CN101731458A - Yolk powder containing double-titer yolk antibody of anti-porcine CCK/Urease, and preparation method thereof - Google Patents
Yolk powder containing double-titer yolk antibody of anti-porcine CCK/Urease, and preparation method thereof Download PDFInfo
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- CN101731458A CN101731458A CN200810217587A CN200810217587A CN101731458A CN 101731458 A CN101731458 A CN 101731458A CN 200810217587 A CN200810217587 A CN 200810217587A CN 200810217587 A CN200810217587 A CN 200810217587A CN 101731458 A CN101731458 A CN 101731458A
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Abstract
The invention discloses yolk powder containing double-titer yolk antibody (Immunoglobulin yolk, IgY) of cholecystokinin/intestinal bacterial urease. A preparation method of the yolk powder comprises the following steps: 1) preparing vaccine by mixing purified cholecystokinin 39 peptide (CCK39)/urease B subunit (UreB) fusion protein with Freund's adjuvant well, immunizing healthy laying hens, determining yolk IgY titer through enzyme-linked immunosorbent assay and collecting hyperimmune eggs; and 2) separating yolk from hyperimmune eggs and preparing the yolk into yolk powder. The yolk powder containing double-titer IgY, which is prepared by the method, is added to feed for feeding pigs, and the satiety of the pigs is not produced or delayed due to CCK in the IgY and secreted by porcine intestinal canals so as to increase feed intake. The IgY can also neutralize urease secreted by intestinal bacteria so as to reduce the production of ammonia, protect porcine intestinal canals and respiratory tract, reduce the waste of feed protein and improve culture environment.
Description
Technical field
The present invention relates to the biologic product field, relate in particular to a kind of plain (Cholecystokinin of anti-swine gallbladder contraction that contains, CCK)/economic benefits and social benefits valency Yolk antibody (Immunoglublin yolk, powdery yolk IgY) and preparation method thereof of enteric bacteria urase (Urease).
Background technology
How to improve the livestock and poultry feed intake, improve breeding environment, reduction ammonia is problem anxious to be solved to the impact of environment always.Improve at present the approach of livestock and poultry feed intake common have following several: the raw material that (1) is selected high-quality for use and is easy to digest; (2) determine the trophic level that daily ration is suitable; (3) feed supplement lactation; (4) feed liquid feed and fermented feed; (5) use the food calling conditioning agent; (6) improve livestock and poultry cultivation environment etc.Utilizing modern biotechnology and product to improve animal feed intake is the focus of studying at present, is one of method wherein and utilize anti-CCK Yolk antibody from the feed intake of the angular adjustment animal of animal physiological adjusting.
On the other hand, along with the raising of aquaculture scale, intensification degree, livestock and poultry self metabolic process and the ammonia that discarded object produced also roll up, and plant and surrounding enviroment are brought great negative impact.The ammonia of livestock and poultry farm mainly is urea and the hydrolysate of uric acid under microorganism urease (Urease) effect that produces owing to gastrointestinal tract of livestock and fowls protein metabolism process, and causes the waste of protein, has reduced feed efficiency; The accumulation of ammonia not only can endanger the health of livestock and poultry, reduces its production performance, also can pollute surrounding enviroment.Therefore, must take the generation and the discharging of corresponding measure control ammonia in the Production of Livestock and Poultry process.
Can adopt urease inhibitor to suppress urease activity at present, thereby reduce the generation of ammonia.This type of inhibitor comprises heavy metallic salt class, natural steroids Sa wine saponin (Yucca Sapanion), diamines, three aminated compounds, hydroxamic acid compounds etc.These inhibitor or cost an arm and a leg, perhaps the growth to livestock and poultry itself has negative effect, perhaps is not approved for livestock and poultry cultivation etc., and it is necessary therefore to develop a kind of safe, novel urease inhibitor.
Summary of the invention
The invention provides a kind of preparation method of powdery yolk of the economic benefits and social benefits valency yolk antibody IgY that contains anti-porcine CCK/Urease, this powdery yolk can improve pig feed intake only, the discharging that reduces its feedstuff-meat ratio and reduce ammonia.
Second purpose of the present invention is to provide a kind of powdery yolk that contains the economic benefits and social benefits valency yolk antibody IgY of anti-porcine CCK/Urease.
For achieving the above object, technical scheme of the present invention is:
The preparation method of the powdery yolk of a kind of economic benefits and social benefits valency IgY that contains anti-porcine CCK/Urease may further comprise the steps:
1) swine gallbladder that purifying is concentrated shrinks plain 39 peptides (CCK39)/enteric bacteria urase B subunit (UreB) fusion and Freund and mixes and make vaccine, open with this vaccine immunity health and to produce hen, tire and collect height and exempt from egg with IgY in the enzyme linked immunosorbent detection test determination yolk;
2) exempt to separate yolk the egg from height, make powdery yolk.
The CCK39/UreB fusion that purifying concentrates mixes with the Freund equal-volume.
Described immune step is specially:
Step 1) at first health open laying hen chest muscle both sides inject respectively 100 μ L concentration be 1mg/mL contain the Freund's complete adjuvant vaccine; After 10 days the chest muscle both sides inject respectively again 200 μ L concentration be 1mg/mL contain the incomplete Freund vaccine; That injects 300 μ L concentration after 25 days more respectively and be 1mg/mL contains the incomplete Freund vaccine; After 55 days injection 400 μ L concentration be 1mg/mL do not contain Adjuvanted vaccines; That injects 500 μ L concentration after 85 days again and be 1mg/mL does not contain Adjuvanted vaccines, and immunity is 5 times altogether.Carry out titration with enzyme linked immunosorbent detection test afterwards, tire to reach 1: 1600 and collect height when above when economic benefits and social benefits valency Yolk antibody and exempt from egg to prepare the high-immunity yolk powder.
Step 2) described powdery yolk preparation process is specially: separate egg white and yolk, collect yolk, spray-drying behind the homogeneous, or vacuum freeze drying, or mix with quality such as adsorbent behind the homogeneous, in 40-50 ℃ of drying wait powdery yolk.
Described adsorbent is cornstarch, precipitated calcium carbonate or silica.
A kind of powdery yolk of the economic benefits and social benefits valency Yolk antibody that contains anti-porcine CCK/Urease fusion by above-mentioned preparation method preparation.
By containing of method for preparing anti-CCK39/UreB fusion economic benefits and social benefits valency Yolk antibody just can distinguish in and a pig CCK and enteric bacteria urase.This economic benefits and social benefits valency Yolk antibody adds application in the pig feed to as feed addictive.
Add to by the powdery yolk of the economic benefits and social benefits valency IgY that contains anti-CCK/Urease of the method for the invention preparation and only to feed pig in the feed, utilize on the one hand anti-CCK39 on intestines and stomach in CCK, pig is not only produced or postpone to produce to satisfy and feel, thereby improve its feed intake; On the other hand, this economic benefits and social benefits valency IgY can also combine with the urase specificity that coliform in the chitling road produces, thereby the urease activity that suppresses enteric microorganism, stop urea and uric acid to change into ammonia, reduce the generation of ammonia, can reduce the concentration of culturing the place ammonia, improve breeding environment, can reduce loss, the waste of protein again, improve feed efficiency.
Description of drawings
Fig. 1 is CCK39 gene PCR product electrophoretic analysis figure, 1:DNA marker DL2000; 2-7:CCK39 PCR product;
Fig. 2 is UreB gene PCR product electrophoretic analysis figure, 1:DNA marker DL2000; 2-5:UreB PCR product;
Fig. 3 is the structure flow chart of the dual-gene fusion expression vector of CCK39/UreB of the present invention.
The specific embodiment
The preparation of embodiment 1CCK39/UreB Expression of Fusion Protein and vaccine
(1) template preparation
(1) preparation of CCK39 cDNA: collect the fresh pig duodenum, extract kit according to RNA and extract the total RNA of pig duodenum, after detect confirming that it possesses integrality, requiring according to cDNA synthetic agent box total RNA reverse transcription to be corresponding cDNA, placed-20 ℃ of preservations standby.
(2) preparation of urease-producing coliform DNA: from fresh pig manure, separate and turn out urease-producing coliform list bacterium colony; spend the night to enlarge and cultivate; collect this fresh cultured thing then; reclaim kit according to plasmid and extract this coliform plasmid, be dissolved among the TE buffer and place-20 ℃ of preservations standby
(2) acquisition of CCK39 gene
Go up the sequence of announcing [gi:47523555] according to GenBank, utilize a pair of special primer of Primer Premier5.0 design design of primers (primer is synthetic by Shanghai Ying Jun Bioisystech Co., Ltd, and is as follows),
Upstream primer is shown in sequence SEQ ID NO:2:
5 '-GATGGATCCTACATCCAGCAGGCTCGAAAAGC-3 ' contains the BamHI restriction enzyme site;
Downstream primer is shown in sequence SEQ ID NO:3:
5 '-GATAAGCTTAAAATCCATCCAGCCCATGTAGTC-3 contains the HindIII restriction enzyme site, and does not contain terminator codon.
With CCK39 cDNA is masterplate, pcr amplification pig duodenum CCK39 genes of interest, reaction: 94 ℃ of 5min, 94 ℃ of 30s, 53 ℃ of 30s, 72 ℃ of 30s, 72 ℃ of 10min, 30 circulations.Carry out the gel imaging system analysis behind the PCR product electrophoresis, consult Fig. 1.
(3) structure of pET43a (+)/CCK39 prokaryotic expression plasmid
After reclaiming the above-mentioned amplified production CCK39 of purifying genes of interest, BamHI and HindIII be double digestion amplified production and pET43a (+) prokaryotic expression plasmid respectively, and gel reclaims.16 ℃ of connections are spent the night, Transformed E .coli DH5 α again, the amicillin resistance screening, choose 6 positive bacterium colonies, carry out PCR and BamH I, the evaluation of Hind III double digestion respectively, the strain number that meets expected results is E.coliDH5 α/pET43a (+)/CCK39, being sent to Shanghai Ying Jun Bioisystech Co., Ltd checks order, the sequence of CCK39 gene such as SEQ ID NO:6, the result analyzes its homology in the Blastn of GenBank comparison.
(4) the UreB gene obtains
Go up the sequence of announcing [gi:148150] according to GenBank, utilize a pair of special primer of Primer Premier5.0 design design of primers,
Upstream primer is shown in sequence SEQ ID NO:4:
5 '-GGGAAGCTTATGATCCCCG GTGAAATTAA-3 ' contains Hind III restriction enzyme site,
Downstream primer is shown in sequence SEQ ID NO:5:
5 '-AAA GCGGCCGC TTAATTCTCACTCTCTAA-3 ' contains Not I restriction enzyme site;
With the urease-producing e. coli plasmid dna is masterplate, pcr amplification UreB genes of interest, reaction: 94 ℃ of 5min, 94 ℃ of 30s, 53 ℃ of 30s, 72 ℃ of 30s, 72 ℃ of 10min, 30 circulations.Carry out the gel imaging system analysis behind the PCR product electrophoresis, consult Fig. 2.
(5) structure of pET43a (+)/dual-gene fusion expression plasmid of CCK39/UreB/
See also Fig. 3, behind the above-mentioned amplified production UreB of the recovery purifying genes of interest, Hind III and Not I be double digestion amplified production and pET43a (+)/CCK39 prokaryotic expression plasmid respectively, and gel reclaims.16 ℃ of connections are spent the night, Transformed E .coli DH5 α again, and 6 positive bacterium colonies are chosen in the amicillin resistance screening, carry out PCR and Hind III respectively, Not I double digestion is identified.The strain number that meets expected results is E.coli DH5 α/pET43a (+)/CCK39/UreB, being sent to Shanghai Ying Jun Bioisystech Co., Ltd checks order, the sequence of CCK39/UreB double-fusion gene is shown in SEQ ID NO:1, the result compares at the Blastn of GenBank, analyzes its homology.
(6) preparation of CCK39/UreB Expression of Fusion Protein and vaccine
Expression engineering bacteria E.coli BL-21 (DE3)/pET43a (+)/CCK39/UreB of being cultured to exponential phase is inoculated in the fermentation medium 2 * YT fluid nutrient medium that contains ampicillin in the ratio of 1% (V/V), 28 ℃ cultivate 4h after, as zymotic fluid A
600During ≈ 0.8, lactose to the final concentration that adds 200g/L is 10g/L, 28 ℃ induce 5h after, 4 ℃, the centrifugal 10min of 10000r/min, collecting precipitation, precipitation is behind PBS washing and resuspended thalline, ultrasonication (300W, ultrasonic time 6s, blanking time, 7s was total to ultrasonic 40 times), 4 ℃, the centrifugal 15min of 10000r/min collect the cracking supernatant.Through Ni
2+SDS-PAGE gel electrophoresis analysis behind the-NTA ni-sepharose purification again through dialysis, concentrated, adopts the Bradford detection method to measure fusion content then.1: 1 by volume ratio and Freund are mixed and made into the vaccine that concentration is 1mg/mL.
Selected for 22 weeks open ages and produce healthy bird inlay, establish two groups of immune group and control groups, and the egg of leaving and taking before the immunity compares, raise by this kind fowl raising requirement.The recombinant C CK39/UreB fusion of purifying and equal-volume Freund mixed make vaccine, adopt the mode of chest multiple spot intramuscular injection to carry out immunity.Immune programme for children and dosage of inoculation are as shown in table 1.
Table 1 laying hen immune programme for children and immunizing dose
| The immunity time (my god) | Vaccine | Immunization route | Inoculum concentration/only |
| ??1d | CCK39/UreB fusion+Freund's complete adjuvant | The chest muscle multiple spot | ??100μg |
| ??10d | CCK39/UreB fusion+incomplete Freund | The chest muscle multiple spot | ??200μg |
| ??25d | CCK39/UreB fusion+incomplete Freund | The chest muscle multiple spot | ??300μg |
| ??55d | The CCK39/UreB fusion | The chest muscle multiple spot | ??400μg |
| ??85d | The CCK39/UreB fusion | The chest muscle multiple spot | ??500μg |
Begin to collect every day egg and numbering after head exempts from, 4 ℃ of preservations are standby.Knock out earlier eggshell when getting yolk, separate yolk and egg white, wash the yolk surface repeatedly, remove egg white as far as possible with sterile purified water, use the punctures membrane of yolk, sucking-off yolk is measured the yolk volume, adds 9 times of sterile purified water dilutions, and regulate the pH value between the 5.0-5.5 with HCl, dilution leaves standstill more than the 6h in 4 ℃, wait to occur obvious layering after, draw supernatant; 4 ℃, the centrifugal 30min of 12000r/min get supernatant, and metered volume adds 0.01% mercuric sulphide, little packing ,-20 ℃ of preservations.
1, the mensuration of enzyme labelled antibody working concentration is selected
Carbonate buffer solution with 0.05moL/L pH9.6 is done dilution in 1: 10,1: 20,1: 40,1: 80,1: 160,1: 320,1: 640,1: 1280 with the positive IgY of the anti-CCK39/UreB of chicken, coated elisa plate 1-8 row, 100 μ L/ holes change 4 ℃ over to behind 37 ℃ of constant temperature 1h and spend the night; After washing, the sealing, with pH7.4 PBS with enzyme labelled antibody do diluted in 1: 200,1: 400,1: 800,1: 1000 after, it is capable to add ELISA Plate 1-4 respectively, behind 37 ℃ of water-bath 1h, detects by indirect ELISA method, measures each hole OD
490Value, work as OD
490Value is about 1.0 and the positive IgY dilution factor of the anti-CCK39/UreB of chicken pairing enzyme labelled antibody concentration when maximum, as enzyme labelled antibody best effort concentration.Best enzyme labelled antibody concentration working dilution is 1: 12800.
2, the suitableeest bag of CCK39/UreB antigen determining by the optimum dilution degree of concentration and yin and yang attribute IgY
With the carbonate buffer solution of 0.05mol/LpH9.6 antigen is done the dilution of 1: 20,1: 40,1: 80 and 1: 160,1-4 is capable for coated elisa plate, and the 100uL/ hole changes 4 ℃ over to behind 37 ℃ of 1h and spends the night; After washing, the sealing, the CCK39/UreB positive, negative IgY are done dilution in 1: 20,1: 40,1: 80,1: 160,1: 320 simultaneously, then positive IgY is joined the 1st, 3,5,7,9 row of ELISA Plate respectively, negative IgY joins ELISA Plate the 2nd, 4,6,8,10 row, the 11st row add IgY dilution (pH7.4 PBS) and make blank, behind 37 ℃ of water-bath 1h, detect by indirect ELISA method.OD when positive serum
490Value is near 1.0, and negative serum is less than 0.2, and the P/N value is when maximum, pairing antigen diluent degree be best bag by concentration, the diluted concentration of the yin and yang attribute serum of correspondence is best yin and yang attribute IgY working concentration, the suitableeest bag of antigen is 3.125 μ g/mL by concentration.
3, indirect ELISA detects
It is as follows that indirect ELISA detects the concrete operations flow process:
Antigen coated → as to wash plate → seal → wash plate → add to treat sample → wash plate → add enzyme labelled antibody → wash plate → substrate colour developing → cessation reaction → result of determination
The highest the tiring of antibody of the economic benefits and social benefits valency IgY of this Research Institute reaches more than 1: 51200.
It (is the powdery yolk preparation of the economic benefits and social benefits valency Yolk antibody of anti-porcine CCK/Urease) that embodiment 5 contains anti-porcine CCK 39/UreB fusion.
Choose qualified immunity eggs, separate egg white and yolk, collect yolk, by spray-drying behind the homogeneous, or vacuum freeze drying, or mix with quality such as adsorbent behind the homogeneous, in 40-50 ℃ of drying powdery yolk.
Described sorbing material is cornstarch, precipitated calcium carbonate, and in the silica any one.
Contain the described economic benefits and social benefits valency Yolk antibody that can improve feed intake with the discharge capacity that reduces pig house ammonia of pig in this powdery yolk.
The powdery yolk that experimental example contains the economic benefits and social benefits valency Yolk antibody of the anti-porcine CCK/Urease fusion piglet test of feeding
(1) experimental animal and design
1, the assorted piglet of binary of 54 birth weights close (about 20kg) is selected in test, adopts the single factor experiment design, is divided into 3 processing, and each handles 3 repetitions, and each repeats 6 piglet;
A, processing 1: blank group (basal diet);
B, processing 2: basal diet+negative egg, i.e. positive controls
C, handle 3: basal diet+the contain positive egg of anti-CCK/Urease Yolk antibody, i.e. test group.
2, test daily ration
This pig farm autogamy powder: in the 1000g powder, corn: 290g, dregs of beans: 145g, premix: 65g.
3, test method
I: feed intake is measured
1) raises the place and carry out the cleaned at regular intervals sterilization.The test piglet freely drinks water, and feeds every day 5 times, repeats specifically the situation of searching for food according to each and replenishes feed.From morning 8:30 to 6:30 in afternoon, every 2h checks the situation of searching for food 1 time, writes down the feed consumption rate of each processing every day, the test period is 22d.When on-test, end, piglet claims individual weight behind the 24h on an empty stomach, adds up total feed consumption rate of every repetition each week, calculates piglet average daily gain, average daily ingestion amount and material anharmonic ratio etc.
2) data are handled
Data are expressed as average ± standard deviation, adopt the SAS statistical software that data are carried out ANOVA variance analysis and DUNCAN multiple ratio.
4, feed the powdery yolk of the economic benefits and social benefits valency Yolk antibody that contains anti-porcine CCK/Urease fusion to the influence of piglet growth performance and efficiency of feed utilization
A test group pig feed intake improves 6.24% and 15.29% than positive controls and blank group respectively.A pig average daily gain is the highest with test group (handling 3 groups), is significantly higher than positive controls (handling 2 groups) and blank group (handling 1 group), improves 16.07% and 23.98% respectively; The feedstuff-meat ratio of test group, positive controls and blank group is respectively 1.6755,1.8200 and 1.8167, has reduced by 7.97% and 7.77% respectively, referring to table 2.
At 30 days duration of test by a definite date, the death rate of three processed group is zero, and does not suffer from diarrhoea.Except that causing each pig of blank group, positive controls and test group to take place arthritis other diseases not to take place with there was dampness in the air because of cold weather.
Table 2 contains the powdery yolk of economic benefits and social benefits valency Yolk antibody of anti-porcine CCK/Urease fusion to the influence of piglet growth performance and efficiency of feed utilization
| The blank group | Positive controls | Test group | |
| An initial piglet number | ??30 | ??30 | ??30 |
| A last piglet number | ??30 | ??30 | ??30 |
| The average starting weight of piglet (kg/ head) | ??6.0 | ??6.6 | ??6.1 |
| The average end of piglet heavy (kg/ head) | ??17.00 | ??18.37 | ??19.75 |
| Average total augment weight (kg) | ??11.00 | ??11.77 | ??13.65 |
| Average daily gain (kg/ head) | ??0.367 | ??0.392 | ??0.455 |
| Average daily ingestion amount (kg/ head) | ??0.6614 | ??0.7177 | ??0.7625 |
| Feedstuff-meat ratio (F/G) | ??1.8167 | ??1.8200 | ??1.6755 |
| The diarrhoea head | ??0 | ??0 | ??0 |
II: the mensuration of urease activity in the ight soil
Respectively 0,2,4,6,8,10,12,14,16,18,20 and the 22d fresh excreta sample of collecting each processing place proper container standby.Adopt the pH value-added approach to measure urease activity.
Concrete operation method is as follows:
Accurately take by weighing two parts in 2.0g sample, place two 25ml test tubes respectively, wherein one adds the 20ml phosphate buffer, as blank test (A pipe); Another adds 20ml urea phosphate buffer solution, as developmental tube (B pipe), building the test tube plug immediately also acutely shakes, place 30 ± 0.5 ℃ of waters bath with thermostatic control, every the 5min jolting once, accurately timing is behind the maintenance 30min, every pipe adds 4 saturated mercuric chloride solutions immediately respectively, with cessation reaction.Measure its pH value respectively.
The result calculates: enzymatic activity=B-A
In the formula: B---the pH value of B pipe
The pH value of A---A pipe
5, contain the powdery yolk of economic benefits and social benefits valency Yolk antibody of anti-porcine CCK/Urease fusion to the influence of intestine of young pigs microorganism urease activity
Table 3 has been listed the microorganism urease activity of different disposal group pig manure sample.As known from Table 3, the pH of feces difference is respectively 0.342,0.322 and 0.269 in the test tube of the test group of blank, positive control and interpolation Yolk antibody.Because the pH value difference is big more, urease activity is strong more, therefore adds Yolk antibody group urease activity and has descended 21.35% and 16.46% respectively than blank, positive controls.This result of the test explanation: add that the urasin activity to intestine of young pigs has the obvious suppression effect behind the powdery yolk of economic benefits and social benefits valency Yolk antibody of anti-CCK/Urease fusion, thereby effectively stop urea to change ammonification, reduced the loss of nitrogenous source, provide condition for improving protein utilization.
Urasin activity in each group test piglet ight soil of table 3
| Handle group | Urease activity Δ pH |
| The blank group | ??0.342 |
| Positive controls | ??0.322 |
| Add the Yolk antibody test group | ??0.269 |
SEQU?ENCE?LISTING
＜110〉Wen Feng, recklessly
＜120〉powdery yolk of a kind of economic benefits and social benefits valency Yolk antibody that contains anti-porcine CCK/Urease and preparation method thereof
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<170>PatentIn?version?3.5
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<210>3
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caaatgggtc?tggtccccgg?ggcagcgcgg?gttctggtac?gattgatgac?gacgacaaga?????60
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tgggctggat?ggattttaag?cttgcggccg?cacagctgta?tacac
Claims (7)
1. the preparation method of the powdery yolk of an economic benefits and social benefits valency Yolk antibody that contains anti-porcine CCK/Urease may further comprise the steps:
1) to shrink plain 39 peptides be that CCK39/ enteric bacteria urase B subunit is that UreB fusion and Freund mix and make vaccine to the swine gallbladder that purifying is concentrated, open with this vaccine immunity health and to produce hen, carry out titration and collect height and exempt from egg with the enzyme linked immunosorbent detection test;
2) exempt to separate yolk the egg from height, make powdery yolk.
2. the preparation method of the powdery yolk of a kind of economic benefits and social benefits valency Yolk antibody that contains anti-porcine CCK/Urease according to claim 1 is characterized in that: fusion CCK39/UreB and Freund equal-volume that purifying concentrates are mixed and made into vaccine.
3. the preparation method of the powdery yolk of a kind of economic benefits and social benefits valency Yolk antibody that contains anti-porcine CCK/Urease according to claim 1, it is characterized in that: described immune step is specially: step 1) at first health open laying hen chest muscle both sides inject respectively 100 μ L concentration be 1mg/mL contain the Freund's complete adjuvant vaccine; After 10 days the chest muscle both sides inject respectively again 200 μ L concentration be 1mg/mL contain the incomplete Freund vaccine; That injects 300 μ L concentration after 25 days more respectively and be 1mg/mL contains the incomplete Freund vaccine; After 55 days injection 400 μ L concentration be 1mg/mL do not contain Adjuvanted vaccines; That injects 500 μ L concentration after 85 days again and be 1mg/mL does not contain Adjuvanted vaccines, and immunity is 5 times altogether, carries out titration with the enzyme linked immunosorbent detection test afterwards, tires to reach 1: 1600 and collect height when above when economic benefits and social benefits valency Yolk antibody and exempts from egg to prepare the high-immunity yolk powder.
4. the preparation method of the powdery yolk of a kind of economic benefits and social benefits valency Yolk antibody that contains anti-porcine CCK/Urease according to claim 1, it is characterized in that: step 2) described powdery yolk preparation process is specially: separate egg white and yolk, collect yolk, spray-drying behind the homogeneous, or vacuum freeze drying, or mix with quality such as adsorbents behind the homogeneous, in 40-50 ℃ of drying powdery yolk.
5. the preparation method of the powdery yolk of a kind of economic benefits and social benefits valency Yolk antibody that contains anti-porcine CCK/Urease according to claim 4, it is characterized in that: described adsorbent is cornstarch, precipitated calcium carbonate or silica.
6. by the powdery yolk of the economic benefits and social benefits valency Yolk antibody that contains anti-porcine CCK/Urease fusion of the described preparation method of claim 1 preparation.
7. the powdery yolk of the described economic benefits and social benefits valency Yolk antibody that contains anti-porcine CCK/Urease fusion of claim 6 is as the application of feed addictive.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008102175876A CN101731458B (en) | 2008-11-10 | 2008-11-10 | Yolk powder containing double-titer yolk antibody of anti-porcine CCK/Urease, and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008102175876A CN101731458B (en) | 2008-11-10 | 2008-11-10 | Yolk powder containing double-titer yolk antibody of anti-porcine CCK/Urease, and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101731458A true CN101731458A (en) | 2010-06-16 |
| CN101731458B CN101731458B (en) | 2012-11-21 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN2008102175876A Active CN101731458B (en) | 2008-11-10 | 2008-11-10 | Yolk powder containing double-titer yolk antibody of anti-porcine CCK/Urease, and preparation method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102429097A (en) * | 2011-07-25 | 2012-05-02 | 大连赛姆生物工程技术有限公司 | Food therapy yolk antibody yolk liquid pasty preparation for animals and production method thereof |
| CN102696887A (en) * | 2012-06-20 | 2012-10-03 | 湖南海谷生物技术有限公司 | Feed active egg powder with nutrition, healthcare and growth promotion functions and production method thereof |
| CN103357011A (en) * | 2013-07-10 | 2013-10-23 | 深圳市宝舜泰生物医药股份有限公司 | Pharmaceutical composition and composite burn cream for treating burns and scalds and preparation method thereof |
| CN105367659A (en) * | 2015-11-18 | 2016-03-02 | 李昕阳 | Preparation method and application of urease activity inhibition yolk antibody |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996004933A2 (en) * | 1994-08-05 | 1996-02-22 | Wisconsin Alumni Research Foundation | Cck antibodies used to improve feed efficiency |
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2008
- 2008-11-10 CN CN2008102175876A patent/CN101731458B/en active Active
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102429097A (en) * | 2011-07-25 | 2012-05-02 | 大连赛姆生物工程技术有限公司 | Food therapy yolk antibody yolk liquid pasty preparation for animals and production method thereof |
| CN102696887A (en) * | 2012-06-20 | 2012-10-03 | 湖南海谷生物技术有限公司 | Feed active egg powder with nutrition, healthcare and growth promotion functions and production method thereof |
| CN102696887B (en) * | 2012-06-20 | 2013-07-31 | 湖南海谷生物技术有限公司 | Feed active egg powder with nutrition, healthcare and growth promotion functions and production method thereof |
| CN103357011A (en) * | 2013-07-10 | 2013-10-23 | 深圳市宝舜泰生物医药股份有限公司 | Pharmaceutical composition and composite burn cream for treating burns and scalds and preparation method thereof |
| CN105367659A (en) * | 2015-11-18 | 2016-03-02 | 李昕阳 | Preparation method and application of urease activity inhibition yolk antibody |
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| CN101731458B (en) | 2012-11-21 |
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