CN104686845A - Nature feed additive for microorganism-derived aquatic animals, and application of nature feed additive - Google Patents
Nature feed additive for microorganism-derived aquatic animals, and application of nature feed additive Download PDFInfo
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Abstract
The invention discloses a nature feed additive for microorganism-derived aquatic animals, and application of the nature feed additive. A preparation method of the nature feed additive comprises the following steps: constructing a recombined engineering bacterium comprising a CpG sequence; carrying out enlarged culture on the recombined engineering bacterium; and collecting thallus, crushing and drying to obtain the nature feed additive for the aquatic animals. According to the preparation method, the CpG sequence is recombined for five times by an isocaudarner technology, and high-copy CpG DNA recombinant plasmid with 90 motifs can be constructed, so that the immune systems of the aquatic animals such as fish and shrimp can be effectively activated, and the nature feed additive has broad-spectrum applicability; a lot of amplification immune stimulation sequences can be obtained by self propagation of the engineering bacterium, so that the problems that the artificial synthesis of the CpG DNA is high in cost and low in production efficiency, is not suitable for large-scale production, and the like can be effectively solved, and the nature feed additive is convenient for industrial popularization; the nature feed additive for the aquatic animals can be directly fed into feed for breeding the aquatic animals, is easy and simple to use and operate, and has the advantages of being good in safety, low in toxicity, high in efficiency, etc.
Description
Technical field
The present invention relates to molecular biology and bio-fermentation engineering field, particularly relate to a kind of microbial source aquatic livestock natural feed addictive and application.
Background technology
Along with developing rapidly of China's culture fishery, aquatic animal disease is on the rise, the use that various antibiotic, agrochemical medicine are a large amount of, has not only threatened the safety of aquatic products, has also created serious water pollution.The farming disease harms and environmental problem have become one of key factor of restriction China culture fishery development.For prawn, China starts wild virus venereal disease evil from early 1990s, and so far still without effectively preventing method, this makes the shrimp culture industry of China suffer huge loss, and the economic loss that therefore country causes every year is up to tens yuan.But, the bird of current China, particularly disease control of aquatic animal level is also lower, and antibiotic etc can only be relied on to treat some bacteriosises, there is no solution for the anti-rules for the treatment of endangering even more serious viral disease, this is also global a great problem.
CpG ODN is mainly present in specific bacterium and viral genome, also engineer can synthesize, has and activate the immune effect of body.CpG ODN has certain architectural feature usually, is namely core with Cytosine-phosphate-guanine, 5 ' end immediately 2 purine, and 3 ' end immediately 2 pyrimidines, generally use X
1x
2cpGY
1y
2(X
1for purine, X
2for purine or thymidine, Y is pyrimidine) represent.Due to the requirement of immune activation to oligonucleotide structure of different animals different (mainly the both wings sequence of CpG), therefore can for the immune feature of different animals, by Prof. Du Yucang containing non-methylated oligonucleotide sequence, MOLECULE DESIGN and transformation are carried out to oligonucleotides, to produce the CpG ODN with best immune activation effect.
Animal immune system is just by identifying that the feature structure of oligonucleotides identifies microbial DNA and self DNA, and induction produces antibacterial polypeptide material, thus forms the specific immune preventing mechanism for pathogen, plays immune activation and antibacterial action.CpG ODN has excellent immunostimulatory activity, can the direct antigen presenting cell such as activated mononuclear cell, macrophage, BMDC, secrete multiple Th1 cytokines, as materials such as interferon (IFN), interleukins (IL-1, IL-12), promote that body produces the immune response of antigentic specificity, and strong antibody response and Th1 type HI can be caused, its significant immune activation effect be traditional polysaccharide medicine incomparable.Meanwhile, CpG ODN, as natural drug, has no side effect, compared with now widely used various antibiotic etc., and drug residue free and the problem such as to develop immunity to drugs, free from environmental pollution.In addition, CpG ODN is a kind of immune activation preparation, it can improve overall diseases prevention and the resistance against diseases of body, the pathogenic microorganisms such as various bacterium, virus are had to the resistivity of wide spectrum, and in the animals such as people, animal fowl, fish, shellfish, shrimp, all there is obvious effect, use wide, effect is comprehensive, is a kind of new bio goods with great Development volue.
Calendar year 2001 Eastoott etc. are by immunostimulatory sequence DNA and tetanus toxin and aluminium agent coupling, by gastric intubation and salivary gland hypodermic injection two kinds of approach immune rats, detect the antibody finding to produce higher than only tetanus toxin and aluminium agent coupling through serum IgG and the secretion IgA of mucosal route immunity far away.The antibody horizontal that this and subcutaneous injection immunity produce is suitable.McCluskie etc., by immunostimulatory sequence DNA and tetanus toxin coupling, by oral route immune mouse, find that immunostimulatory sequence can bring out strong Th1 type immune response, produce the IgG of high titre, and tetanus toxin can not induce immune responses.These researchs show, CpG ODN can be entered by oral mode and to play a role in body and effect is unaffected, and this is the development of CpG ODN as disease-resistant drug feed, provides scientific basis and guarantee.
At present, in the world to the research of CpG ODN mainly in medical application exploitation, and emphasis is control for mankind's major disease and common vertebrate control and prevention of disease, and the R and D application for aquatic livestocks such as fishes and shrimps is very few.In addition, the invertebrates such as shrimp crab are because lacking acquired immune system, the material having bioactive material or extract from organism produced in main dependence biological living or bio-metabolic process, as the protective agents of the farming disease harms, does not have safe and effective immune regulator.Therefore, develop and a kind ofly have the biological products that are nuisanceless, noresidue of remarkable enhancement effect most important to aquatic livestock immune system.
Summary of the invention
The invention provides a kind of microbial source aquatic livestock natural feed addictive and application, this aquatic livestock feed addictive is used for the disease control of aquiculture animal, can significantly enhancement antigen humoral and cellular immune response reaction, the immunity of organisms of comprehensive raising aquatic livestock, tool high immunological activity.
A kind of aquatic livestock feed addictive, prepare by the following method:
(1) recombination engineering comprising CpG sequence is built;
(2) described recombination engineering is expanded cultivation;
(3) thalline is collected, broken, dry, obtained described aquatic livestock feed addictive.
Particularly, in step (1), the construction method of described recombination engineering comprises:
(1) the CpG sequence clone of Prof. Du Yucang is entered in pMD19-T carrier, construction recombination plasmid pMD19-T-CpG; Described CpG sequence is as shown in SEQ ID NO.1;
(2) utilize isocaudarner Xho I and Sal I double digestion recombinant plasmid pMD19-T-CpG, obtain the CpG fragment that two ends have identical cohesive terminus,cohesive termini, and this CpG fragment is recombined in the pYES2 carrier cut through Xho I enzyme, construction recombination plasmid pYES2-CpG;
(3) repeat the operation of step (2), CpG sequence repeated for five times to be reconstituted in carrier pYES2, construction recombination plasmid pYES2-5CpG, electricity is transformed in Pichia pastoris, obtains recombination engineering.
Wherein, described recombination engineering is Pichia pastoris (Pichia pastpris) GC01.This bacterial strain is deposited in the China Committee for Culture Collection of Microorganisms's common micro-organisms center being positioned at No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on July 20th, 2012, called after Pichia pastoris (Pichia pastpris) GC01, preserving number is: CGMCC NO.6368.
Described recombination engineering expands the method for cultivating and comprises: seed culture, initial incubation and feed-batch culture.Wherein, initial incubation is fermented and cultured, and the culture medium constituent of fermented and cultured is: 25g/L glucose, 10g/L peptone, 10g/L (NH
4)
2sO
4, 7g/L KH
2pO
4, 1g/L K
2hPO
4, 0.5g/LMgSO
47H
2o, 0.3g/L MnSO
4, 0.3g/L FeSO
47H
2o, pH5.0.
As preferably, the temperature of fermented and cultured is 28 ~ 30 DEG C, and the time is 10 ~ 15h, can promote strain growth.
The method preparing dusty yeast has a variety of, as preferably, the method preparing dusty yeast described in the present invention is: get zymotic fluid, the centrifugal 20min of 5000r/min collects thalline, 100 DEG C of water bath processing 15min, 4 DEG C of ice bath coolings, carry out ultrasonication, ultrasonication condition is: ultrasonication power 450W, and broken time 20min, the time interval (working time: intermittent time) is 10
:8 (s/s); Through 25 ~ 30 DEG C of pneumatic conveying dryings after ultrasonication, make dusty yeast; The dusty yeast prepared under this condition is better as effect during aquatic livestock feed addictive.
Present invention also offers a kind of aquatic animal feed comprising described aquatic livestock feed addictive.
As preferably, in every kilogram of aquatic animal feed, the addition of aquatic livestock feed addictive is 30 ~ 40g/kg.
Compared with prior art, the beneficial effect that the present invention has, as follows:
(1) utilize isocaudarner technology 5 recombinant C pG sequences, construct the height copy CpG DNA recombinant plasmid with 90 CpG motifs, effectively can activate the immune system of the aquatic livestocks such as fishes and shrimps, there is wide spectrum applicability;
(2) to be increased in a large number immunostimulatory sequence by the self-reproduction of engineering bacteria, efficiently solve Prof. Du Yucang CpG DNA cost intensive, production efficiency low, be unsuitable for the problems such as large-scale production, be convenient to industrialization promotion;
(3) aquatic livestock feed addictive of the present invention, namely high immunological activity CpG DNA dusty yeast, can be added directly in the feed of aquaculture of aquatic animal, uses easy and simple to handle, and have that security is good, low toxicity, the advantage such as efficient.
Accompanying drawing explanation
Fig. 1 is the HindIII/XbaI restriction enzyme digestion and electrophoresis figure of the restructuring pYES2 containing different CpG motif, and wherein, swimming lane 1 is pYES2-CpG; Swimming lane 2 is pYES2-2CpG; Swimming lane 3 is pYES2-3CpG; Swimming lane 4 is pYES2-4CpG; Swimming lane 5 is pYES2-5CpG; Swimming lane M is DL2000DNA Marker;
Fig. 2 is the bacterium colony PCR qualification result of the restructuring E.coli Top10 containing different CpG motif, and wherein, swimming lane 0 is the E.coli Top10 bacterium colony containing pYES2; Swimming lane 1 is the E.coli Top10 bacterium colony containing pYES2-CpG; Swimming lane 2 is the E.coli Top10 bacterium colony containing pYES2-2CpG; Swimming lane 3 is the E.coli Top10 bacterium colony containing pYES2-3CpG; Swimming lane 4 is the E.coli Top10 bacterium colony containing pYES2-4CpG; Swimming lane 5 is the E.coli Top10 bacterium colony containing pYES2-5CpG, and swimming lane M is DL2000DNA Marker;
Fig. 3 is Yeast engineering bacteria GC01 high-density culture process curve.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in detail.
Experimental technique described in the embodiment of the present invention if no special instructions, is conventional method.
The nutrient media components that following examples use and concentration as follows:
10 × YNB:13.4g YNB is dissolved in 100mL deionized water, is heated to YNB and dissolves completely, filtration sterilization, 4 DEG C of preservations.
10 × D:200g glucose is dissolved in 1000mL deionized water, autoclaving 15min or filtration sterilization, 4 DEG C of preservations.
500 × biotin: 20mg biotin is dissolved in 100mL deionized water, filtration sterilization, 4 DEG C of preservations.
MD is dull and stereotyped: 800mL deionized water (if flat board processed adds 20g agar powder), and 121 DEG C of sterilizing 20min, treat that temperature is cooled to 60 DEG C, the 10 × D of the 500 × biotin of 2mL, the 10 × YNB of 100mL, 100mL, and mixing, is down flat plate.
Seed culture medium: i.e. YPD fluid nutrient medium: peptone 20g, yeast extract 10g, be dissolved in 900mL deionized water (if flat board processed adds 20g agar powder), 121 DEG C of sterilizing 30min, add the 10 × D of 100mL after cooling, 4 DEG C of preservations.
Fermentation medium: glucose 25g, peptone 10g, (NH
4)
2sO
410g, KH
2pO
47g, K
2hPO
41g, MgSO
47H
2o 0.5g, MnSO
40.3g, FeSO
47H
2o 0.3g, adds deionized water to 1000mL, adjusts pH5.0 with ammoniacal liquor, 121 DEG C of sterilizing 30min.
Fermentation feed medium: glucose 500g, peptone 70g, MgSO
47H
2o 10g, adds deionized water 1000mL, 105 DEG C of sterilizing 40min.
The preparation of embodiment 1 aquatic livestock CpG DNA recombinant plasmid
(1) engineer's synthesis has the CpG sequence of immune activation effect to aquatic livestock, and carries out thio-modification and polyacrylamide gel electrophoresis purifying (purity 99.99%) to full chain nucleotides.This sequence comprises 18 CpG motifs, and introduces Xho I restriction enzyme site at 3 ' end, forms the DNA fragmentation containing 18CpG motif.CpG sequence is as shown in SEQ ID NO.1.
(2) by CpG DNA fragmentation directed cloning in pMD19-T carrier, construction recombination plasmid pMD19-T-CpG, and proceed in DH5 α bacterial strain carry out expansion cultivate; Utilize alkaline lysis small lot to extract DNA, use agarose gel electrophoresis to detect.
(3) utilize isocaudarner Xho I and Sal I double digestion recombinant plasmid pMD19-T-CpG, obtain the CpG DNA fragmentation that two ends have identical cohesive terminus,cohesive termini; Utilize Xho I single endonuclease digestion pYES2 carrier, make its linearisation; Electroresis appraisal also rubber tapping reclaims object fragment, carries out coupled reaction;
Linked system is as follows: 2.5 μ l 10 × T4DNA ligase buffer, 4 μ l target DNAs (CpG DNA), 1 μ l carrier (pYSE2), 1 μ l T4DNA ligase, add sterilized water to 25 μ l.16 DEG C of reactions are spent the night.In linked system, the mol ratio of plasmid and target DNA is about 1: 10 ~ 1: 100;
Connect rear Plastid transformation E.coli Top10 competent cell, Amp resistance screening transformant; Bacterium colony PCR, HindIII/XbaI restriction enzyme digestion and electrophoresis qualification clone.
(4) repeat the operation of step (3), CpG DNA fragmentation is repeated for five times to be reconstituted in carrier pYES2, construction recombination plasmid pYES2-5CpG; Recombinant plasmid pYES2-5CpG is transformed in E.coli Top10, with Amp resistance screening transformant, and cuts and electroresis appraisal clone with bacterium colony PCR, Hind III/Xba I enzyme, finally by Invitrogen company, pYES2-5CpG is checked order.
The conversion of embodiment 2 CpG DNA recombinant plasmid and the expansion of engineering bacteria are cultivated
(1) the pYES2-5CpG plasmid electricity built is transformed Pichia pastoris (Pichia pastpris) GS115 bacterial strain, electric Transformation Parameters is V=1.5kV, 25uF, 200Ohms, 4 ~ 10msec; Bacterial strain coating MD flat board (containing 2mg/mL G418) after electricity transforms, is placed in 30 DEG C of cultivations, until there is single bacterium colony; Through plasmid extraction, enzyme is cut and is determined that pYES2-5CpG plasmid has proceeded in Pichia pastoris with the qualification such as electrophoresis, obtains engineering bacteria;
(2) seed growth phase
The engineering bacteria getting above-mentioned structure is inoculated in 50mL seed culture medium, 30 DEG C, 220r/min cultivates 16h as first order seed, by 10% inoculum concentration access 200mL seed culture medium, and 30 DEG C, 220r/min cultivates 24h as secondary seed.
(3) the initial incubation stage
Secondary seed is inoculated into the fermentation cylinder for fermentation that 3L fermentation medium is housed, inoculum concentration is 10%, and fermentation time controls at about 12h, and during fermentation, dissolved oxygen amount is more than 30%, temperature is 30 DEG C, and pH controlled in 5.0 (adding 20% ammoniacal liquor adjust ph by stream).
(4) the feed-batch culture stage
After initial incubation terminates, carry out flow feeding culture medium.Regulate stream rate of acceleration according to remaining sugar concentration in zymotic fluid, concentration of glucose is controlled at below 1.0g/L.The feed supplement time is 30h, and feed supplement cumulative volume is 1.6L, and during fermentation, dissolved oxygen amount is more than 30%, and temperature is 30 DEG C, and pH controlled in 5.0 (adding 20% ammoniacal liquor adjust ph by stream).During fermentation ends, dry cell weight reaches 69.4g/L.
(5) after fermentation ends, getting zymotic fluid centrifugal 20min under 5000r/min condition and collect thalline, after cleaning 3 times, is prepare yeast cream at 1: 4 by the weight ratio of yeast paste and water with distilled water; Yeast cream is placed in 100 DEG C of water-baths and processes 15min, 4 DEG C of ice bath coolings, carry out ultrasonication, ultrasonication condition is: ultrasonication power 450W, broken time 20min, the time interval (working time: intermittent time) is 10: 8 (s/s); Yeast cream after ultrasonication, through 25 ~ 30 DEG C of pneumatic conveying dryings, makes dusty yeast.Get appropriate dusty yeast yeast plasmid Mini Kit and extract DNA, determined by ultraviolet spectrophotometry concentration.Result shows, and the content of pYES2-5CpG recombinant plasmid can reach 0.6 μ g/mg dusty yeast, and this dusty yeast is aquatic livestock feed addictive.
The disease-resistant test of feed addictive to perch of embodiment 3 aquatic livestock
Juvenile seabass: get Ninghai, Zhejiang woman's bow sea-farming Co., Ltd perch seedling 7200 tail.Juvenile fish body weight 20-30g, the long 10-15cm of body.
Dusty yeast containing CpG DNA recombinant plasmid: provided by Zhejiang Province crown Technology Co., Ltd.
Test is divided into 2 groups (test group and control groups), often organizes 3 repetitions, eachly repeats 1200 tails that breed fish fry.Test group is fed the disease-resistant feed (dusty yeast addition is 30g/kg feed by weight) added containing the aquatic products additive for animal feed dusty yeast of CpG DNA recombinant plasmid (namely containing), control group fed conventional feed (adding normal dusty yeast).Maintain water temperature 20 ~ 25 DEG C.Normally to throw something and feed every day feed, throw something and feed continuously 4 weeks, the statistics natural occurrence death rate, Computation immunity protective rate RPS.
Result shows, the dusty yeast disease-resistant feed added containing CpG DNA recombinant plasmid can improve perch average survival 21.70%, improves RPS 66.42%.Concrete condition is in table 1.
Relative immunity protective rate (Relative Percent Survival, RPS), RPS=(the 1-immune group death rate/control group death rate) × 100%.
The disease-resistant test of feed addictive to perch of table 1 aquatic livestock
The disease-resistant test of feed addictive to Penaeus Vannmei of embodiment 4 aquatic livestock
Penaeus Vannmei: juvenile prawn 6000 tail getting green source, Zhoushan water cultivation Co., Ltd Penaeus Vannmei, average weight is 0.69 ± 0.31g, and average body length is 4.50 ± 0.55cm.
Feed: the basal feed of prawn is made up of dregs of beans, fish meal, shrimp med, flour, fish oil, wheat bran, soybean lecithin.
Dusty yeast containing CpG DNA recombinant plasmid: provided by Zhejiang Province crown Technology Co., Ltd.
6000 tail prawn random experiments be divided into 2 groups (test group and control groups), often organize 3 repetitions, each repetition puts juvenile prawn 1000 tail in a suitable place to breed.Test group is fed containing the bait (dusty yeast addition is 40g/kg bait by weight) of aquatic products additive for animal feed (namely containing the dusty yeast of CpG DNA recombinant plasmid), the normal bait of control group fed (adding normal dusty yeast).Every day respectively feeds intake 1 time (feeding intake by 5% of prawn body weight) sooner or later, throws something and feeds continuously 30 days.Add up death rate of the onset after off-test, calculate relative immunity protective rate RPS.
The disease-resistant test of feed addictive to Penaeus Vannmei of table 2 aquatic livestock
Result shows, adds high immunological activity CpG DNA dusty yeast and can improve Penaeus Vannmei average survival 20.64%, improve RPS 66.06%.Concrete condition is in table 2.
Claims (9)
1. an aquatic livestock feed addictive, is characterized in that, prepares by the following method:
(1) recombination engineering comprising CpG sequence is built;
(2) described recombination engineering is expanded cultivation;
(3) thalline is collected, broken, dry, obtained described aquatic livestock feed addictive.
2. aquatic livestock feed addictive as claimed in claim 1, it is characterized in that, the construction method of described recombination engineering comprises:
(1) the CpG sequence clone of Prof. Du Yucang is entered in pMD 19-T carrier, construction recombination plasmid pMD 19-T-CpG; Described CpG sequence is as shown in SEQ ID NO.1;
(2) utilize isocaudarner Xho I and Sal I double digestion recombinant plasmid pMD 19-T-CpG, obtain the CpG fragment that two ends have identical cohesive terminus,cohesive termini, and this CpG fragment is recombined in the pYES2 carrier cut through Xho I enzyme, construction recombination plasmid pYES2-CpG;
(3) repeat the operation of step (2), CpG sequence repeated for five times to be reconstituted in carrier pYES2, construction recombination plasmid pYES2-5CpG, electricity is transformed in Pichia pastoris, obtains recombination engineering.
3. aquatic livestock feed addictive as claimed in claim 1, it is characterized in that, described recombination engineering is Pichia pastoris (Pichia pastpris) GC01.
4. aquatic livestock feed addictive as claimed in claim 1, is characterized in that, recombination engineering expands in incubation, and the constituent of fermentation medium is: 25g/L glucose, 10g/L peptone, 10g/L (NH
4)
2sO
4, 7g/L KH
2pO
4, 1g/L K
2hPO
4, 0.5g/L MgSO
47H
2o, 0.3g/L MnSO
4, 0.3g/L FeSO
47H
2o, pH 5.0.
5. aquatic livestock feed addictive as claimed in claim 4, it is characterized in that, the temperature of fermented and cultured is 28 ~ 30 DEG C, and the time is 10 ~ 15h.
6. aquatic livestock feed addictive as claimed in claim 1, is characterized in that, described in be broken for ultrasonication, broken condition is power 450W, time 20min, the working time: the intermittent time is 10s: 8s.
7. aquatic livestock feed addictive as claimed in claim 1, it is characterized in that, the temperature of described drying is 25 ~ 30 DEG C.
8. one kind comprises the aquatic animal feed of aquatic livestock feed addictive as claimed in claim 1.
9. aquatic animal feed as claimed in claim 8, it is characterized in that, in every kilogram of aquatic animal feed, the addition of aquatic livestock feed addictive is 30 ~ 40g/kg.
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| CN106490361A (en) * | 2016-10-31 | 2017-03-15 | 南宁学院 | A kind of preparation method of the CpG ODN for having immune-enhancing activity to pig |
| CN106519010A (en) * | 2016-11-07 | 2017-03-22 | 上海海洋大学 | Preparation of luteinizing hormone recombinant protein feed additive and application method thereof |
| CN108142730A (en) * | 2016-12-06 | 2018-06-12 | 屏东科技大学 | Shrimp with immunoloregulation function feed addictive and the shrimp fodder compound containing this |
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| CN108203697A (en) * | 2017-12-25 | 2018-06-26 | 杭州皇冠农业生物工程技术研究中心有限公司 | A kind of Yeast engineering bacteria of fish natural killer cell enhancement factor and its application |
| CN108203697B (en) * | 2017-12-25 | 2021-01-05 | 杭州皇冠农业生物工程技术研究中心有限公司 | Yeast engineering bacterium of fish natural killer cell enhancement factor and application thereof |
| CN108949802A (en) * | 2018-06-25 | 2018-12-07 | 浙江皇冠科技有限公司 | A kind of high yield CpG ISS engineering bacteria construction method and purposes |
| CN108998384A (en) * | 2018-06-25 | 2018-12-14 | 浙江皇冠科技有限公司 | Recombinate the engineering bacteria of fish interferon and its preparation method of product |
| CN108949598A (en) * | 2018-09-13 | 2018-12-07 | 浙江皇冠科技有限公司 | Recombinate the engineering bacteria of Fish leukocyte interleukin -2 and its preparation method of product |
| CN112080504A (en) * | 2020-07-23 | 2020-12-15 | 浙江理工大学 | CG island oligonucleotide with immunostimulation capability of aquaculture animals and application thereof |
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