CN101007845B - Method for screening high-yield egg-laying poultry - Google Patents
Method for screening high-yield egg-laying poultry Download PDFInfo
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- CN101007845B CN101007845B CN2006100016999A CN200610001699A CN101007845B CN 101007845 B CN101007845 B CN 101007845B CN 2006100016999 A CN2006100016999 A CN 2006100016999A CN 200610001699 A CN200610001699 A CN 200610001699A CN 101007845 B CN101007845 B CN 101007845B
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Abstract
The present invention provides an effective method for screening egg-producing poultry, which is characterized in that insulin-like growth factor I (IGF-I) is used as an index. The method comprises the steps of collecting a tested sample, and detecting the total amount of the insulin-like growth factor I in the sample, wherein the judgment of the high-egg-yield poultry comprises the step of judging whether the insulin-like growth factor I detected in the sample is higher or not. The invention also provides an epitope of insulin-like growth factor I and antibodies directed against the epitope.
Description
Technical field
The present invention relates to a kind of method of screening high egg-yield fowl, the lay eggs method of native chicken of particularly a kind of screening height.
Background technology
The raising of soil chicken is to act on one's own and move at open environment by native chicken to improve its meat, owing to the fine and tender taste of native chicken receives liking of part Asian countries and European countries greatly, is commonly used to prepare multiple nourishing tonic especially in Chinese society.Yet native chicken has lower egg laying performance (Li Yuanbai, 1992 than commercial laying hen haply.The native chicken in Taiwan.Animal husbandry system of National Chung Hsing University, in the platform, Taiwan.56 pages).
Common native chicken has complicated and more irregular assortment of genes (is worn modest, bell show branch, Zhang Xiuluan, Huang Xiangji.1997。Close relative's breeding of Taiwan soil chicken: VI. inbred line two-way cross descendant's a growth and reproductive performance field assessment.Middle poultry meeting will 26 (2): 187-196); on most poultry farming farm; no matter be small-scale or commercial-scale farm; the soil chicken all is not to preserve its kind with systematized mutual breeding (cross-mating), and often is to select the preferable native chicken of meat as the kind chicken of cultivating from the chicken group.Yet these kinds chicken generally has lower egg laying performance than other laying hen, and some is planted chicken even only give birth to a term and is less than 70 eggs (bell show branch, 1998.Taiwan Province's livestock products test 40 all festival domestic animal fowl genetic breeding Conference Papers collection pp.121-132).When the world market of native chicken still continues to increase at present, but suffer from can't increase native chicken output with the demand of maintaining market.
Summary of the invention
In view of the demand, the invention provides a kind of effective ways that screen high egg-yield fowl.
One aspect of the present invention provides a kind of method of screening high egg-yield fowl, it is characterized by with quasi-insulin growthing factor I (insulin like growth factor I (IGF-I)) as index, this method comprises a corpse or other object for laboratory examination and chemical testing of collecting the test bird, detect the total amount of quasi-insulin growthing factor I in a corpse or other object for laboratory examination and chemical testing, wherein the judgement of high egg-yield fowl is according to the quasi-insulin growthing factor I content that is detected in the corpse or other object for laboratory examination and chemical testing.
The present invention also provides the antigen determining area of quasi-insulin growthing factor I on the other hand, comprises the amino-acid residue shown in SEQ ID NO:1 75 to 89 at least.
The present invention also provides antibody more on the other hand, but its specificity comprises the amino-acid residue shown in SEQ ID NO:1 75 to 89 at least in conjunction with the antigen determining area of quasi-insulin growthing factor I.
Another aspect of the invention provides a kind of method of screening high egg-yield fowl, it is characterized by with quasi-insulin growthing factor I as index, and wherein this high egg-yield fowl is to be decided by the quasi-insulin growthing factor I content that detected in the corpse or other object for laboratory examination and chemical testing.The specific embodiment one of according to the present invention, wherein eliminate quasi-insulin growthing factor I content among the tested fowl group lower 5 percent to 50, for example 1 15.Another specific embodiment according to the present invention again, wherein select quasi-insulin growthing factor I content among the tested fowl group higher 2 percent to 20, for example 2 percent.
Further aspect of the present invention provides a kind of method of screening high egg-yield fowl, this method comprises a corpse or other object for laboratory examination and chemical testing of collecting the test bird, with antibody, but its specificity is in conjunction with the antigen determining area of quasi-insulin growthing factor I, comprise the amino-acid residue shown in SEQ ID NO:1 75 to 89 at least, detect the total amount of insulin-like growth factor in the corpse or other object for laboratory examination and chemical testing, wherein the judgement of high egg-yield fowl is whether to contain higher amount according to the quasi-insulin growthing factor I that is detected in the corpse or other object for laboratory examination and chemical testing.
Description of drawings
Fig. 1 is the protein band of the colloid pictorial display quasi-insulin growthing factor I of polyacrylamide gel electrophoresis.Indicating 1,2,5 places among the figure is high-yield egg chicken serum, indicates 3,4,6 places and is low laying hen serum.
Fig. 2 is that a graphic representation shows the analytical results that uses the PONT biosensor to carry out the field test.
Fig. 3 is that a graphic representation shows the analytical results that uses enzyme linked immunosorbent assay to carry out the field test.
Embodiment
For making content of the present invention clear understandable, below will do more detailed explanation to employed proper noun.
" antigen determining area " is meant single antigen position and antibody response on the protein at this.Antigen generally has a plurality of not synantigen determining area, and has different narrow spectrum reactions with antibody.
" immunizator " speech refers to and anyly causes when entering in the body or produce immunoreactive material or life entity.
The invention provides a kind of method of screening high egg-yield fowl, it is characterized by with quasi-insulin growthing factor I (insulin like growth factor I (IGF-I)) as index.
According to one embodiment of the invention, the antigen determining area of quasi-insulin growthing factor I is provided, comprise the amino-acid residue shown in SEQ ID NO:1 75 to 89 at least.This antigen determining area is positioned at quasi-insulin growthing factor I intermediary high-hydrophilic, flexibility and the zone of coming to the surface.
This antigen determining area can synthesize peptide (peptide), this antigen determining area also can be connected with other molecule or in conjunction with to form albumen aggregate (protein aggregates), antigen protein or immunizator, can promote immune response to produce the phase antagonist in entering the host animal body.The judgement of antigen determining area can be sought the lay eggs protein index of the relevant biopathways of research of large numbers of participations by bioinformation software.
The present invention also provides antibody, but its specificity is in conjunction with the antigen determining area of quasi-insulin growthing factor I, this antigen determining area comprises the amino-acid residue shown in SEQ ID NO:1 75 to 89 at least, and wherein this antibody can be polyclonal antibody (polyclonal antibody) or monoclonal antibody (monoclonalantibody).
The present invention also provides a kind of method of screening high egg-yield fowl, and wherein this high egg-yield fowl is decided by the interior quasi-insulin growthing factor I content that is detected of a corpse or other object for laboratory examination and chemical testing.The specific embodiment one of according to the present invention, wherein eliminate quasi-insulin growthing factor I content among the tested fowl group lower 5 percent to 50, for example 1 15.Another specific embodiment according to the present invention again, wherein select quasi-insulin growthing factor I content among the tested fowl group higher 2 percent to 20, for example 2 percent.
The specific embodiment one of according to the present invention, this method comprises by the test bird collects a corpse or other object for laboratory examination and chemical testing, with the antibody of specificity in conjunction with the antigen determining area of quasi-insulin growthing factor I, detect the content of insulin-like growth factor I in the corpse or other object for laboratory examination and chemical testing, this antigen determining area comprises the amino-acid residue shown in SEQ ID NO:1 75 to 89 at least, and wherein whether the judgement of high egg-yield fowl is to contain higher or low amount according to the protein content that is detected in the corpse or other object for laboratory examination and chemical testing.Protein definition in this higher amount is with the antigen determining area of specificity in conjunction with quasi-insulin growthing factor I, comprise the antibody of the amino-acid residue shown in SEQ ID NO:1 75 to 89 at least, in the fowl group, select the institute higher bird of the protein content that detects, or the bird of the superseded low percentages of protein that detects.For example, when farmer's human desires selects among this fowl group 5 percent high egg fowl as stud bird, he will in the fowl group, select a serum corpse or other object for laboratory examination and chemical testing detect quasi-insulin growthing factor I content higher 5 percent as stud bird.And for example, 1 15 low egg fowl in superseded this fowl group of farmer's human desires, he will select a serum corpse or other object for laboratory examination and chemical testing to detect 1 15 lower conduct of quasi-insulin growthing factor I content in the fowl group and eliminate bird.
A corpse or other object for laboratory examination and chemical testing used in the present invention can comprise cell, tissue or the body fluid of testing bird.In one embodiment, a corpse or other object for laboratory examination and chemical testing comprises that by the collected serum of test bird this serum can be prepared by the test whole blood that bird extracted according to known serological technique.
According to the present invention, the test bird comprises chicken class, duck class, goose class, dove class, coloured birds and rare birds.According to specific embodiment, this chicken class is that native chicken class includes but not limited to commercial red plumage soil chicken, comes the red plumage soil of prosperous laying hen and Taiwan chicken.
In a preferred embodiment, carry out western hybridization with the quasi-insulin growthing factor I in the red plumage soil chicken serum of identification Taiwan.As shown in Figure 1, this antibody picks out the quasi-insulin growthing factor I with the about 13.7kDa of precursor and the about 7.0kDa of mature protein in height is laid eggs Taiwan of group red plumage soil chicken serum.Yet, also can carry out other immune analysis method, comprise that having antibody is fixed in wherein enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay (ELISA)) and protein chip, with the content of quasi-insulin growthing factor I in the decision serum.
According to another embodiment, the peptide of antigen determining area of the present invention can further be made amendment with multiple antigenic peptide modification technology before making host animal produce immune response.Then, collect the antibody that is produced from host animal.
In a preferred embodiment, antibody is to form by immunizator to make host animal, and is prepared as the rabbit generation immune response of known generation antibody.The host animal that the present invention produces antibody includes but not limited to ox, horse, donkey, goat, sheep, pig, mouse, rat and other commercially available laboratory animal that is used to produce antibody, as long as this host animal itself does not have the peptide sequence of above-mentioned antigen determining area.
Produce immunoreactive method for known to the person of ordinary skill in the field and comprise subcutaneous or the abdominal injection immunizator.The person of ordinary skill in the field can change the employed immune scale of construction of generation immune response according to antigenicity that will produce immunoreactive animal, immunizator and injection position.Increase method for enhancing antigenicity for known to the person of ordinary skill in the field and include but not limited at heterologous protein (heterologous protein), (β-galactosidase) connects antigen, or by comprise adjuvant (adjuvant) when producing immune response as sphaeroprotein or beta galactase.
According to another embodiment of the present invention, this antibody can be polyclonal antibody or monoclonal antibody.
About obtaining of monoclonal antibody, the spleen cell that produces the host animal of immune response is removed, merge (fuse) with cancer cell of bone marrow, make it become the hybridoma (hybridoma cells) that produces monoclonal antibody.
One of a plurality of methods known to can the person of ordinary skill in the field pick out the hybridoma that produces the antibody with required feature, and these comprise with a stain analyzes (dot blot analysis), an enzyme linked immunosorbent assay, western hybridization or reactive immunoassay.Duplicate the hybridoma of the required antibody of secretion, and judge immunoglobulin (Ig) kind and time kind according to known steps.
About obtaining of polyclonal antibody, separate and comprise sero-fast antibody from producing immunoreactive animal, and have with the screening of one of above-mentioned steps the existing of antibody of specific specificity.
Above-mentioned antibody uses with detectable mark pattern, and antibody can be by using radio isotope (radioisotope), affinity marker thing (as vitamin H, avidin etc.), enzyme labelling thing (as horseradish peroxidase (horseradish peroxidase) or alkaline phosphatase (alkalinephosphatase) etc.), fluorescent marker (as fluorescent yellow or rhodamine (rhodamine) etc.), paramagnetic atom (paramagnetic atoms) etc.The step of finishing mark is for known to the person of ordinary skill in the field, that the antibody of institute of the present invention mark can be used for is external, in the body and original position analysis (in situassay) in case in the serum of bird or a corpse or other object for laboratory examination and chemical testing liquid identification quasi-insulin growthing factor I (or its fragment), preferable immunoassay is known different sorts enzyme linked immunosorbent assay and reactive immunoassay.Antibody itself can be directly used in treatment or other diagnostic method.
Above-mentioned antibody can be fixed on the solid support thing, the example of solid support thing comprises plastics, as polycarbonate (polycarbonate), complex carbohydrates (complex carbohydrates) as agarose (agarose) and sepharose (sepharose), acrylic resin (acrylic resin) and as polyacrylamide (polyacrylamide) and latex bead (latex beads).Antibody is connected technology on the solid support thing for known to the person of ordinary skill in the field, and that institute's fixed antibody can be used for is external, in the body and original position analysis (in situ assay) and be used in identification quasi-insulin growthing factor I in the serum of bird.Therefore antibody can be fixed or is combined in protein chip or Portable induction installation to detect the intravital quasi-insulin growthing factor I of inspection of test bird, so that according to the protein content screening high egg-yield fowl that is detected in the corpse or other object for laboratory examination and chemical testing.
The present invention is further illustrated with following example.Following example belongs to illustrative, is not intended to limit the present invention's category.
Example one is searched the lay eggs albumen index of native chicken of height
The Taiwan in 35 weeks red plumage soil chicken is used in this experiment, be divided into height lay eggs group and the low group of laying eggs according to the red plumage in the Taiwan soil sum of laying eggs in 26 to 48 weeks of chicken, as shown in Table 1, the lay eggs average egg number of chicken in 26 to 48 weeks of group of height is approximately 117, and the chicken that is approximately 69 at the average egg number in 26 to 48 weeks then is listed in the low group family of laying eggs.
Table 1
| Group | Sample number | Egg number (individual) |
| The high group of laying eggs | 19 | 117±1 * |
| The low group of laying eggs | 18 | 69±4 * |
*Significant difference degree p＜0.05
Be the albumen index of finding out and laying eggs relevant, red plumage soil chicken extracts preadolescence (pre-pubertal) stage (the 14th week) and (mature) stage in ripening stage (the 35th a week) blood corpse or other object for laboratory examination and chemical testing from each gulf of organizing a performance, get supernatant liquor behind the centrifugal blood, quantitative, dry back adds lysate (lysis buffer) and comprises:
7M urea (urea), 2M Thio Urea (thiourea), 4% dimethylamino propanesulfonic acid (3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulphonate, CHAPS), 2% dithiothreitol (DTT) (Dithiothreitol, DTT) and 2%pH 4-7 immobilization pH gradient buffering liquid (immobilized pH gradient buffer, IPG buffer) with stripping protein.
After centrifugal 15 minutes, take out the supernatant liquor of 400 micrograms with 10,000 centrifugal force values (g) in 4 ℃, for carrying out the electrophoretic analysis of amphitropic protein matter.
Amphitropic protein matter electrophoretic analysis work be divided into first dimension isoelectric focusing electrophoresis (isoelectricfocusing, IEF) and the sds polyacrylamide gel electrophoresis analysis of second dimension (SDS-polyacrylamide gel electrophoresis, SDS-PAGE).Use immobilization pH gradient, adhere to the acrylamide molecule and make gel with carrier ampholyte to form fixed pH gradient, use IPGphor system (Amersham Biosciences) that protein is carried out 100,000 volt of hour (voltage-hour, VH) electric field, comprise 50mM three (methylol) aminomethane hydrochloride (Tris-HCl) pH8.8 with SDS level pad (SDSequilibration buffer) afterwards, 6M urea, 30% glycerine (glycerol) and 0.02%SDS water and adhesive tape, again equilibrated IPG adhesive tape is put into 10-15% gradient SDS running gel upper limb, use Daltsix vertical type electrophoresis system (Amersham Biosciences) to contain 1% gel embedding of SDS damping fluid, carry out the electrophoretic analysis of second dimension, the voltage that uses is 200 volts (V), electric current is 30 milliamperes (mA), stops electrophoresis work when staining agent marches to the sheet glass lower edge.
Second gel finished of dimension electrophoretic analysis is soaked in fixed solution (fixation solution) comprised 10% acetic acid and 40% methyl alcohol 10 minutes, be soaked in again in water and the solution (rehydration solution) at least 5 minutes, and used redistilled water (secondary water) to clean afterwards 3 times.Gel is immersed in contains a solution A (Solution A; 80 mg/ml sodium sulfate (Na
2SO
4)) 100 milliliters of secondary water in 10 minutes, clean 2 times with secondary water, add and contain 1 ml soln B (Solution B; 100 mg/ml Silver Nitrate (AgNO
3)) 50 milliliters of secondary water effect 10 minutes, clean 1 time with secondary water, add and contain 40 milliliters of secondary water, 10 ml soln C (Solution C; 150 mg/ml yellow soda ash (Na
2CO
3)) and a solution D (Solution D; 37.5% formaldehyde (formaldehyde)) 50 milliliters of effects of solution were outwelled after 10 seconds, add identical solution colour developing again, after gel is with Silver Nitrate colour developing fully, add 2 milliliters of acetic acid (acetic acid) neutralization reaction, afterwards gel is moved in the secondary water, be stored in 4 ℃.
To dye that the target protein particle downcuts one by one on the gel through amphitropic protein matter electrophoresis and silver dyeing silver later, be soaked in decolouring damping fluid (destaining buffer) (15mM yellow prussiate of potash (potassium ferricyanide[K
3Fe (CN)
6]), 50mM Sulfothiorine (sodiumthiosulfate[Na
2S
2O
3])) in decolour fully up to gel.Gel is placed 1 milliliter of 25mM bicarbonate of ammonia (NH
4HCO
3) in slight concussion 10 minutes, remove supernatant liquor, inserted in 1 milliliter of 25mM bicarbonate of ammonia/50% acetonitrile (acetonitrile) slight concussion again 10 minutes, remove supernatant liquor, the shrinkage a little of gel this moment, it is dried to utilize Vacuumdrier to take off fully again.Add 50 milliliters of 25mM bicarbonate of ammonia and 1 milliliter of beta-mercaptoethanol (β-mercaptoethanol) act on 20 minutes under the lucifuge, add 50 microlitre 25mM bicarbonate of ammonia/50% acetonitriles and 5 microlitre 4-vinylpridines (4-vinylpyridine) again, effect is 20 minutes under the lucifuge, remove supernatant liquor, repeat again to place 50 microlitre 25mM bicarbonate of ammonia and 50 nanograms to modify Trypsin (modified trypsin) gel in the above-mentioned steps (Promega) in 37 ℃ of effects 18 hours, collect supernatant liquor, and clean gel with 200 microlitres, 0.1% formic acid (formic acid), mixed gently 20 minutes, collect supernatant liquor and mix with before supernatant liquor, to be kept at-20 ℃ after the Vacuumdrier drying, wait for liquid chromatography tandom mass spectrometer (LC/MS/MS) analysis, the liquid chromatography tandom mass spectrometer combines the solute separating force of high pressure liquid chromatograph (HPLC) and the detectivity of mass spectrograph acumen, high pressure liquid chromatograph is according to the single-minded characteristic of the kind of a plurality of particular peptide, as electric charge, size, specific label of hydrophobicity and existence (tag) or amino acid isolated peptides.
Example two antibody are synthetic
The antigen determining area that use-case one is predicted is 15 amino acid peptides that come from the amino-acid residue 75 to 89 shown in SEQ ID NO:1.This peptide has sequence KPTGYGSSSRRLHHK and is positioned at quasi-insulin growthing factor I intermediary high-hydrophilic, flexibility and the zone of coming to the surface, and preferablely has a secondary protein structure, as α spiral (α-helix) and βPing Mian (β-sheet).
The synthetic of peptide is synthetic with multiple antigenic peptide (multiple antigenic peptide) form.This kind peptide molecular weight is approximately 8～18kDa, and extend outward for comprising 8 peptide chains of 7 Methionins (lysine) bond the central zone of multiple antigenic peptide, in the manufacturing of antibody, does not need and carries combination of proteins and just can independently be used as immunizing antigen.All synthetic peptides must need to be confirmed its quality via high pressure liquid chromatograph and mass spectrometric analysis.Then peptide is expelled to rabbit to produce the antigen determining area that polyclonal antibody specificity connection peptides comprises the amino-acid residue 75 to 89 shown in sequence number 1.Contain sero-fast antibody and use the Analysis and Screening of known some stain to have specific narrow spectrum antibody from producing to separate in the immunoreactive rabbit.
Analyze antibody with the quasi-insulin growthing factor I in the red plumage soil chicken serum of identification Taiwan by the western hybrid method, behind the intraserous protein of above-mentioned sds polyacrylamide gel electrophoresis analytical separation, protein transduction on the gel is moved on to nitrocotton (nitrocellulose) or poly-difluoroethylene (PVDF) film, at blocking-up liquid (blocking solution; 100 milliliters of cleaning buffer solutions (20mM three (methylol) aminomethane hydrochloride pH7.4,500mM sodium-chlor, 0.05% polysorbas20 (Tween-20)) and 3% gelatin) interior with above-mentioned film blocking-up one hour, clean with cleaning buffer solution again.Adding combines with the film generation with the above-mentioned polyclonal antibody of cleaning buffer solution and the dilution of 1% gelatin, then add alkaline phosphorus and separate secondary antibody that enzyme engages anti-rabbit immunoglobulin G (alkaline phosphataseconjugated anti-rabbit IgG) with in conjunction with polyclonal antibody, its conjugated antigen determining area comprises the amino-acid residue shown in sequence number 1 75 to 89 at least.Film is transferred to alkaline phosphorus separates the enzyme developing solution developing, the film after the air-dry development and with the scanning of laser intensity instrument to quantize protein content.Please refer to Fig. 1, this antibody picks out the quasi-insulin growthing factor I (the 1st road of running gel, the 2nd road and the 5th road) with the about 13.7kDa of precursor and the about 7.0kDa of mature protein in height is laid eggs Taiwan of group red plumage soil chicken serum.
The example three screening height native chicken of laying eggs
Raise commercial red plumage soil chicken at two different farms (breeding farm 1 and breeding farm 2), come the bird inlay of the red plumage soil of prosperous laying hen and Taiwan chicken.
Collect whole bloods with preparation serum in 8,14,18,19,20,22,23,26,27,32 weeks in laying hen vegetative period.By enzyme linked immunosorbent assay according to the quasi-insulin growthing factor I content in the following steps tests bird serum.
Coating in the step of porose disc with suitable antigen, the protein standard/serum that has diluted of getting 150 microlitres/hole adds in 96 porose discs, places 4 ℃ of cultivations overnight.The protein standard is coating damping fluid (4.3 gram sodium bicarbonates, 5.3 gram sodium carbonates and fill it up with to 1 liter with distilled water pH9.4) serial dilution and dilute serum suitably, outwells that liquid in 96 porose discs is removed unconjugated antigen and with cleaning buffer solution (8.0 gram sodium-chlor, the 1.42 gram Sodium phosphate dibasic (Na of 200 microlitres
2HPO
4.2H
2O), 0.2 gram dipotassium hydrogen phosphate, 0.2 gram Repone K, 1 milliliter of polysorbas20 and fill it up with to 1 liter with distilled water pH 7.4) clean porose disc, the blocking-up liquid (8.0 gram sodium-chlor, 1.42 gram Sodium phosphate dibasics, 0.2 gram dipotassium hydrogen phosphate, 0.2 gram Repone K, 10 gram foetal calf serum albumen (bovine serum albumin (BSA) fraction V) are also filled it up with to 1 liter with distilled water pH7.4) that then utilizes 200 microlitres room temperature reaction 45 minutes blocking non-specificity combination, and as above-mentioned wash-out hole dish.
Adding is with 100 microlitre rabbit polyclonal antibodies of antibody diluent (8.0 gram sodium-chlor, 1.42 gram Sodium phosphate dibasics, 0.2 gram dipotassium hydrogen phosphate, 0.2 gram Repone K, 10 gram foetal calf serum albumen are also filled it up with to 1 liter with distilled water pH7.4) dilution and cultivated 45 minutes in 37 ℃ of incubators, repeats above-mentioned cleaning step.Preparation is connected to alkaline phosphorus and separates the anti-rabbit immunoglobulin G secondary antibody of enzyme or horseradish peroxidase and dilute with antibody diluent, add the anti-rabbit immunoglobulin G secondary antibody after the 100 microlitres dilutions again and in 37 ℃ of incubators, cultivated 30 minutes, repeat above-mentioned cleaning step.Then, what add 100 microlitres again is subjected to matter liquid (tetramethyl benzidine (tetramethyl benzidine)) lucifuge as in 37 ℃ of incubators 20 minutes, add 100 microlitre stop buffers (16 milliliters of 1N hydrochloric acid are filled it up with to 1 liter with distilled water) termination reaction, and read porose disc to judge optical density (OD) (Optical Density) in wavelength 450 nanometers at enzyme linked immunosorbent assay dish reading machine.Obtain typical curve by measured protein standard, and calculate intraserous protein concn according to typical curve.Table 2 has been enumerated red plumage soil chicken and leghorn at the collected intraserous quasi-insulin growthing factor I average content of different time points.
Table 2
aAbove-mentioned mean value is with the protein content (nanograms/milliliter) of enzyme linked immunosorbent assay detection with respect to antigen peptide
In the chicken group of red plumage soil chicken, serum quasi-insulin growthing factor I concentration is higher in 22 to 32 weeks, and the expression chicken reaches the ripening stage of laying eggs.On the other hand, leghorn group's serum quasi-insulin growthing factor I concentration is higher in 19 to 23 weeks, and the expression leghorn reaches the ripening stage than red plumage soil chicken is Zao.
It is total and carry out statistical study with the quasi-insulin growthing factor I content that is detected to write down every hen egg of being produced in 55 weeks in addition.Listed as table 3, for Different Chicken, relation conefficient (correlation coefficient) is measured positively related degree between two parameters (lay eggs sum and serum quasi-insulin growthing factor I concentration).
Table 3
aAnalyzed with enzyme linked immunosorbent assay
bRelation conefficient (r)
*Significant difference degree p＜0.01
Represent that near 1 high relation conefficient serum quasi-insulin growthing factor I concentration is very relevant with the chicken egg number, as shown in table 3, no matter all showing to produce in serum quasi-insulin growthing factor I concentration and 55 weeks, most its breeding farms of native chicken has high relation conefficient between the egg sum, therefore the invention provides a kind of effective screening high egg-yield fowl, particularly the method for the high native chicken of laying eggs of screening.
Example four uses the PONT biosensor to carry out the field test
Collecting whole blood 25 to 37 weeks (25-37wk) in the native chicken vegetative period of laying eggs with a preparation serum corpse or other object for laboratory examination and chemical testing.The Eppendorf tube mixing for standby use that the diluent of the serum corpse or other object for laboratory examination and chemical testing of 5 microlitres and 995 microlitres is added 1.5 milliliters, get the corpse or other object for laboratory examination and chemical testing that 100 microlitres have diluted, add the reagent strip that contains the rabbit polyclonal antibody that example two synthesized by collecting window, make a corpse or other object for laboratory examination and chemical testing and antibody response after about 60 minutes, utilize scanner in the PONT biosensor to detect quasi-insulin growthing factor I content in the native chicken serum.
Then, in having 1197 serum corpse or other object for laboratory examination and chemical testing altogether, carry out the analysis of egg laying performance by choosing 10% a minimum serum corpse or other object for laboratory examination and chemical testing of quasi-insulin growthing factor I content concn and other 90% serum corpse or other object for laboratory examination and chemical testing among the native chicken group.As shown in Figure 2, before 29 weeks, all can observe out the laying rate difference between 10% a minimum serum corpse or other object for laboratory examination and chemical testing of quasi-insulin growthing factor I content concn and other the 90% serum corpse or other object for laboratory examination and chemical testing.And the analytical results of field test can be judged lower its laying rate of native chicken of quasi-insulin growthing factor I content concn obviously than lower with other native chicken in the group thus, therefore the invention provides a kind of effective ways that screen high egg-yield fowl.
Example five uses enzyme linked immunosorbent assay to carry out the field test
Collecting whole blood 25 to 50 weeks (25-50wk) in the native chicken vegetative period of laying eggs with a preparation serum corpse or other object for laboratory examination and chemical testing.Detect quasi-insulin growthing factor I content in the native chicken serum corpse or other object for laboratory examination and chemical testing according to example three described enzyme linked immunosorbent assay steps.
Then, in having 1560 serum corpse or other object for laboratory examination and chemical testing altogether, carry out the analysis of egg laying performance by choosing 10% a minimum serum corpse or other object for laboratory examination and chemical testing of quasi-insulin growthing factor I content concn and other 90% serum corpse or other object for laboratory examination and chemical testing among the native chicken group.In addition, the laying rate in 25 to 27 weeks in this example is calculated with following formula according to example four test-results:
10% corpse or other object for laboratory examination and chemical testing that concentration is minimum: Y=-0.0103X
2+ 0.666X-10.124
90% corpse or other object for laboratory examination and chemical testing of other concentration: Y=-0.052X
2+ 0.3284X-4.483
Wherein, Y is a laying rate, and X is age in week.For example, 10% a minimum corpse or other object for laboratory examination and chemical testing of quasi-insulin growthing factor I content concn in this example, the laying rate in 26 weeks is:
Y＝-0.0103(26)
2+0.666(26)-10.124＝0.22
And by that analogy class in minimum 10% corpse or other object for laboratory examination and chemical testing of insulin-like growth factor I content concn and all the other 90% corpse or other object for laboratory examination and chemical testing laying rate in 25 to 27 weeks.
As shown in Figure 3, before 29 weeks, can observe out laying rate difference between 10% a minimum corpse or other object for laboratory examination and chemical testing of quasi-insulin growthing factor I content concn and other 90% corpse or other object for laboratory examination and chemical testing equally.And the analytical results of field test further proves lower its laying rate of native chicken of quasi-insulin growthing factor I content concn obviously than lower with other native chicken in the group thus, therefore the invention provides a kind of effective ways that screen high egg-yield fowl.
The person of ordinary skill in the field should promptly understand, and can make amendment and unlikely departing from its broad sense inventive concept to aforementioned each specific embodiment.Thereby should be appreciated that the present invention is not limited to every certain specific embodiments that this specification sheets discloses, and be to contain ownership as the spirit of the present invention that claim defined and the modification project of category.
Sequence table
＜110〉Taiwan Animal Sci. ﹠ Tech. Inst.
＜120〉method of screening high egg-yield fowl
<130>6P04010-TW
<160>1
<170>PatentIn version 3.3
<210>1
<211>153
<212>PRT
<213>Chicken
<400>1
Met Glu Lys Ile Asn Ser Leu Ser Thr Gln Leu Val Lys Cys Cys Phe
1 5 10 15
Cys Asp Phe Leu Lys Val Lys Met His Thr Val Ser Tyr Ile His Phe
20 25 30
Phe Tyr Leu Gly Leu Cys Leu Leu Thr Leu Thr Ser Ser Ala Ala Ala
35 40 45
Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Ieu Gln Phe
50 55 60
Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Lys Pro Thr Gly Tyr Gly
65 70 75 80
Ser Ser Ser Arg Arg Leu His His Lys Gly Ile Val Asp Glu Cys Cys
85 90 95
Phe Gln Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Ile
100 105 110
Lys Pro Pro Lys Ser Ala Arg Ser Val Arg Ala Gln Arg His Thr Asp
115 120 125
Met Pro Lys Ala Gln Lys Glu Val His Leu Lys Asn Thr Ser Arg Gly
30 135 140
Asn Thr Gly Asn Arg Asn Tyr Arg Met
145 150
Claims (7)
1. the antigen determining area of a quasi-insulin growthing factor I is characterized in that the sequence shown in SEQ IDNO:1 amino-acid residue 75 to 89.
2. antibody, but it is characterized in that the antigen determining area of specificity in conjunction with the quasi-insulin growthing factor I of claim 1.
3. the antibody according to claim 2 is characterized in that polyclonal antibody.
4. the antibody according to claim 2 is characterized in that monoclonal antibody.
5. one kind is screened the lay eggs method of native chicken of height, it is characterized in that comprising:
Collect a corpse or other object for laboratory examination and chemical testing by the native chicken of test;
With the antibody of specificity, detect the content of quasi-insulin growthing factor I in the corpse or other object for laboratory examination and chemical testing in conjunction with the antigen determining area of the quasi-insulin growthing factor I of claim 1;
Whether the judgement of the wherein high native chicken of laying eggs is higher according to the content of the quasi-insulin growthing factor I that is detected in the corpse or other object for laboratory examination and chemical testing.
6. the method according to claim 5 is characterized in that this antibody is the antibody of claim 3 or 4.
7. the method according to claim 5 is characterized in that this corpse or other object for laboratory examination and chemical testing is a serum.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2006100016999A CN101007845B (en) | 2006-01-24 | 2006-01-24 | Method for screening high-yield egg-laying poultry |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2006100016999A CN101007845B (en) | 2006-01-24 | 2006-01-24 | Method for screening high-yield egg-laying poultry |
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| Publication Number | Publication Date |
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| CN101007845A CN101007845A (en) | 2007-08-01 |
| CN101007845B true CN101007845B (en) | 2010-08-11 |
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| CN2006100016999A Expired - Fee Related CN101007845B (en) | 2006-01-24 | 2006-01-24 | Method for screening high-yield egg-laying poultry |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103497886B (en) * | 2013-09-13 | 2015-08-19 | 江苏省家禽科学研究所 | The test kit of duck IGF-I and IGF-I R gene mRNA expression amount joint-detection and using method |
| CN110423825B (en) * | 2019-08-26 | 2022-05-10 | 浙江省农业科学院 | Poultry egg yield molecular marker and application thereof |
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2006
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Non-Patent Citations (4)
| Title |
|---|
| Genbank accession No: ABA02291.2005,全文. |
| Genbank accession No: ABA02291.2005,全文. * |
| J.S. Yun et al.Relationships of circulating concentrations of insulin-likegrowth factor (IGF-Ⅰ)-Ⅰ and -Ⅱto egg production and growth rate in the korean nativeogolchicken.Asian-Australasian Journal of Animal Sciences16 4.2003,16(4),481-488. |
| J.S. Yun et al.Relationships of circulating concentrations of insulin-likegrowth factor (IGF-Ⅰ)-Ⅰ and-Ⅱto egg production and growth rate in the korean nativeogolchicken.Asian-Australasian Journal of Animal Sciences16 4.2003,16(4),481-488. * |
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| CN101007845A (en) | 2007-08-01 |
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