CN104673830A - Method for improving calcium and phosphorus utilization ratio of livestock and poultry - Google Patents
Method for improving calcium and phosphorus utilization ratio of livestock and poultry Download PDFInfo
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- CN104673830A CN104673830A CN201410042182.9A CN201410042182A CN104673830A CN 104673830 A CN104673830 A CN 104673830A CN 201410042182 A CN201410042182 A CN 201410042182A CN 104673830 A CN104673830 A CN 104673830A
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- phytase
- coccidia
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Abstract
The invention provides a method for improving the calcium and phosphorus utilization ratio of livestock and poultry. The method comprises the following steps: (1) obtaining full-length DNA of a phytase gene; (2) preparing a coccidium transfection vector pMDEA-phytase; (3) performing transfection on the vector and screening transgenic coccidia; and (4) inoculating the transgenic coccidia for expressing phytase into bodies of the livestock and poultry in an oral administration manner to ensure that the coccidia express and release phytase while developing in intestinal tracts, and degrading phytic acid. By adopting the method provided by the invention, phytase genes with different sources are expressed in the coccidia for the first time, so that a recombinant micro biological reactor is obtained and can generate phytase directly applied to the livestock and poultry, and equipment for production and purification and related operations are not needed.
Description
Technical field
The present invention relates to biological technical field, specifically, relate to and a kind ofly improve the method for poultry calcium phosphorus utilization at poultry expression in vivo phytase gene.
Background technology
Phytic acid is as the major storage form of phosphorus in various plants tissue (particularly seed), and it exists with the form of calcium magnesium sylvite.A large amount of phytic acid is all contained in the livestock and poultry being Main Components with the cereal such as corn, wheat (chicken, ox, sheep, pig etc.) feed.The existence of phytic acid greatly reduces the content of available calcium phosphorus in feed.But can not phytic acid be digested without the normal microflora of Expressing Recombinant Phytase in poultry (chicken, duck etc.) and non-ruminant animal (pig, rabbit etc.) self enteron aisle, therefore want the utilising efficiency improving Calcium in Feed phosphorus, just need to utilize phytase to phytic acid of degrading in livestock and poultry body.
Phytase mainly through adding vivoexpression purifying in feed in current Production of Livestock and Poultry solves the problems referred to above.And the cost producing phytase is higher, its non-refractory in feed making, its application is subject to certain restrictions.According to prediction, the ratio that China in 2014 adds the feed of feeding phytase will bring up to 80% by about 50%, and the adding proportion by 0.013% calculates, and within 2014, feeding phytase demand can reach 4.59 ten thousand tons, increase by 92.76% than 2010 annual production.
Thus develop and a kind ofly improve the novel method of poultry and livestock feed calcium phosphorus utilization at poultry expression in vivo phytase gene, to replace at livestock and poultry breeding industry or the interpolation of phytase in part substitute feed, produce huge economic benefit, for Animal husbandry production provides power.
Summary of the invention
In order to solve problems of the prior art, the object of this invention is to provide and a kind ofly improve the method for poultry calcium phosphorus utilization at poultry expression in vivo phytase gene.
In order to realize the object of the invention, first the present invention provides a kind of method improving poultry calcium phosphorus utilization, and described method comprises the steps:
1) phytase gene full length DNA is obtained;
2) preparation of the coccidia transfection carrier containing phytase gene:
Above-mentioned phytase full genome is built up in coccidia expression vector pMDEAAssA, obtain coccidia transfection carrier pMDEA-phytase;
3) transfection of coccidia carrier and the screening of transgenosis coccidia:
By carrier pMDEA-phytase transfection coccidia sporozoite, and be seeded in livestock and poultry body, utilize drug resistance gene to carry out pressure screening, collect the offspring's egg capsule in ight soil, after carrying out Screening and Identification, obtain the transgenosis coccidia of expressing described phytase;
4) the transgenosis coccidia of Expressing Recombinant Phytase is seeded in livestock and poultry body by oral way, expresses while making coccidia carry out Endogenous stages in enteron aisle and discharge phytase, degraded phytic acid.
As preferably, described livestock and poultry are chicken, duck, pig or rabbit.
Wherein, described step 1) utilizes PCR method to obtain phytase gene full length DNA from genus bacillus, black-koji mould or intestinal bacteria.
Further, described step 2) be specially the phytase gene that step 1) to be increased and obtain and coccidia expression vector pMDEAAssA restriction enzyme A geI, SacII enzyme are cut, endonuclease bamhi is carried out DNA fragmentation recovery, connect and transform, then little upgrading grain is identified, the plasmid that qualification is positive is carried greatly, the coccidia transfection carrier pMDEA-phytase that must build.
Present invention also offers according to step 2) build the coccidia transfection carrier pMDEA-phytase and the application of described carrier in raising poultry calcium phosphorus utilization that obtain.
Beneficial effect of the present invention is:
(1) the present invention first by the phytase gene expression of different sources in coccidia, thus obtain the miniature organism reactor of restructuring, the phytase directly applying to livestock and poultry can be produced, without the need to producing and the equipment of purifying and relating operation.
(2) the invention provides a kind of novel method utilizing phytase to improve livestock and poultry calcium phosphorus utilization, in animal and bird intestines, directly can produce phytase, thus help digest the phosphoric acid in feed.
(3) the transgenosis coccidia of Expressing Recombinant Phytase that the present invention obtains uses simple, with low cost, has a extensive future.
Accompanying drawing explanation
Fig. 1 is the qualification of the transgenosis coccidia of Expressing Recombinant Phytase of the present invention.Wherein: A is the expression of reporter gene; B utilizes the many anti-results of coccidia sporozoite being carried out to immunofluorescence dyeing of anti-phytase.
Fig. 2 is the present invention transgenosis coccidia of inoculating Expressing Recombinant Phytase to the effect of chicken weightening finish and calcium phosphate use.Wherein: A is after oral vaccination coccidia, the body weight gain situation of chicken; B is the measurement result to the calcium phosphorus content in the ight soil of chicken discharge.
Fig. 3 is the luminous efficiency of the coccidian oocyst that after coccidia transfection pMDEA-phytase plasmid, continuous passage obtains.
The transgenosis coccidia of Fig. 4 application Expressing Recombinant Phytase utilizes the effect of Calcium in Feed phosphorus to broiler chicken.A is determination of calcium content in ight soil, and B is phosphorus detection in ight soil.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Embodiment 1
1, vector construction
Use Standard PCR technology by colibacillary phytase full-length gene (its nucleotide sequence is as shown in SEQ ID No.1) amplification and add AgeI and SacII restriction enzyme site sequence at two ends respectively, the correct PCR primer restriction enzyme A geI of order-checking and SacII enzyme are cut, be connected into AgeI and SacII enzyme to cut carrier pMDEAAssA(and formed based on pBR322 vector construction by laboratory, contriver place, mic2 promotor containing Eimeria tenella, drug screening gene DHFR-TS3m, reporter gene yellow fluorescence protein EYFP, the actin promotor of Eimeria tenella and 3 ' regulating and controlling sequence, the signal peptide sequence of toxoplasma gondii dense granule protein 8, its nucleotide sequence is as shown in SEQ ID No.2) after in the skeleton that obtains, then the positive colony obtained is identified.Carried greatly by the correct recombinant plasmid of qualification, its nucleotide sequence of coccidia transfection carrier pMDEA-phytase(that must build is as shown in SEQ ID No.3).
2, the screening of consideration convey dye and transgenosis coccidia
Coccidia expression vector pMDEA-phytase SnaBI is carried out enzyme cut, then the linearizing DNA segment of alcohol settling.Get 1 × 10
7the sporozoite of individual Eimeria tenella, the linearization plasmid of 5-10 μ g, the consideration convey dye damping fluid of 5 μ L NdeI restriction enzymes and 100 μ L, move in electric shock cup after mixing, electric shock cup is inserted in the electric shock tank of consideration convey dye instrument Nucleofector II, select core transfection procedures U-033 to carry out consideration convey dye; In electric shock cup, 1000 μ L DMEM nutrient solutions are added, by coccidia sporozoite inoculation to the 7 age in days non-ball worm after transfection immediately after transfection.2nd day starts after inoculation, adds Pyrimethamine hcl medicine (150 micro-gs/kg) and carry out drug pressure screening in feed.Collect the egg capsule in 6 ~ 8 days ight soil, with the polypide that flow cytometer screening is luminous after Sporulated, inoculation chicken, then collect egg capsule, inoculate chicken and repeat said medicine screening process, repeatedly 6 generations, obtaining the progeny population of stably express reporter gene yellow fluorescence protein.
3, indirect immunofluorescence (IFA) qualification
Get the Sporulated Oocysts 2 × 10 of expressing yellow fluorescence protein
7individually add equal-volume granulated glass sphere, to vibrate 5min with turbula shaker 1000rpm, add after most of sporocyst disengages and within 40 minutes, discharge sporozoite with excystation containing the pancreatin of 1.5% and the excystation damping fluid of 10% bile 42 DEG C of effects.Sporozoite is crossed after post removing impurity through DE-52 cellulose column, and the sporozoite polypide obtained is for indirect IF staining.Drip on slide glass by sporozoite suspension, methyl alcohol fixes how anti-the anti-phytase mouse of rear preparation is hatches as primary antibodie, hatches rear washing 3 times, then adds that sheep anti mouse two that Texas Red marks is anti-hatches.At fluorescence microscopy Microscopic observation coloration result after 5 washings.
4, the calcium phosphate use test of the transgenosis coccidia of SPF chicken inoculation Expressing Recombinant Phytase
30 28 age in days SPF chickens are divided into 3 groups, are respectively blank group, wild coccidia group and transgenosis coccidia group.All chickens are all fed at experimental session and are not added the full-valence pellet feed of phytase.Within 1st day, namely give wild coccidia group and the transgenosis coccidia group wild coccidia of oral vaccination and transgenosis coccidia respectively, dosage of inoculation is 5 × 10
3.Within after inoculation every 3 days, monitor chicken body weight, detect the content of phosphorus in ight soil.
5, result
Dyeing through indirect immunofluorescence (for phytase protein) confirms, expresses coccidia (Figure 1A) Expressing Recombinant Phytase (Figure 1B) simultaneously of yellow fluorescence reporter protein.
After the coccidia per oral inoculation of Expressing Recombinant Phytase the 6th day, phosphorus content in coccidia group chicken ight soil is just lower than blank group and wild-type coccidia inoculation group, and from after inoculation the 12nd day, the phosphorus content in transgenosis coccidia group chicken ight soil was significantly lower than other two groups.To off-test, transgenosis coccidia group chicken body weight is significantly higher than two other control group, and the price of deed is also significantly higher than two control groups.In chicken body weight (28-45 age in days) and ight soil, the data of calcium phosphorus content (45 age in days) are shown in Fig. 2.
Embodiment 2
The step 2 of embodiment 1 and 3, we compared for continuous passage algebraically and the relation obtaining stably express phytase transgenosis coccidia.After offspring's coccidian oocyst of every generation is collected, through purifying and Sporulated, detect its luminous efficiency under fluorescent microscope, its calculation formula is: Sporulated Oocysts × 100% of the Sporulated Oocysts/all that luminosity factor=yellow fluorescence protein is expressed.We find, the luminous efficiency in the 4th generation and the 5th generation is respectively 54% and 83%, and drug pressure can obtain 100% expression yellow fluorescence reporter protein transgenosis coccidia when screening for the 6th generation, continuous passage (7-9 generation) subsequently is still 100% express fluorescent protein (see figure 3).By the qualification of step 3 in embodiment 1, confirm that continuous drug pressure 6 generations of going down to posterity are the optimal conditions of transgenosis coccidia obtaining Expressing Recombinant Phytase.
Embodiment 3
In a transgenosis coccidia simultaneous test, we compare commercial recombinant phytase and add with the transgenosis coccidia that obtains of application the present invention the contrast of Calcium in Feed phosphate use effect.
1 aa broiler chicken is divided at random blank group, phytase interpolation group and transgenosis coccidia use group (often organizing 10 chickens).Feed is the full-valence pellet feed (entrusting Beijing Hua Dou feed corporation,Ltd small-scale production) added without phytase.Phytase interpolation group is then omnidistance in feed adds the production of phytase 5000(Su Kehan biotechnology company limited), addition is 600g/kg.The transgenosis Eimeria mitis of the chicken of the transgenosis coccidia group Expressing Recombinant Phytase described in 1 age in days oral administration inoculation, every chicken inoculation dosage is for being 5 × 10
3sporulated Oocysts.Measure calcium phosphorus content in the ight soil during 1-42 age in days (measuring once for every 7 days).
Detected result is shown in Fig. 4.The phytase adding restructuring in visible feed obviously can reduce the content of Calcium in Feed phosphorus; And after the transgenosis coccidia of oral vaccination Expressing Recombinant Phytase, the calcium phosphorus content in chicken ight soil also has remarkable reduction, although it is lower than phytase interpolation group.
This measurement result shows, the phytic acid that the transgenosis coccidia of Expressing Recombinant Phytase can effectively be degraded in feed, reaches the object improving Calcium in Feed phosphate use, can the interpolation of Some substitute recombinant phytase.Based on the effective application of coccidia living vaccine in livestock and poultry, the transgenosis coccidia of this Expressing Recombinant Phytase both as vaccine prevention coccidiosis of livestock and poultry, can reduce the pollution to environment, achieved many things at one stroke while the utilization carrying high calcium and phosphorus.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (6)
1. improve a method for poultry calcium phosphorus utilization, it is characterized in that, described method comprises the steps:
1) phytase gene full length DNA is obtained;
2) preparation of the coccidia transfection carrier containing phytase gene:
Above-mentioned phytase full genome is built up in coccidia expression vector pMDEAAssA, obtain coccidia transfection carrier pMDEA-phytase;
3) transfection of coccidia carrier and the screening of transgenosis coccidia:
By carrier pMDEA-phytase transfection coccidia sporozoite, and be seeded in livestock and poultry body, utilize drug resistance gene to carry out pressure screening, collect the offspring's egg capsule in ight soil, after carrying out Screening and Identification, obtain the transgenosis coccidia of expressing described phytase;
4) the transgenosis coccidia of Expressing Recombinant Phytase is seeded in livestock and poultry body by oral way, expresses while making coccidia carry out Endogenous stages in enteron aisle and discharge phytase, degraded phytic acid.
2. method according to claim 1, is characterized in that, described livestock and poultry are chicken, duck, pig or rabbit.
3. method according to claim 1, is characterized in that, described step 1) utilizes PCR method to obtain phytase gene full length DNA from genus bacillus, black-koji mould or intestinal bacteria.
4. method according to claim 1, it is characterized in that, described step 2) be specially the phytase gene that step 1) to be increased and obtain and coccidia expression vector pMDEAAssA restriction enzyme A geI, SacII enzyme are cut, endonuclease bamhi is carried out DNA fragmentation recovery, connect and transform, then little upgrading grain is identified, the plasmid that qualification is positive is carried greatly, the coccidia transfection carrier pMDEA-phytase that must build.
5. step 2 in method according to claim 4) build the coccidia transfection carrier pMDEA-phytase obtained.
6. carrier according to claim 5 is improving the application in poultry calcium phosphorus utilization.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109133962A (en) * | 2018-08-13 | 2019-01-04 | 中国科学院城市环境研究所 | A kind of compound carbon aerogels of electrostatic spinning nano fiber and preparation method thereof |
| CN109730208A (en) * | 2019-02-14 | 2019-05-10 | 广东恒兴饲料实业股份有限公司 | A kind of meat rabbit feed and preparation method thereof without calcium monohydrogen phosphate |
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| CN101024076A (en) * | 2007-03-29 | 2007-08-29 | 中国农业大学 | Novel use of coccidium |
| CN101683123A (en) * | 2008-09-25 | 2010-03-31 | 河南农业大学 | Application of transgenic Lactobacillus with phytase gene as pig feed additive |
| CN102657865A (en) * | 2012-04-28 | 2012-09-12 | 中国农业大学 | Method for conveying proteins or antigens to mammal intestinal tract in targeted way by using transgenic coccidian |
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2014
- 2014-01-28 CN CN201410042182.9A patent/CN104673830A/en active Pending
Patent Citations (3)
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| CN101024076A (en) * | 2007-03-29 | 2007-08-29 | 中国农业大学 | Novel use of coccidium |
| CN101683123A (en) * | 2008-09-25 | 2010-03-31 | 河南农业大学 | Application of transgenic Lactobacillus with phytase gene as pig feed additive |
| CN102657865A (en) * | 2012-04-28 | 2012-09-12 | 中国农业大学 | Method for conveying proteins or antigens to mammal intestinal tract in targeted way by using transgenic coccidian |
Non-Patent Citations (1)
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| 楼洪兴、许尧兴: "植酸酶在饲料中的应用", 《中国饲料》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109133962A (en) * | 2018-08-13 | 2019-01-04 | 中国科学院城市环境研究所 | A kind of compound carbon aerogels of electrostatic spinning nano fiber and preparation method thereof |
| CN109730208A (en) * | 2019-02-14 | 2019-05-10 | 广东恒兴饲料实业股份有限公司 | A kind of meat rabbit feed and preparation method thereof without calcium monohydrogen phosphate |
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Application publication date: 20150603 |