CN104357548B - A kind of molecule labelling method indicating the general premunition of sheep - Google Patents

A kind of molecule labelling method indicating the general premunition of sheep Download PDF

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CN104357548B
CN104357548B CN201410498552.XA CN201410498552A CN104357548B CN 104357548 B CN104357548 B CN 104357548B CN 201410498552 A CN201410498552 A CN 201410498552A CN 104357548 B CN104357548 B CN 104357548B
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杨华
杨永林
钟发刚
赵文娟
康立超
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Xinjiang Academy of Agricultural and Reclamation Sciences
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Abstract

The present invention is animal molecular genetics field, relates to sheep genetic breeding and Molecular Marker Assisted Selection Technology.A kind of molecule labelling method indicating the general premunition of sheep, including: according to sheep indoleamine 2,3 dioxygenase 1 (IDO1) gene order design pair of primers;From Blood In Sheep, extract genomic DNA, utilize above-mentioned primer I NF and primer I NR that the genomic DNA of sheep is carried out PCR amplification;Pcr amplification product restricted enzyme Nhe I enzyme action, digestion products is separated by electrophoresis through agarose gel, carries out genotype judgement according to electrophoretic separation result;Build genotype effects statistical model;According to genotype, Sheep Populations is divided into three types, selects AA genotype individuals to carry out group, complete the molecular marker of the general premunition of sheep.The present invention is easy to operate, economical and practical, be a kind of more effectively, the molecule labelling method of the general premunition of sheep accurately, the accuracy of seed selection can be improved.

Description

A kind of molecule labelling method indicating the general premunition of sheep
Technical field
The invention belongs to animal molecular genetics field, belong to sheep genetic breeding and Molecular Marker Assisted Selection Technology, It is specifically related to screening and the application of sheep general premunition genetic marker, particularly relates to a kind of indication sheep the most disease-resistant The molecule labelling method of power.
Background technology
Currently, the main purpose of Sheep Breeding is according to specific breeding objective, and fully meat, the Mao Yong of lifting etc. are specific Production performance, to obtain maximum production economy benefit.Along with popularizing of intensive farm mode, and selection-breeding journey The raising of degree, applies to select the increasing of pressure to sheep important economical trait, and sheep premunition decreases, and causes disease The increase of incidence rate, especially infectious disease.At present, the control measure of livestock and poultry are mainly used vaccination Treat with antibiotics, but, owing to pathogenic bacteria wide variety and variation are very fast, immunity inoculation, Drug therapy, The means of prevention such as health isolation can not fully effective control disease popular, the abuse of antibiotic also results in animal products Carrying a large amount of left drug, cause food pollution, healthy to the mankind produces harmful effect, has a strong impact on modern farming enterprise's Industry is healthy, sustainable development.According to statistics, disease the economic loss caused accounts for the 12-15% of the animal husbandry output value, This huge economic loss is dead mainly due to sheep, production efficiency reduces, medical expenses increase, administration fee is used Increase and product loss cause.Therefore, while cultivating high yield sheep variety, a safe and reliable and warp is found The feasible diseases prevention and treatment controlling soil moist that helps has been the task of top priority.
The result that the genetic background being usually animal and the interphase interaction of its environment occur of disease, if sheep is in heredity There is susceptibility to some disease in aspect, the Therapeutic Method of employing may not cure the disease that environmental condition is caused completely Sick.In the case of this, the effect of breeding for disease resistance is just particularly important, and the premunition for sheep selects Property breeding has become a kind of more efficiently method that may replace conventional therapy.Result of study finds different cultivars, same to product Planting the hereditary difference that there is premunition between Different Individual, this hereditary difference is caused by hereditary variation, thus Constitute the hereditary basis that disease resistance is selected by body.Therefore, it can by modern molecular genetic thremmatology method pin Sheep premunition is selected, to improve immunity and the premunition of body.
Premunition is also by the common effect of h and E, and premunition can be divided into general anti-according to the difference of hereditary basis Sick power and special premunition.General premunition is not limited to a certain pathogen, and it is affected by polygenes and environment are common, The antigenic specificity of pathogen is the least on the impact of general premunition.Special premunition refers to that sheep is to certain specified disease Or the resistance of pathogen, mainly controlled by a major gene resistance, be likely to simultaneously in various degree by other gene loci (including controlling element) and the impact of environmental factors, special premunition can pass through the direct challenge test of specific disease substance It is measured.Owing to general premunition embodies the body total defense ability to disease, it is to maintain body at bad ring The essential condition survived in border and produce, it is thus determined that the genetic marker of the general premunition of sheep has become cultivation has one As the key factor of premunition sheep.Research shows, the overall immune function improving body can improve general premunition, Therefore, the inherent immunity of body is just particularly important in improving general premunition.
Animal defines fairly perfect immune system in long-term evolutionary process, to resist exotic disease pathogenic microorganism Infect and invasion and attack, concurrently disinfect the cell of internal aging death, maintain stable machine state.Research shows, immunity should Answering can be as the indirect indexes of premunition, and the individuality that immunity is strong shows stronger premunition, at clear and definite immune character Carry out multidirectional selection-breeding on the basis of inherited character, be expected to cultivate the stronger sheep variety of general premunition (being).Cell The factor plays important assosting effect in immune response, and the secretion level of cytokine is immune response Ability and the important symbol of general premunition.Research shows, IFN-α and IFN-γ have immunomodulating and antiviral, Antineoplastic action, plays a significant role in animal innate immunity is reacted, can directly reflect under animal normal condition Immune state.IgG is the immunoglobulin that in humans and animals serum, content is the highest, and the most not only content is high And the persistent period is long, the immunologic competences such as antibacterial, antiviral, antitoxin can be played.IL-1 β be mononuclear cell- The cytokine that macrophage produces, participates in immunoreation and inflammatory reaction.IL-2 is main, the strongest internal T Cell growth factor, is the core substance in Organism immunoregulation network, all has important in cell and humoral immunization Regulation effect, can promote T cell and the propagation of B cell and differentiation, promotes cytokine profiles and the expression of receptor thereof. IL-5 can promote B cell proliferation and differentiation, the synthesis of induction IgA.IL-6 can promote IL-2 and receptor thereof Express, play an important role in immunne response, acute phase response, hemopoietic regulation.IL-8 has chemotaxis and inflammation Reagentia, chemotactic and activation neutrophilic granulocyte and chemotactic basophilic granulocyte.IL-10 is an anti-inflammatory factor, Participate in body natural immunity regulation, macrophage, Th1 cell secretion of cytokines can be suppressed, promote that B cell increases Grow and antibody tormation, promote thymus and proliferation of mast cells.IL-12 work in coordination with IL-2 promote cytotoxic T cell (TC), NK and LaK cell breaks up, and induction Th1 cell, suppression Th2 cell and promotion B cell Ig produce, and exempt from cell Epidemic disease and the cell-mediated antineoplastic immune of I type work.
Indoleamine 2,3 dioxygenases (indoleamine-2,3-diogenase, IDO) are a kind of intracellular containing ferrous iron The enzyme of haemachrome, is uniquely to be catalyzed indole ring oxicracking in tryptophan modules beyond liver, is along kynurenine approach Catabolic rate-limiting enzyme.In recent years, research finds, the express cell of IDO or its associated metabolites is mainly distributed In the T cell district of thymic medulla and secondary lymphatic organ, and the most specifically express in macrophage or dendritic cell On (dendritic cells, DC).IDO wide expression, in immune system, is glued including thymus, Placenta Hominis, digestive tract Immunologic tolerance or the immunologic privileged sites, especially table on some inhibition antigen presenting cells such as film, camera oculi anterior, epididymis Reach rise, such as some mononuclear phagocyte and the subgroup of dendritic cell (DC).In the research of transplantation immunity, IDO T cell, B cell, NK cell, macrophage and dendritic cell all there is immunoregulation effect.The expression of IDO The immune response of body, protection cell and tissue grafts can be reduced, participate in immunologic tolerance in allograft, Play important in maintaining periphery immunologic tolerance, autoimmune disease, organ transplantation, tumour immunity and immunity of pregnancy Regulation effect.
IDO is distributed widely in people and other mammal extrahepatic tissues, in low expression level under normal condition, in inflammation In disease or course of infection, the expression of IDO dramatically increases.Cytokine interferon gamma (IFN-γ) is the strongest IDO Induced expression thing, tumor necrosis factor α (TNF-α), tumor necrosis factor β (TNF-β) and lipopolysaccharide (LPS) Also the generation of IDO can be induced to a certain extent.
At present, mainly carried out mankind's IDO gene polynorphisms to grind to the relevant of resistance/susceptibility of disease Study carefully, it was demonstrated that IDO gene rs9657182 site is relevant with the depression that Caucasian's IFN-α is induced.IDO gene IVS3+562del C, IVS6+61G → A and IVS9+2431G → A site mutation is not to cause Iranian to repeat certainly The reason of the property sent out miscarriage.The sudden change of IDO gene affects Treg function, can induce autoimmune disease.And for IDO The correlational study of gene polynorphisms and the resistance/susceptibility of sheep disease is not reported.
In recent years, the Recent Progresses In The Development of gene SNP detection method and pertinent instruments quickly, has been reported and has established multiple SNP Detection method.Taqman method is to design specific probe for SNP site variation, and probe labelling is relatively costly, Cause testing cost the highest.DHPLC technology and mass spectral analysis are required to use high performance liquid chromatograph and mass spectrograph, Apparatus expensive, it is difficult to large-scale application.Single-strand conformation polymorphism analysis (SSCP) technology is difficult to meet the need of automatization Want, it is difficult to large-scale application.Allele specific oligonucleotide oligonucleotide hybridization (ASO) is rarely employed at present.DNA Chip is widely applied in SNP detects due to advantages such as high fluxs, but the condition when carrying out many sites and detecting simultaneously Be difficult to control to, repeatable poor, false positive and false negative result easily occur.Micrometering sequence is as a kind of based on array prominent Variation is analysed, and also faces at present and how to reduce false negative rate and the key issue such as variation of target nucleotide sequences composition.High Temperature Ligase detection reaction (ligase detection reaction, LDR) is that a kind of novel gene in recent years is many State property detection method, reliable results, but also need to verify further.SNaPshot method is based on fluorescent labeling Single base extension The typing method of principle, is commonly available to 10-30 SNP site analysis.DNA sequencing method is relatively accurate, and price is relatively Expensive.High-resolution melting curve method (HRM) is the SNP research tool the method risen in recent years, it is not necessary to design is visited Pin, low cost, but result precision is relatively low.And restriction fragment length polymorphism (RFLP) detection accuracy is high, Expense is low, is particularly suitable for the detection of unit point.Therefore, this patent, for IDO1 gene polynorphisms site, sets up one Plant the PCR-RFLP method for selecting molecular marker of the energy general premunition of Accurate Prediction sheep.
Summary of the invention
It is an object of the invention to provide a kind of easy to operate, economical and practical, more effectively, accurately sheep the most anti- The molecule labelling method of sick power, provides a kind of effective molecule for improving the general premunition of sheep and immunne response ability Labelling breeding technique, can assist-breeding adaptability and the strong high-quality kind sheep of premunition, accelerate breeding process, improve seed selection Accuracy.
The present invention is achieved through the following technical solutions:
A kind of molecule labelling method indicating the general premunition of sheep, its feature mainly comprises the steps that
(1) described according to sheep indoleamine 2,3-dioxygenase 1 (IDO1) gene order design pair of primers:
INF:5’-TGCTGCTGAATTTCTCCAGG-3’;
INR:5’-CACCTTGGATTGCCGGCTTGCAGGAATCATGATGTATAG-3’;
For meeting the detection analysis of force-RFLP, according to restricted enzyme Nhe I recognition sequence GCTAGC, under Trip primer I NR is forcibly introduced into GC base mutation, i.e. IDO1 gene coding region 1076 and two alkali of AA of 1077 Base sports GC base, and the mutating alkali yl of introducing represents with frame, thus forms Nhe I forced-RFLP-PCR detection Analyze.The primer I NF of IDO1 gene extron 9 as shown in SEQ ID NO:1 in sequence table, primer I NR such as sequence In table shown in SEQ ID NO:2;
(2) from Blood In Sheep, extract genomic DNA, utilize above-mentioned primer I NF and the primer I NR gene to sheep Group DNA carries out PCR amplification;
(3) pcr amplification product restricted enzyme Nhe I enzyme action, digestion products carries out electrophoresis through agarose gel Separate, according to electrophoretic separation result carry out genotype judgement, the standard that genotype judges as: electrophoresis presents a band, Size is 232bp, then undergo mutation in site, sheep IDO1 gene coding region 1072, and PCR primer can not be by restricted interior Cut enzyme Nhe I incision, by its named AA genotype;Electrophoresis presents two bands, and size is 232bp and 191bp, Then site, sheep IDO1 gene coding region 1072 is in heterozygous state, and PCR primer can not be by restricted enzyme Nhe I cuts completely through, by its named AG genotype;Electrophoresis presents a band, and size is 191bp, then sheep IDO1 Site, gene coding region 1072 is without sudden change, and PCR primer can be cut completely through by restricted enzyme Nhe I, be named For in GG genotype;
(4) according to experimental sheep colony feature, structure genotype effects statistical model: Y=μ+G+B+A+S+F+e, its Middle μ is colony's average, and G is genotype effects, and B is variety effect, and A is age effect, and S is sex-effects, F For the other effect in field, e is random error;Utilize JMP4.0 statistical software by genotype effects and sheep immune indexes character Being associated analyzing, and estimate the least square average of character, analysis result shows, IDO1 gene coding region 1072 The genotype effects that site mutation causes and IFN-α, IL-2 and IL-12 level pole significant correlation in sheep peripheral blood (P<0.01);The result of multiple comparisons shows, IFN-α and the IL-2 water mean pole of AA and AG genotype individuals are notable Higher than GG genotype individuals, the IL-12 level pole of AA genotype individuals is significantly higher than AG and GG genotype individuals;
(5) according to genotype, Sheep Populations is divided into three types, selects AA genotype individuals to carry out group, thus Realize the molecular mark of the general premunition of sheep, i.e. complete the molecule labelling method of the general premunition of sheep;
Wherein in step (2), PCR amplification condition is 94 DEG C of degeneration 5min;94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 35 circulations;72 DEG C extend 5min;4 DEG C of insulations.
The base sequence that wherein in step (3), restricted enzyme Nhe I is identified is GCTAGC.
Described agarose gel electrophoresis separation digestion products, the agarose gel concentration used is 2.5%-3%.
The present invention provides effective molecule mark for the raising general premunition of sheep and immunne response in terms of breeding for disease resistance Note.
Advantages of the present invention is embodied in:
(1) present invention is that the molecular marker assisted selection that sheep breeding for disease resistance works provides one more efficiently, accurately Molecular genetic marker, provide a kind of effective molecular marker breeding means for improving the general premunition of sheep, with should Genetic marker premunition general to sheep is marked assisted Selection, carries out the breeding for disease resistance of sheep, improves exempting from of sheep Epidemic disease responsibility, can significantly more efficient reduce sheep hereditary generation, reduce the sickness rate of many diseases, Improve sheep environmental suitability, increase herd with confined farming under the conditions of sheep survival rate.
(2) using 1 variant sites as labelling, forced PCR-RFLP is for predominantly detecting method in application, has Low cost, practical, accuracy is high, more easy-operating advantage, educates for accelerating to improve the disease-resistant molecular genetic of sheep Plant work to lay a good foundation, thus accelerate breeding process.
(3) detection method of the present invention is simple to operate, and testing cost is low, and accuracy is high, it is possible to realize automatization Directly detection, the present invention will play a significant role in the breeding for disease resistance of sheep.
Accompanying drawing explanation
Fig. 1 is the agarose gel electrophoresis figure of IDO1 gene PCR product;Wherein M is pUC19DNA/Msp I (Hpa II) marker, C swimming lane is blank;1-7 swimming lane is different sheep individuality IDO1 gene PCR product.
Fig. 2 is that IDO1 gene PCR product is through restricted enzyme Nhe I enzyme action, the agarose gel electricity of digestion products Swimming figure;Wherein M is pUC19DNA/Msp I (Hpa II) marker, and the 1st, 3,7,9,12,15 swimming lane is GG genotype;4th, 8,10,14 swimming lane is AG genotype;2nd, 5,6,11,13,16 swimming lane is AA Genotype.
Detailed description of the invention
Embodiment: the present invention is described in more detail by example below:
One, the detection analysis in sheep IDO1 gene the 9th Varia nt in exon site
1, sample collecting
Use disposable syringe to extract the blood of about 1mL from sheep jugular vein, inject through autoclaving and equipped with about The 1.5mL of the 200 aseptic EDTA of μ L 2% (Ethylene diamine tetraacetic acid, EDTA) anticoagulant In centrifuge tube, shaking up gently, record sheep number ,-20 DEG C save backup.
2, medicine and enzyme
Trishydroxymethylaminomethane (Tris), Sigma Chemicals Co;The saturated phenol of Tris, EDTA-Na2Beijing ancient cooking vessel State's biotech development center;E.C. 3.4.21.64 (Proteinase K), MERCK Co;dNTP(dATP;dTTP;dCTP; DGTP), Taq enzyme (supporting 10 × Taq buffer, 25mmol/L Mg2+), Dalian treasured biotech firm;Agarose (Agarose H), pUC19DNA/Msp (Hpa II) Marker, Sangon Biotech (Shanghai) Co., Ltd.; Restricted enzyme Nhe I, Fermentas (MBI) company.
3, key instrument
Icycler PCR instrument (Bio-Rad company), Gel2000 gel imaging system, Ultrospec 1000 ultraviolet Spectrophotometer, Sigma 3K30 High speed refrigerated centrifuge, Milli-Q ultra-pure water instrument, Bio-Rad voltage stabilizing electrophresis apparatus And supporting electrophoresis tank.
4, the design of primer and synthesis
According to sheep IDO1 gene (GenBank accession number: NM_001141953) the 9th exon sequence and restriction Property restriction endonuclease Nhe I recognition sequence GCTAGC, design forward primer INF and downstream primer INR, primer is raw by giving birth to work The synthesis of thing engineering (Shanghai) Co., Ltd.:
INF:5’-TGCTGCTGAATTTCTCCAGG-3’;
INR:5’-CACCTTGGATTGCCGGCTTGCAGGAATCATGATGTAGCTAG-3’。
5, a small amount of of ovine genome DNA is extracted
Method one:
(1) take 20 μ L anticoagulated bloods, add 500 μ L hemocyte lysates, add E.C. 3.4.21.64 to final concentration of 100-200 μ g/mL, mixes 55 DEG C of digestion 12h, until no longer there being the agglomerate of thickness in solution.
(2) solution is cooled to room temperature, adds 5M NaCl to final concentration 1.5M, mix 10min.The bodies such as addition Long-pending phenol/chloroform, overturns centrifuge tube mixing 10min repeatedly.
(3) 12,000rpm, room temperature is centrifuged 10min.Take supernatant, add equal-volume chloroform mixing 10min.
(4) 12,000rpm, room temperature is centrifuged 10min.Take 2 times of volume dehydrated alcohol precipitation DNA of supernatant.
(5) DNA is chosen it is put in 1.5mL centrifuge tube, wash 1 time with 70% ethanol.
(6) 7,500rpm, room temperature is centrifuged 5min, abandons supernatant.
(7) DNA is dissolved in after drying in 200 μ L TE.
Method two:
(1) 20 μ L whole bloods are equipped with in the 1.5mL centrifuge tube of 700 μ L 1 × SET, mix gently.
(2) addition E.C. 3.4.21.64 (10mg/mL) is to the SDS of final concentration 100-200 μ g/ μ L and 10% to the denseest Degree 0.5%, 55 DEG C of digestion 12h.
(3) after digestion completely, add the saturated phenol of isopyknic Tris, overturn back and forth so that it is mixing.
(4) 12,000rpm is centrifuged 10min, with the suction nozzle cutting off tip, upper strata aqueous phase is carefully moved into another and is centrifuged Guan Zhong, discards organic facies.Repeat the third and fourth step once.
(5) in aqueous phase, add isopyknic phenol, chloroform, isoamyl alcohol mixed liquor (volume ratio is 24:23:1), Mixing 10min.12,000rpm, centrifugal 10min, removal aqueous phase is to another centrifuge tube.
(6) in aqueous phase, add isopyknic chloroform, isoamyl alcohol mixed liquor (23:1), back and forth reverse mixing 10min, 12,000rpm, centrifugal 10min, removal aqueous phase is to another centrifuge tube.
(7) in aqueous phase, 1/10 volume NaAc (3M, pH5.2) and the dehydrated alcohol of 2 times of volumes is added, back and forth Reverse, precipitate DNA.
(8) DNA is chosen it is put in 1.5mL centrifuge tube, wash 1 time with 70% ethanol.
(9) 7,500rpm is centrifuged 5min.Carefully outwell ethanol in pipe, will be upside down on filter paper, allow ethanol flow to end, It is placed in air drying.
(10) add the TE of 200 μ L, put in 50 DEG C of water-baths overnight dissolving DNA.Be stored in after dissolving-20 DEG C standby With.
Method three:
Ezup pillar genome DNA extraction test kit description according to Sangon Biotech (Shanghai) Co., Ltd. Or the genome DNA extracting reagent kit description of other biological technology company operates (SK8252).
6, PCR reaction
(1) carrying out PCR amplification with ovine genome DNA for template, genes of interest PCR primer size is 232bp. In pcr amplification product, ovine genome sequence is as shown in SEQ 3.
25 μ L reaction systems comprise following solution or reagent:
(2) by the mixing of above-mentioned solution and PCR reaction is carried out by following condition.
94 DEG C of degeneration 5min;94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 35 circulations;72 DEG C are prolonged Stretch 5min.
(3), after reaction terminates, use 2% agarose gel that PCR primer (5~10 μ L) is carried out electrophoresis detection, Electrophoresis result is shown in Fig. 1.
7, the rflp analysis of IDO1 gene
Application Fermentas (MBI) restricted enzyme Nhe I carries out enzyme action, and enzyme action system is 10 μ L, reaction System and condition are as follows:
Above-mentioned reactant liquor is mixed, in 37 DEG C of water-baths, digests 3~5h, after digestion, total overall reaction liquid is carried out 2.5% Agarose gel electrophoresis, detection PCR primer enzyme action effect and segment polymorphism.
8, the analysis in IDO1 genovariation site
The PCR primer of IDO1 gene 232bp is through restricted enzyme Nhe I enzyme action, through 2.5% agarose gel electrophoresis Separate, detect in IDO1 gene extron C.1072G the base transition variant sites of A, according to restriction enzyme digestion and electrophoresis figure Different genotype sheep individuality is named by spectrum, respectively AA genotype (232bp), AG genotype (232bp And 191bp) and three kinds of genotype (Fig. 2) of GG genotype (191bp).
Two, the mensuration of the general anti-disease ability of sheep:
Gather 343 10 monthly age lamb vena cava anterior blood 2mL that different cultivars is healthy, wherein Suffolk 62 (26 ♂, 36 ♀), polyembryony Suffolk 118 (36 ♂, 82 ♀), Kazakh sheep 64 (25 ♂, 39 ♀), lake Sheep 59 (13 ♂, 46 ♀), polyembryony Suffolk and 40 (hereinafter referred to as Sahas of Kazakh sheep F1 hybrid lamb F1;7 ♂, 33 ♀).Conventional method separation serum, is centrifuged 15min in 2500r/min, takes supernatant subpackage.Application Sheep IgG, IFN-α, IFN-γ, IL-1 β, IL-2, IL-5, IL-6, IL-8, IL-10 and IL-12ELISA Detection kit (upper sea blue base bio tech ltd, China) carries out immunne response index determining, in strict accordance with Test kit description process serum sample, standard substance are done 5 concentration dilution gradients, microplate reader (Model 550, U.S. Bio-Rad) detect absorbance (OD value) at 450nm wavelength, then according to IgG in standard curve calculating serum, IFN-α, IFN-γ, the concentration of IL-1 β, IL-2, IL-5, IL-6, IL-8, IL-10 and IL-12.
Three, sheep IDO1 gene the 9th exon polymorphism and and general anti-disease ability between relation analysis and resistance Labelling determines.
Respectively to Suffolk, polyembryony Suffolk, Kazakh sheep, sheep and Saha F1 sheep IDO1 gene the 9th Exon PCR-RFLP banding pattern carries out statistical analysis, and carries out the calculating (table 1) of genotypic frequency and gene frequency.
Table 1 sheep IDO1 gene C .1072G > genotypic frequency in A site and gene frequency
Note: χ2 0.05(2)=5.991, χ2 0.01(2)=9.21.
According to experimental sheep colony feature, structure genotype effects statistical model: Y=μ+G+B+A+S+F+e, wherein μ For colony's average of character, G is genotype effects, and B is variety effect, and A is age effect, and S is sex-effects, F For the other effect in field, e is random error.Utilize the JMP4.0 statistical software different genes respectively to sheep IDO1 gene Type effect and partial immunity index character are associated analyzing, and estimate the least square average of character.Analysis result table Bright (being shown in Table 2), the genotype effects that IDO1 gene coding region 1072 site mutation causes and IFN-in sheep peripheral blood α, IL-2 and IL-12 level pole significant correlation (P < 0.01), do not cause IFN-γ in peripheral blood, IgG, IL-6, The notable change of IL-1 β, IL-5, IL-8 and IL-10 content.The result of multiple comparisons shows, AA and AG genotype Individual IFN-α and IL-2 water mean pole are significantly higher than GG genotype individuals (P < 0.01), AA genotype individuals IL-12 level pole is significantly higher than AG and GG genotype individuals (P < 0.01).
The impact on immune indexes of the table 2 sheep IDO1 gene different genotype
Note: colleague's lower case difference represents that difference is extremely notable (P < 0.01).
IFN-α has immunomodulating and antiviral, antineoplastic action, plays weight in animal innate immunity is reacted Act on, can directly reflect the immune state under animal normal condition.IL-2 is that main, the strongest internal T is thin The intracellular growth factor, is the core substance in Organism immunoregulation network, all has important tune in cell and humoral immunization Joint effect, can promote T cell and the propagation of B cell and differentiation, promotes cytokine profiles and the expression of receptor thereof. IL-12 can work in coordination with IL-2 and promote that cytotoxic T cell (TC), NK and LaK cell break up, induction Th1 cell, Suppression Th2 cell and promotion B cell Ig produce, and act as in the antineoplastic immune that cellular immunization and I type are cell-mediated With.IFN-α in sheep peripheral blood, the colony that IL-2 and IL-12 level content is higher have higher general premunition, Therefore, IDO1 gene can be as one of main candidate of the general premunition of sheep, and AA genotype can be as individuality The general effective genetic marker of premunition, for predicting and identify the sheep that general premunition is stronger, and can be used for quickly Set up the Sheep Populations that general premunition is stronger.

Claims (5)

1. indicating a molecule labelling method for the general premunition of sheep, its feature mainly comprises the steps that
(1) according to sheep indoleamine 2,3-dioxygenase 1IDO1 gene order design pair of primers:
INF:5’-TGCTGCTGAATTTCTCCAGG-3’;
INR:5’-CACCTTGGATTGCCGGCTTGCAGGAATCATGATGTAGCTAG-3’;
(2) from Blood In Sheep, extract genomic DNA, utilize above-mentioned primer I NF and the primer I NR gene to sheep Group DNA carries out PCR amplification;
(3) pcr amplification product restricted enzyme Nhe I enzyme action, digestion products carries out electrophoresis through agarose gel Separate, according to electrophoretic separation result carry out genotype judgement, the standard that genotype judges as: electrophoresis presents a band, Size is 232bp, then undergo mutation in site, sheep IDO1 gene coding region 1072, and PCR primer can not be by restricted interior Cut enzyme Nhe I incision, by its named AA genotype;Electrophoresis presents two bands, and size is 232bp and 191bp, Then site, sheep IDO1 gene coding region 1072 is in heterozygous state, and PCR primer can not be by restricted enzyme Nhe I cuts completely through, by its named AG genotype;Electrophoresis presents a band, and size is 191bp, then sheep IDO1 Site, gene coding region 1072 is without sudden change, and PCR primer can be cut completely through by restricted enzyme Nhe I, be named For GG genotype;
(4) according to experimental sheep colony feature, structure genotype effects statistical model: Y=μ+G+B+A+S+F+e, its Middle μ is colony's average, and G is genotype effects, and B is variety effect, and A is age effect, and S is sex-effects, F For the other effect in field, e is random error;Utilize JMP4.0 statistical software by genotype effects and sheep immune indexes character Being associated analyzing, and estimate the least square average of character, analysis result shows, IDO1 gene coding region 1072 The genotype effects that site mutation causes and IFN-α, IL-2 and IL-12 level pole significant correlation in sheep peripheral blood P<0.01;The result of multiple comparisons shows, IFN-α and the IL-2 water mean pole of AA and AG genotype individuals are significantly higher than GG genotype individuals, the IL-12 level pole of AA genotype individuals is significantly higher than AG and GG genotype individuals;
(5) according to genotype, Sheep Populations is divided into three types, selects AA genotype individuals to carry out group, thus Realize the molecular mark of the general premunition of sheep, i.e. complete the molecular marker of the general premunition of sheep.
2. the molecule labelling method indicating the general premunition of sheep as claimed in claim 1, it is characterised in that Qi Zhongbu Suddenly in (2), PCR amplification condition is 94 DEG C of degeneration 5min;94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 35 circulations;72 DEG C extend 5min;4 DEG C of insulations.
3. the molecule labelling method indicating the general premunition of sheep as claimed in claim 1 or 2, it is characterised in that its The base sequence that in middle step (3), restricted enzyme Nhe I is identified is GCTAGC.
4. the molecule labelling method indicating the general premunition of sheep as claimed in claim 1 or 2, it is characterised in that institute Stating agarose gel electrophoresis separation digestion products, the agarose gel concentration used is 2.5%~3%.
5. the molecule labelling method indicating the general premunition of sheep as claimed in claim 3, it is characterised in that described fine jade Sepharose electrophoretic separation digestion products, the agarose gel concentration used is 2.5%~3%.
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