/4, fell the pond once in per 10 days.Raising experiment is added up each pond stichopus japonicus gross weight after finishing, sampling statistics stichopus japonicus weight and counting.
Testing data adopts the one-way analysis of variance (ONE-WAY ANVOA) among the SPSS18.0 to carry out statistical study, and average adopts the LSD method to carry out multiple comparisons, and test-results sees Table 3
Table 3 stichopus japonicus pseudomonas HS1 bacterial strain is to the influence of stichopus japonicus growth performance and survival rate
| Group | Initial body weight (g) | Whole opisthosoma heavy (g) | Rate of body weight gain (%) | Initial specification (head/jin) | Whole last specification (head/jin) | Survival rate (%) |
| Handle | 1397 | 2675.33±103.78* | 91.51±7.43* | 495 | 236 | 91.30* |
| Contrast | 1397 | 2201±164.67 | 57.55±11.79 | 495 | 275 | 87.53 |
Annotate: * represents significant difference (p＜0.05)
As can be seen from Table 3, stichopus japonicus pseudomonas HS1 bacterial strain can significantly improve stichopus japonicus growth rate (P＜0.05) after adding, average growth rate improves 33.96 % on year-on-year basis, and two groups of survival rate of Apostichopus japonicus are all higher, but still can improve the surviving rate (P＜0.05) of stichopus japonicus behind the interpolation pseudomonas HS1.
Test example 4: stichopus japonicus pseudomonas HS1 bacterial strain is to the influence of stichopus japonicus immunity function
Experiment is divided into two groups, and one group is that test group is namely by 10
9Cfu/g feed ratio adds bacterium throws something and feeds, and another group is only used feedstuff feeding for control group, and every group arranges three repetitions.Select the bubble chamber of 70 L capacity for use, with each 30 experiment of feeding that place 6 casees to carry out 28 days by a definite date of 9 g left and right sides specification stichopus japonicus.The control of experiment water temperature is at 17 ~ 20 ℃, and continuous charge is uninterruptedly adopted an amount of fresh water to keep the skin wet and evaporated the water yield that reduces.Daily ration, feeding quantity control and according to the stichopus japonicus situation adjustment of ingesting, is thrown the bacterium amount by 10 about 3 % of stichopus japonicus body weight
9Cfu/g feed ratio.Test per seven days once sampling, measure and respectively organize the stichopus japonicus immune indexes.During sampling, randomly draw three stichopus japonicus for every group, the abdominal cavity is dissected and is got coelomic fluid, and drawing antithrombotics is l:1 to the volume ratio with coelomic fluid, the coelomic fluid that the gained antithrombotics diluted, and a part is directly used in the mensuration of coelomocyte counting, respiratory burst activity; Another part obtains serum with 4 ℃ of centrifugal 10 min of low temperature ultracentrifuge 3000 g, is stored in-20 ℃ of mensuration that are used for humoral immunization index acid phosphatase (ACP), serum superoxide dismutases (SOD), serum lysozyme (LZM).The mensuration of humoral immunization index all adopts Nanjing to build up the corresponding index detection kit mensuration that bio-engineering research is produced, and operating process is carried out according to operation instruction.Cellular immunization index determining method is as follows:
1. cytometry: get 50 μ L coelomic fluid vortex mixings, utilize blood counting chamber in 400 times of following direct census of opticmicroscope, calculate every milliliter of coelomic fluid mesenteron cell number.
2. the mensuration of activate the phagocytic capacity: each is handled and gets two stichopus japonicus at random, sterilised yeast suspension 0.2 mL at its back injection 0.2mg/mL, four hours pneumoretroperitoneums are dissected stichopus japonicus and are got its coelomic fluid, with isopyknic antithrombotics dilution, directly observe counting under 400 times of opticmicroscopes.Coelomocyte number/selected coelomocyte the number (100) of phagocytic rate=engulf.
3. the mensuration of respiratory burst vigor: the respiratory burst vitality test is taked the NBT reduction method.At first, with RPMI 1640 substratum coelomic fluid is diluted to 2 * 10
6The concentration of cell/mL; Coelomic fluid, 0.5 mL NBT, 11 μ L phorbol-12-myristin-13-ethyl ester (PMA), the 89 μ L substratum of 0.5 mL dilution are added in the 1.5 aseptic mL centrifuge tubes together; Behind 25 ℃ of placement 1 h, supernatant is removed in the 10 min hypsokinesis of 540 g low-temperature centrifugations.After adding 1 mL, 70 % methyl alcohol termination reactions, the moon first that reaction is generated is dissolved among 600 μ L, 2 M KOH and the 700 μ L DMSO for (NBT oxidation products), and measures the OD value under 630 nm wavelength.
Testing data adopts the one-way analysis of variance (ONE-WAY ANVOA) among the SPSS18.0 to carry out statistical study, and average adopts the Tukey method to carry out multiple comparisons, and test-results sees Table 4
The influence of the stichopus japonicus immune indexes of table 4 stichopus japonicus pseudomonad strain HS1
Annotate: * represents significant difference (p＜0.05)
As can be seen from Table 4, stichopus japonicus pseudomonad strain HS1 can improve stichopus japonicus cellular immunization and the every index level of humoral immunization, strengthens stichopus japonicus immunizing power.
Sequence table
＜110〉Dalian HaiBao Fishery Company Ltd.
＜120〉stichopus japonicus pseudomonas HS1 bacterial strain and purposes
<160> 3
<210> 1
<211>21
<212> DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400> 1
CAGGCCTAACACATGCAAGTC 21
<210> 2
<211>18
<212> DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400> 2
GGGCGGWGTGTACAAGGC 18
<210> 3
<211> 1433
<212> DNA
＜213〉the stichopus japonicus pseudomonas (
Pseudomonas elyakovii)
<400> 3
CAGGGGCGGGCGGGCCTAACACATGCAAGTCGAGCGGTAACAGAAAGTAGCTTGCTACTTTGCTGACGAGCGGCGGACGGGTGAGTAATGCTTGGGAACATGCCTTTAGGTGGGGGACAACAGTTGGAAACGACTGCTAATACCGCATAATGTCTACGGACCAAAGGGGGCTTCGGCTCTCGCCTAAAGATTGGCCCAAGTGGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCAACGATCCCTAGCTGGTTTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGTCAGGAGGAAAGGTTAGTAGTTAATACCTGCTAGCTGTGACGTTACTGACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTACGCAGGCGGTTTGTTAAGCGAGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATTTCGAACTGGCAAACTAGAGTGTGATAGAGGGTGGTAGAATTTCAGGTGTAGCGGTGAAATGCGTAGAGATCTGAAGGAATACCGATGGCGAAGGCAGCCACCTGGGTCAACACTGACGCTCATGTACGAAAGCGTGGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAACGATGTCTACTAGAAGCTCGGCTCTTCGGAGTTGTTTTTCAAAGCTAACGCATTAAGTAGACCCGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACACTTGACATACAGAGAACTTTCTAGAGATAGTTTGGTGCCTTCGGGAACTCTGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCCTTAGTTGCTAGCAGGTAATGCTGAGAACTCTAAGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGTGTAGGGCTACACACGTGCTACAATGGCGCATACAGAGTGCTGCGAACTCGCGAGAGTAAGCGAATCACTTAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGTATCAGAATGACGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTAGATAGTCTAACCTTAGGGAGGACGTTACCACGGGAGGGTTCCCTGA 1433
Claims (3)
1. stichopus japonicus pseudomonas HS1 bacterial strain, classification called after stichopus japonicus
ElyacoviiPseudomonas HS1 (
Pseudomonas elyacovii), being preserved in Chinese typical culture collection center, preserving number is CCTCC M 2012512.
2. stichopus japonicus pseudomonas HS1 bacterial strain according to claim 1, it is characterized in that: the gene fragment order of the 16S rDNA of described stichopus japonicus pseudomonas HS1 bacterial strain is as follows:
CAGGGGCGGGCGGGCCTAACACATGCAAGTCGAGCGGTAACAGAAAGTAGCTTGCTACTTTGCTGACGAGCGGCGGACGGGTGAGTAATGCTTGGGAACATGCCTTTAGGTGGGGGACAACAGTTGGAAACGACTGCTAATACCGCATAATGTCTACGGACCAAAGGGGGCTTCGGCTCTCGCCTAAAGATTGGCCCAAGTGGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCAACGATCCCTAGCTGGTTTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGTCAGGAGGAAAGGTTAGTAGTTAATACCTGCTAGCTGTGACGTTACTGACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTACGCAGGCGGTTTGTTAAGCGAGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATTTCGAACTGGCAAACTAGAGTGTGATAGAGGGTGGTAGAATTTCAGGTGTAGCGGTGAAATGCGTAGAGATCTGAAGGAATACCGATGGCGAAGGCAGCCACCTGGGTCAACACTGACGCTCATGTACGAAAGCGTGGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAACGATGTCTACTAGAAGCTCGGCTCTTCGGAGTTGTTTTTCAAAGCTAACGCATTAAGTAGACCCGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACACTTGACATACAGAGAACTTTCTAGAGATAGTTTGGTGCCTTCGGGAACTCTGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCCTTAGTTGCTAGCAGGTAATGCTGAGAACTCTAAGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGTGTAGGGCTACACACGTGCTACAATGGCGCATACAGAGTGCTGCGAACTCGCGAGAGTAAGCGAATCACTTAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGTATCAGAATGACGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTAGATAGTCTAACCTTAGGGAGGACGTTACCACGGGAGGGTTCCCTGA 。
3. a stichopus japonicus pseudomonas HS1 bacterial strain as claimed in claim 1 or 2 is as the purposes of stichopus japonicus fodder additives.
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