CN105039518B - The screening technique of Anhui's local pig kind weanling pig premunition candidate genetic label - Google Patents
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Abstract
The invention discloses a kind of screening techniques of Anhui's local pig kind weanling pig premunition candidate genetic label, include the following steps：(1) control-animal model is selected；(2) sample collection；(3) ear tissue DNA is extracted；(4) PCR amplification TLR4 target fragments；(5) sscp analysis；(6) pcr amplification product bidirectional sequencing；(7) Population Genetics specificity analysis；(8) measurement of sero-immunity index；(9) relevance of TLR4 the gene genetics variation and sero-immunity index detected by mathematics model analysis is used, and analyzes the otherness of sero-immunity index between kind.It is intended to searching and the relevant effective genetic marker of Anhui's local pig kind disease resistance trait, molecular biology reference is provided for Anhui's local pig germ plasm resource characteristic and breeding for disease resistance research.
Description
Technical field
The present invention relates to biotechnologies, are lost more particularly to a kind of Anhui's local pig kind weanling pig premunition candidate
Pass the screening technique of label.
Background technology
With the development of the social economy, China's pig-breeding large-scale degree is higher and higher, the prevention and control of various diseases
System becomes more important and severe.A variety of diseases bring serious harm to the development of pig-breeding industry, cause economic loss, and from
The angle of genetic breeding is weighed, and the genetic improvement of some important economical traits of pig kind has also been seriously affected.Therefore swinery is improved
General premunition can help to reduce production cost, be more advantageous to reduction medicament residue, improves pork product quality, public defends
Raw safety and animal welfare etc..The study found that the pathogenesis of some swine diseases is relevant with the genetic background of pig itself, therefore
Applied genetics method, raising pig has effects that effect a permanent cure to the resistance of disease from hereditary basis.It assists selecting with molecular labeling
It is the molecular breeding technology indicated to select (MAS) and candidate gene approach, makes it possible that pig disease resistant breeding is studied.Molecule is disease-resistant to educate
Kind mainly improves the accuracy of selection by MAS, and therefore, it is crucial to find suitable molecular labeling and candidate gene.
TLR4 is TLRs families important member, is the major receptors of bacteria lipopolysaccharide or lipid A identification, can identify a variety of leather
The pathogenic microorganisms such as Lan Shi negative pathogenics bacterium, Chlamydia, conveyor screw and virus, triggering activate special signal pathway, start and
The immune response of body is adjusted, the hereditary difference of acceptor gene can cause body to differ multiple pathogens resistance.Pig
The gene coding regions TLR4 overall length 2526bp encodes the polypeptide being made of 841 amino acid residues, is made of 3 exons, wherein
Exon 3 longest, containing 2265bp, exons 1 and 2 length are respectively 216 and 167bp.Since being cloned, being sequenced from it, grind
The person of studying carefully just using pig TLR4 as the important candidate gene of disease resistance, study its respiratory system, intestinal tract disease and
Effect in innate immune reaction.The pig TLR4 assignments of genes gene mapping in SSC1q2.9-q2.13, and are shown TLR4mRNA by Qiu little Tian etc.
There are expression and the expression quantity highest in lung in the heart, liver, spleen, lung, the various tissues of kidney, it is believed that high expression of the TLR4 in lung
It is related with pig pulmonary disease.Bao Wenbin etc. is research shows that the downward of TLR4 gene expression amounts has one with F18 E. coli resistances
Determine relationship.Yang etc. determines that g.1605G ＞ T variations cause TLR4 downstream to transmit letter to pig TLR4 genes into the cell in PK-15
Number ability significantly reduce (P ＜ 0.01).In view of importance of the TLR4 genes in animal body immune response, to its polymorphism
Network analysis is carried out with hereditary effect to have great theoretical and practical significance.
Diarrhea of weaned piglets and oedema are a kind of common diseases in current large-scale pig farm cultivation.In the feeding of piglet
It supports in management process, wean is an extremely important link.After weaned piglet, due to weaning stress, piglet autodigestion system
System not yet development is complete, adds the influence of the extraneous undesirable elements such as the antibody sources termination from parent, usually easily leads to son
Diarrhea (Post-weaning diarrhea, PWD) and oedema (Oedema disease, OD) occurs after pig wean, wherein
F18 Escherichia coli are most important etiologies.Up to now, the harm for caused by enteropathogenic E. Coli, still without effective
Precautionary measures and therapy, bring huge economic loss to pig breeding industry.
Research of the present invention is external with three, Anhui land race (country fair pig, the black pig in Anhui south and six white pig of Anqing) and one
Kind Large White is subjects, detects the hereditary variation of the TLR4 genes in four groups.By measuring in easy infection F18
The sero-immunity index of the wean cultivation period piglet of Escherichia coli, carries out between interracial variance analysis and genotype
Correlation analysis inquires into difference and TLR4 gene pleiomorphism of the weanling pig sero-immunity index between different cultivars to difference
The hereditary effect of population weanling pig sero-immunity index.It is intended to find relevant with Anhui's local pig kind weanling pig premunition
Candidate effective genetic marker provides molecular biology ginseng for Anhui Native pig kind germplasm resource characteristics and breeding for disease resistance research
It examines.
Invention content
The present invention provides a kind of screening technique of Anhui's local pig kind weanling pig premunition candidate genetic label, this hairs
Bright method has the advantages that detection result is ideal, at low cost, easy to operate.
A kind of screening technique of Anhui's local pig kind weanling pig premunition candidate genetic label, includes the following steps：
(1) selection of control-animal model：Select Anhui Native pig kind country fair pig, the black pig in Anhui south, six white pig of Anqing with it is outer
The piglet for carrying out the wean of 28 age in days of pig kind Large White is the control-animal model for studying immune indexes breed difference；
(2) sample collection：When 35 age in days of piglet, acquisition ear sample tissue is simultaneously taken a blood sample on an empty stomach；
(3) ear sample tissue DNA extracts；
(4) PCR amplification Toll-like receptor 4 is TLR4 gene target fragments；
(5) target fragment single-strand conformation polymorphism, that is, sscp analysis；
(6) pcr amplification product bidirectional sequencing；
(7) Population Genetics specificity analysis；
(8) measurement of piglet sero-immunity index；
(9) hereditary variation of TLR4 genes and being associated with for sero-immunity index detected by mathematics model analysis are used
Property, and analyze the otherness of sero-immunity index between kind；
According to TLR4 gene mononucleotide polymorphisms, that is, single nucleotide polymorphism：SNP with it is immune
The correlation analysis of index, inquires into whether detected SNP site has shadow to the index of different swinery weanling pig disease resistances
It rings, to which can the detected SNP site of judgement as the candidate significant notation site of piglet disease-resistant breeding.
The screening technique of Anhui's local pig kind weanling pig premunition candidate genetic label of the present invention, wherein institute
State being selected as control-animal model described in step (1)：Country fair pig, the black pig in Anhui south, six white pig of Anqing and the Large White of 35 ages in days
28 age in days weanling pigs select 100,105,92,132 respectively, and wherein country fair pig picks up from the limited public affairs of the safe and sound agricultural development in Guangde Anhui
Department, the black pig in Anhui south pick up from the plump eco-agricultural development Co., Ltd in Jixi Anhui, and it is good that six white pig of Anqing picks up from Wangjiang Anhui modern times
Kind cultivation Co., Ltd, Large White pick up from the safe and sound agricultural development Co., Ltd in Feidong Anhui.
The screening technique of Anhui's local pig kind weanling pig premunition candidate genetic label of the present invention, wherein institute
Stating step (2) middle ear sample tissue sampling process is：In advance 70% volume-fraction concentration is injected in the Eppendorf pipes of 1.5mL
Alcohol, then every pig adopt ear tissue block 1.5g, be fitted into the Eppendorf pipes, saved backup in -20 DEG C.
The screening technique of Anhui's local pig kind weanling pig premunition candidate genetic label of the present invention, wherein institute
Stating step (3) middle ear sample tissue DNA extraction process is：1. with eye scissors clip 0.2g ear tissues, its surface hair and wine are removed
Essence is fitted into 1.5mL Eppendorf pipes, and as far as possible shreds ear tissue；2. the extraction of DNA is using the full formula gold biology in Beijing
The tissue DNA extracts kit of Science and Technology Ltd. is extracted；3. obtained DNA tune final concentration to be placed on 2-8 and take the photograph to 100ng/ μ L
Family name's degree preserves；4. DNA purity detectings：It takes 1 μ L DNA to measure the ratio of OD260/OD280 on SMA 1000, works as OD260/
OD280 ratios illustrate that carried DNA purity is higher between 1.8-2.0；5. quality testing：Carried DNA is coagulated with 1.5% agarose
Gel electrophoresis detects, and observation extracts the quality of product to carry out subsequent experimental.
The screening technique of Anhui's local pig kind weanling pig premunition candidate genetic label of the present invention, wherein institute
The process for stating PCR amplification target fragment in step (4) is：Using the ear tissue sample DNA of step (3) acquisition as template, according to
3 sequence of GenBank pig TLR4 gene extrons, accession number AY753179 are set using 5.0 softwares of Primer Premier
2 pairs of primers are counted, carry out the clone of 3 partial sequence of TLR4 gene extrons, and using the detection clone's production of 2% agarose gel electrophoresis
Whether object is purpose segment；Wherein SEQ ID NO in the nucleotide sequence of primer TLR4 exon3-1 such as sequence table：1 and SEQ
ID NO：Shown in 2, SEQ ID NO in the nucleotide sequence of primer TLR4 exon3-2 such as sequence table：3 and SEQ ID NO：4 institutes
Show, annealing temperature and target fragment length are shown in Table 1.
The screening technique of Anhui's local pig kind weanling pig premunition candidate genetic label of the present invention, wherein institute
The process for stating target fragment single-strand conformation polymorphism in step (5) is：First described TLR4-exon3-1, TLR4-exon3-2 are drawn
The pcr amplification product of object pair carries out native polyacrylamide gel electrophoresis respectively, is used after then carrying out cma staining respectively
Gel imaging system observation electrophoresis result obtains the weanling pig TAP1 gene single-strand conformation polymorphisms；
Wherein, the native polyacrylamide gel electrophoresis specifically comprises the following steps：
(A) apparatus such as glass plate, clip, tightrope, the sample comb used in glue are cleaned, are dried；
(B) making sheet：Upward by clean and dry bottom plate flour milling, the round end on ground glass side seals glass downward, with tightrope
Three faces of glass plate, clamp；
(C) the PAGE 24mL of 10% polyacrylamide gel 72mL, distilled water 42mL, 30% are prepared, 10 ×
50 330 μ L of μ L, AP of TBE6mL, TEMED, quick encapsulating after mixing；
(D) glue is filled to the edge of glass plate, is inserted into comb, pay attention to checking in glue whether there is bubble at this time, in room
Temperature is lower to place, and is used after glue polymerization；
(E) after gel polymerisation is good, 1 × TBE is added into electrophoresis tank, removes lower clip, tightrope and comb, glass plate is fixed
In irrigation with syringe well on electrophoresis tank, is used in combination；
(F) prerunning 30min under 300V voltages；
(G) it takes in 3 μ L pcr amplification products to 96 hole PCR plates, 7 μ L sample-loading buffers is added, after blowing and beating mixing with rifle, use
Adhesive tape seals；
(H) it is denaturalized 10min for 98 DEG C, then rapid ice bath 10min, then point sample；
(I) 240V electrophoresis 1h, 150V electrophoresis 12h；
Wherein, the cma staining specifically comprises the following steps：
(a) after the completion of electrophoresis, glass plate is removed, it is careful to take out gel and mark, it is rinsed 2 times with distilled water；
(b) fixer is poured into, the fixer is double steamings containing 10% volume fraction ethyl alcohol and 0.5% volume fraction acetic acid
Aqueous solution, slight oscillatory 5min recycle fixer；
(c) silver nitrate solution for pouring into 2% mass fraction is protected from light slight oscillatory 15min, recycles silver nitrate solution；
(d) after being rinsed 2 times with distilled water, developing solution is poured into, the developing solution is containing 3% volume fraction NaOH and 0.1%
The double steaming solution of volume fraction HCHO, oscillation carries out chromogenic reaction, until band is clear；
(e) distilled water rinses, and electrophoresis result is observed in gel imaging system.
The screening technique of Anhui's local pig kind weanling pig premunition candidate genetic label of the present invention, wherein institute
The process for stating pcr amplification product bidirectional sequencing in step (6) is：According to single-strand conformation polymorphism as a result, selecting different banding patterns
Sample, each banding pattern select 3 samples, and 50 μ L systems of PCR amplification are sent to Shanghai Sheng Gong Co., Ltds and carry out bidirectional sequencing, sequencing
As a result DNAStar or DNAMan, the analysis of Chromas softwares are used.
The screening technique of Anhui's local pig kind weanling pig premunition candidate genetic label of the present invention, wherein institute
Stating Population Genetics specificity analysis process in step (7) is：The SNPs that step (5), (6) are detected using popgene softwares
Site carries out Population Genetics analysis, including gene frequency, genotype frequency, homozygosity, heterozygosity, effective number of alleles
The calculating of number, polymorphism information content judges whether the distribution of genotype in group is in Hardy- with Chi-square Test
Weinberg balances carry out 2 comptibility tests of χ calculating using popgene softwares.
The screening technique of Anhui's local pig kind weanling pig premunition candidate genetic label of the present invention, wherein institute
The continuous mode for stating piglet sero-immunity index in step (8) is：35 age in days weanling pig empty stomach vena cava anteriors are taken a blood sample, conventional point
From serum, immune indexes are measured using pig ELISA kit, mass metering concentration is respectively 1000pg/ at microplate reader 450nm
The standard items of ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.25pg/ml, 15.6pg/ml totally 7 concentration
Absorbance, draw out standard curve, measure the concentration of first sample sheet；And then according to corresponding standard items standard curve,
IL-6, IL-10, IL-12, IFN-γ, the concentration of TNF-α in serum are calculated successively.
The screening technique of Anhui's local pig kind weanling pig premunition candidate genetic label of the present invention, wherein institute
State the mathematical analysis model that uses in step (9) for：1. the difference analysis of sero-immunity index uses SPSS19.0 between kind
Statistical software One-way ANOVA programs carry out variance analysis and significance test, and experiment the data obtained is with " average+SD "
It indicates；2. the mathematical model that different genotype uses the effect variance analysis of each immune indexes for：Yij=μ+Bi+Gj+eij；Its
In, YijFor immune indexes phenotypic number；μ is group's mean value；Bi indicates the wean batch effect of weanling pig；GjIndicate jth kind base
Because of type effect；eijIndicate random error；Using SPSS19.0 statistical softwares GLM programs to the detected hereditary variation of this experiment
It is analyzed with the relevance of sero-immunity index.
The screening technique of Anhui's local pig kind weanling pig premunition candidate genetic label of the present invention is compared with prior art
With as follows a little：
The method of the invention is outer with the local pig kind (country fair pig, six white pig of Anqing, the black pig in Anhui south) in three, Anhui Province and one
It is subjects to carry out pig kind Large White, is detected in four 3 sequences of group's TLR4 gene extrons using the method for clone, sequencing
The hereditary variation in area.By the sero-immunity index (serum for measuring the wean cultivation period piglet of easy infection F18 Escherichia coli
IL-6, IL-10, IL-12, IFN-γ, TNF-α content), carry out the relevance between interracial variance analysis and genotype
It is disconnected to different population to inquire into difference and TLR4 gene pleiomorphism of the weanling pig sero-immunity index between different cultivars for analysis
The hereditary effect of milk piglet sero-immunity index.TLR4 gene polymorphism sites detected by exploration and analysis are disconnected to different swinerys
Whether milk piglet disease-resistant power has an impact, to which can the detected SNP site of judgement be lost as the candidate of piglet disease-resistant breeding
Pass label.So as to provide molecular biology reference for Anhui Native pig kind germplasm resource characteristics and breeding for disease resistance research.This
Invention the method has the advantages that detection result is ideal, at low cost, easy to operate.
Below in conjunction with the accompanying drawings to the screening side of the Anhui's local pig kind weanling pig premunition candidate genetic label of the present invention
Method is described further.
Description of the drawings
Fig. 1 is pig ear tissue genomic DNA quality measurements figure in the present invention；
Fig. 2 is the PCR amplification result figure of pig TLR4 gene exon3-1 primer pairs in the present invention；
Fig. 3 is the PCR amplification result figure of pig TLR4 genes exon3-2 primer pairs of the present invention；
Fig. 4 is the SSCP detection figures of pig TLR4 gene exon3-1 primer PCR amplified productions in the present invention；Wherein, swimming lane 1,
2,3,4,5,8,9 be EE genotype, and swimming lane 6,7,10 is EF types；
Fig. 5 is the SSCP detection figures of pig TLR4 gene exon3-2 primer PCR amplified productions in the present invention；Wherein, swimming lane 1,
2,3,4 be CC genotype, and swimming lane 5,6,7 is CA types, and swimming lane 8,9,10 is AA types；
Fig. 6 is pig TLR4 gene exon3-1 primer PCR amplified fragments sequencing result comparison diagrams in the present invention；Wherein, (a)
Figure is 417 site EE genotype sequencer maps, and (b) figure is 417 site EF genotype sequencer maps；(c) figure is 318 site EE genotype
Sequencer map (backward sequencing), (d) 8 sites Figure 31 are EF genotype sequencer map (backward sequencing)；
Fig. 7 is that pig TLR4 gene exon3-2 primer PCR amplified fragments sequencing results comparison diagram is (reversed to survey in the present invention
Sequence)；Wherein, (a) figure is CC genotype sequencer maps, and (b) figure is CA genotype sequencer maps, and (c) figure is AA genotype sequencer maps；
Specific implementation mode
One, 3 sequence area single nucleotide polymorphism analysis of pig TLR4 gene extrons
1, test material
Experimental animal：
100 country fair pigs pick up from the safe and sound agricultural development Co., Ltd in Guangde Anhui in experimental animal, and the black pig in 105 Anhui south is picked up from
The plump eco-agricultural development Co., Ltd in Jixi Anhui, 92 six white pigs of Anqing pick up from Wangjiang Anhui modern times breeding and cultivate limited public affairs
Department, 132 Large Whites pick up from the safe and sound agricultural development Co., Ltd in Feidong Anhui, and all sampling pigs are 35 age in days weanling pigs.
A fritter ear tissue (about 1.5g) is taken with overbit clamp, is positioned over the 1.5mL equipped with 1mL 70% (volume fraction) ethyl alcohol
In Eppendorf pipes, Cord blood takes back laboratory, and -20 DEG C save backup.Phenol/chloroform method extracts ear sample tissue DNA, ultrapure
Water dissolution, ultramicron ultraviolet specrophotometer survey concentration and purity, set 4 DEG C of spare or -20 DEG C of preservations.
Key instrument：
(1) PCR instrument：American AB I companies
(2) pH analyzers：Italian HANNA companies
(3) electronic balance：Ao Haosi companies of the U.S.
(4) electrophoresis tank：Liuyi Instruments Plant, Beijing
(5) thermostat water bath：The permanent Science and Technology Ltd. in Shanghai one
(6) high-pressure sterilizing pot：Shenan Medical Appliances Factory, Shanghai
(7) gas bath constant temperature oscillator：Jin Cheng Guo Sheng laboratory apparatus factory of Community of Jin Tan County city
(8) micro refrigerated centrifuge：Beckman Coulter companies of the U.S.
(9) whiteness colorimeter：Beijing occasion Tyke Instrument Ltd.
(10) digimatic calipers：Chengdu Chengliang Tools Group Co., Ltd
(11) air dry oven：The permanent Science and Technology Ltd. in Shanghai one
(12) voltage stabilization and current stabilization electrophoresis apparatus：Liuyi Instruments Plant, Beijing
(13) the miniature mini centrifuges of MiniStar：Tomos companies of the U.S.
(14) ultraviolet specrophotometer：Heng Tong Science and Technology Ltd. of Beijing Merrill Lynch
(15) full automatic gel imaging analysis instrument：Shanghai Peiqing Science Co., Ltd
Main agents：
Golden DNA Polymerase, 2xReaction Mix are purchased from Tiangeng biochemical technology Co., Ltd；Agarose is purchased
From Spain YITO Bio-instrument Company；DNA Marker are purchased from Dalian treasured bioengineering Co., Ltd；Albumen
Enzyme K is purchased from Beijing bio tech ltd Ai Delai；10 × Loading Buffer have purchased from the full formula gold biotechnology in Beijing
Limit company；Methylene-bisacrylamide, acrylamide, TEMED are purchased from Sigma companies；Primer, chloroform, Tris saturated phenols,
EDTA, SDS, bromophenol blue, isoamyl alcohol, absolute ethyl alcohol, dimethylbenzene cyanogen, glycerine, sodium acetate, NaCl, KCl, NaOH, Na2HPO4、
KH2PO4, glacial acetic acid, silver nitrate, deionized formamide, formaldehyde, citric acid, sodium citrate, glucose etc. has purchased from Shanghai life work
Limit company.
Main agents are prepared：
(1) Tissue lysates
0.605g Tris-base are added in 200mL ddH2O, adjusts pH value to 8.0,18.61g is added
Na2EDTA.2H2O is added 2.5g SDS, is settled to 200mL, high pressure sterilization is spare after dissolving.
(2) 20mg/mL Proteinase Ks
The Proteinase K of bottled 100mg is dissolved in 5mL sterilizing ultra-pure waters, (1mL/ in autoclaved centrifuge tube is sub-packed in
Pipe), -20 DEG C save backup.
(3) 6x sample-loading buffers
0.25% bromophenol blue, 0.25% dimethylbenzene cyanogen, 30% glycerine water solution.
(4) ethidium bromide (EB)
It takes EB10g to be completely dissolved in 100mL water, is kept in dark place.
(5)0.5M EDTA
It weighs 46.5g EDTA-Na and is dissolved in 200mL ultra-pure waters, with NaOH tune pH to 8.0, be settled to 250mL.
(6)10xTBE
108Tris alkali, 55g boric acid is taken to measure 40mL 0.5M EDTA, ultra-pure water is settled to 1000mL.
(7) 30% acrylamides
It takes 72.5g acrylamides, 2.5g methenes, ultra-pure water to be settled to 250mL, filters, 4 DEG C save backup.
(8) 10% ammonium persulfates
1g ammonium persulfates are dissolved in the distilled water of 10mL, and 4 DEG C save backup.
(9) 0.2%AgNO3
2g AgNO3 are dissolved in 1000mL ddH2O.
(10) 3%NaOH (developing solution)
15g NaOH are dissolved in 500mL ddH2O, and formaldehyde 1.0mL is added.
(11) fixer
100mL ethyl alcohol adds 5mL acetic acid, water is added to be settled to 1000mL.
Test method
DNA is extracted
(1) clip 0.2g or so ear tissue samples are put into 1.5mL EP pipes, are cut to erosion, are added Tissue lysates, 55 DEG C
Place 3h；
(2) 5uL Proteinase Ks (20ug/uL) are added, 55 DEG C of digestion are overnight；
(3) plus 0.6mL saturated phenols mix well, and are layered after placement, then continue reverse mixing, until forming suspension；
(4) 12000rpm centrifuges 10min；
(5) it draws in the limpid DNA solution to another clean centrifuge tube in upper layer (1.5mL)；
(6) 600uL phenol-chloroforms-isoamyl alcohol (25: 24: 1) is added, overturns mixing until forming suspension；
(7) 12000rpm centrifuges 10min；
(8) it draws in the limpid DNA solution to another clean centrifuge tube in upper layer (1.5mL)；
(9) step 6,7,8 are repeated；
(10) 0.6mL chloroform-isoamyl alcohols (24: 1) solution is added, mixes well 30min；
(11) 12000rpm centrifuges 10min, draws in the limpid DNA solution to another clean centrifuge tube in upper layer
(1.5mL) is added 1mL ice absolute ethyl alcohol (2 times of volumes), mixes well, occur cotton-shaped floating material, i.e. DNA in centrifuge tube；
(12) 12000rpm centrifuges 10min, and cotton-shaped suspended matter is drawn in another clean centrifuge tube (1.5mL)；
(13) 0.5-1mL70% ethyl alcohol (being put into -20 DEG C in advance) is added, after 10min, ethyl alcohol is sucked out, then add 70% ethyl alcohol,
It rinses 2-3 times repeatedly；
(14) ambient temperature overnight dry DNA sample, until without ethyl alcohol smell；
(15) be added 150-300uLTE/ddH2O stay overnight or 65 DEG C of water-bath 2h, so that DNA is dissolved；
(16) adjust final concentration to 100ng/uL；
(17) 4 DEG C or -20 DEG C of long-term preservations.
DNA concentration and purity detecting
(1) DNA of 1 μ L is taken to measure the ratio between OD260/OD280 on SMA 1000, ratio between 1.8-2.0,
Illustrate that carried DNA purity is higher；
(2) DNA bands are detected with 1.5% agarose gel electrophoresis method, the quality of observation extraction product carries out follow-up real
It tests.Software analysis tool
DNA databases：http：//www.ncbi.nlm.nih.gov/.
Primer-design software：Primer 5.0, Stamford Photographing On-line software.
Sequence analysis software：DNAStar, DNAMan, Chromas.
Statistical analysis software：SPSS 19.0.
Design of primers
According to GenBank pigs TLR4 genes (accession number AY753179) exon 3 sequence, using Primer Premier
5.0 software Design primers deliver the synthesis of Shanghai Sheng Gong Bioisystech Co., Ltd.
Wherein SEQ ID NO in the nucleotide sequence of primer TLR4exon3-1 such as sequence table：1 and SEQ ID NO：2 institutes
Show, SEQ ID NO in the nucleotide sequence of primer TLR4 exon3-2 such as sequence table：3 and SEQ ID NO：Shown in 4, annealing temperature
Degree and target fragment length are shown in Table 1.
1 pig TLR4 gene primer sequences of table
Newly synthesized primer is solid sprills, and 1min is first centrifuged before dissolving, after being deposited to tube bottom, by dilution ratio plus
Enter the sterilizing ddH of corresponding volume2O is diluted to 10 μ g/ μ L, mixes well, 4 DEG C of preservations.
PCR reaction systems
PCR reaction systems are shown in Table 2.
2 PCR reaction systems of table
PCR amplification program：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, Tm DEG C of (annealing temperature is depending on primer) renaturation
30s, 72 DEG C of extension 30s, 35 cycles；Last 72 DEG C of extensions 8min, 4 DEG C of preservations.
Agarose gel electrophoresis detects
Take 3 μ L pcr amplification products and 2 μ L 6 × Loading Buffer mixings, with 2% agarose gel electrophoresis into
Row detection (voltage 6V/cm) observes the result of PCR amplification under gel imager, takes pictures.By amplified band and Marker bands
Compare, judges whether pcr amplification product is purpose segment.If amplified band be purpose segment, and be single band, brightness compared with
It is good, then pcr amplification product is preserved for -20 DEG C, to carry out next step experiment.
Single-strand conformation polymorphism detects (SSCP)
Native polyacrylamide gel electrophoresis
(1) required instrument, including glass plate, sample comb and rope etc. are tested in cleaning, and are dried, and are selected one and are done
Only, smooth desktop making sheet is spare.
(2) 10% polyacrylamide gels of 72mL (distilled water of the PAGE of 24mL30%, 42mL, 10 × TBE are prepared
6mL, AP：330 μ L, TEMED：50 μ L), quick encapsulating after mixing, being inserted into comb afterwards, (attention checks whether there is bubble, if any answering
Exclude), placing several hours allows its natural coagulation (can be put into baking oven and accelerate solidification).
(3) after fixing the plate for coagulating good glue, 1 × TBE, 300V voltage prerunnings 30min are added into electrophoresis tank.
(4) 7 μ L denaturants are added per Kong Zhongxian in 96 hole PCR plates, it is rear that 3 μ LPCR products are added, after blowing and beating mixing with rifle,
Transparent adhesive tape seal；
After (5) 98 DEG C of denaturation 10min, rapid ice bath cools down 10min, then point sample.
(6) 240V voltages 20min, 120V voltage stay overnight 14-16h.
Cma staining
(1) electrophoresis terminates, and removes glass plate, cuts glue and marks, and clear water is washed 3 times.
(2) fixer (10% ethyl alcohol and 0.5% acetic acid) shakes 3-5min, recycling.
(3) 2%AgNO3 solution is poured into, jog 10-15min is protected from light, is recycled.
(4) distillation washing 1-2 times.
(5) now match developing solution (3%NaOH, 0.1%HCHO), colour developing is until band occurs after rinse.
(6) distilled water rinses, and electrophoresis result, preservation of taking pictures are observed in gel imaging system.
PCR product sequencing
According to SSCP as a result, selecting the sample of different banding patterns, each banding pattern selects 3 samples, 50 μ L systems of PCR amplification send to
Shanghai Sheng Gong Co., Ltds carry out bidirectional sequencing, sequencing result DNAStar or DNAMan, Chromas analysis.
Population Genetics is analyzed
For SSCP the result shows that codominant inheritance is presented, phenotypic response genotype directly calculates gene frequency and gene
Type frequency.
Gene frequency refers to the ratio that a certain gene number accounts for its same gene seat gene number in group.Its calculation formula
For：
Pi=[2 (ii)+(ij1)+(ij2)+...+(ijn-1)+(ijn)]/2n
In formula, Pi：The frequency of i-th of allele；ii：I allele homozygous individual number；iin：Contain i equipotential bases
The heterozygous individual number of cause；n：For sum individual in a group.
Genotype frequency=genotype individuals number/measurement group sum × 100%
Analysis of genetic diversity
(1) homozygosity (homozygosity, Ho)：Refer to the journey of equipotential gene pure on specific markers site in a certain group
Degree, reflects the consistency of population genetic.
In formula, Ho is the homozygosity in a certain site, and Pi is the frequency of i-th of allele, and n is the equipotential base in a certain site
Factor.
(2) heterozygosity (heterozygosity, He)：Opposite with homozygosity, heterozygosity height shows that gene is consistent in group
Property is poor, and the hereditary variation of group is larger, rich polymorphism.
In formula, He：The heterozygosity in a certain site, Pi：I-th of gene frequency, n on a certain site：A certain site
Number of alleles, Ho：Hereditary homozygosity；
(3) effective number of allele (effective number of alleles, Ne)：The inverse of homozygosity, reflection etc.
Influencing each other between the gene of position is an index for weighing gene homozygosity.Allele is distributed more uniform in group,
Effective number of allele is closer to the actually detected allele absolute number arrived.
Ne=1/Ho=1/ ∑s Pi2
In formula, Ne：The effective number of allele in a certain site, Pi：I-th of gene frequency, Ho on a certain site：It loses
Pass homozygosity；
(4) polymorphism information content (polymorphism information content, PIC)：To the more of marker gene
State property is estimated.0.5 polymorphic, 0.5 ＞ PIC ＞, 0.25 moderate polymorphics of height of PIC ＞, 0.25 low polymorphics of PIC ＜.
In formula, n：Allele number；Pi and Pi：Respectively i-th and frequency of i-th of the allele in group.PIC
For value for the estimation to marker gene polymorphism, PIC ＞ 0.5 are that height is polymorphic, and PIC ＜ 0.25 are low polymorphic, 0.25 ＜ PIC
＜ 0.5 is moderate polymorphic.
Population genetic variations are analyzed
(1) Hardy-Weinberg law balance detection：When group is in Hardy-Weinberg equilibrium state, an equity
The gene frequency of position gene and the relationship of genotype frequency have：D=p2, H=2pq, R=q2, wherein D, H, R are genotype frequency
Rate, and p, q are gene frequency, and have p+q=1.
(2) Chi-square Test formula：χ 2=∑s (Oi-Ei) 2/Ei
Wherein, Oi is observed value, and Ei is theoretical value.Because of the degree of freedom df=1 of this research data, when genotype theoretical value
When less than 5, correction formula can be used：
As a result with analysis
The DNA detections of pig ear tissue
DNA after extraction is detected through 1.5% agarose gel electrophoresis, testing result such as Fig. 1.As shown in Figure 1, this experiment
The DNA sample quality of extraction is good, disclosure satisfy that the requirement of experiment.1 μ L DNA extraction samples are taken to be measured on SMA 1000 simultaneously
OD260/OD280, ratio are located at 1.8-2.0, and carried DNA purity is higher, can carry out subsequent experimental.
PCR amplification result
3 partial sequence of PCR amplification TLR4 gene extrons, product are detected with 2% agarose gel electrophoresis (see Fig. 2-figure
3).As shown in Fig. 2, TLR4-exon3-1 primer pair amplifies segments are 283bp, as shown in figure 3, TLR4-exon3-2 primer pairs expand
Increasing segment is 209bp, and pcr amplification product meets the size of expected amplified fragments, as shown, specificity is good and without non-specific
Property band, can be used for follow-up test.
PCR-SSCP testing results
Primer pair TLR4-exon3-1 PCR-SSCP detection display, have in TLR4-exon3-1 amplified fragments 2 it is chain
SNP site, there are 2 kinds of banding patterns, are respectively designated as EE and EF genotype (Fig. 4)；Primer pair TLR4-exon3-2 PCR-SSCP inspections
It surveys and finds, have 1 SNP site in TLR4-exon3-2 amplified fragments, there are 3 kinds of genotype in the site：CC, CA and AA genotype
(Fig. 5).
3 amplified fragments the sequencing results of TLR4 gene extrons
It takes the PCR product of primer TLR4-exon 3-1, TLR4-exon 3-2 differences banding pattern individual to be sequenced, and will survey
Sequence result is compared with pig TLR4 gene DNA sequences (NCBI accession number is AY753179).
As a result, it has been found that：(1) TLR4-exon 3-1 amplified fragments sequencing result is：It is deposited at the 318th bit base of TLR4 genes
There are G → A, (sequencing result is shown in Fig. 6-a, figure at C → A (sequencing result is shown in Fig. 6-c, Fig. 6-d, backward sequencing), 417 bit bases
Linked gene mutation 6-b), it is wild type to define EE (CC/GG), and EF (CA/GA) is heterozygosis subtype, 318,417 site mutations
Do not cause amino acid change, the amino acid of coding is respectively accordingly Thr106Thr, Ala139Ala.(2)TLR4-exon
3-2 amplified fragments sequencing results are：The sense mutations that 1 C → A is found at the 1027th bit base of TLR4 genes, defining CC is
Wild type, CA are heterozygosis subtype, and AA is saltant type (sequencing result is shown in Fig. 7-a, Fig. 7-b, Fig. 7-c, backward sequencing), 1027 sites
Mutation causes amino acid to become Lys from Gln.
TLR4 gene C 318A/G417A, C1027A loci gene type frequencies and gene frequency analysis
By being detected to 4 kinds, 429 pig TLR4 gene Cs 318A/G417A, C1027A mutational sites, it is counted
Gene frequency and genotype frequency, as a result as shown in table 3, table 4.The mutational sites C1027A detect in four strains,
It is protogene that each genotype frequency trend, which is CA ＞ AA ＞ CC, A allele, in the black pig in Anhui south and the black pig groups of Huo Shou, and
Result in Large White, country fair pig groups is on the contrary, genotype frequency CC ＞ CA ＞ AA, C allele is protogene.C318A/
The sites G417A EE genotype frequencies in the black pig in Anhui south, country fair pig, six white pig of Anqing and Large White group are respectively 79.05%,
71.00%, 81.08 and 76.76%, for the preponderant genotype of 4 groups, E allele is protogene.
3 C1027A locus genes frequency of table and genotype frequency analysis
4 C318A/G417A locus genes frequency of table and genotype frequency analysis
Population Genetics is analyzed
The Population Genetics analysis of the sites TLR4 gene C 318A/G417A
PIC values are to weigh the index of genetic variation degree in group, and as shown in Table 5, C318A/G417A linkage sites are in Anhui
PIC values in four the black pig in south, country fair pig, six white pig of Anqing and Large White groups are respectively less than 0.25, are in low polymorphic；And it passes through
Cross χ2Comptibility test, the black pig in Anhui south, country fair pig, six white pig of Anqing and four groups of Large White χ2Value is respectively less than 5.991, place
In Hardy-Weinberg equilibrium state (P ＞ 0.05).
Genetic polymorphism and χ of 5 sites pig TLR4 gene C 318A/G417A of table in group2It examines
Note：PIC ＞ 0.5 are that height is polymorphic, and 0.25 ＜ PIC ＜ 0.5 are moderate polymorphic, and PIC ＜ 0.25 are low polymorphic, χ2
(0.05)=5.991；Following table is same.
The Population Genetics analysis of the sites TLR4 gene C 1027A
As can be seen from Table 6, the sites C1027A are in four the black pig in Anhui south, country fair pig, six white pig of Anqing and Large White groups
In PIC be all higher than 0.25 be less than 0.5, be in moderate polymorphic；And pass through χ2Comptibility test, the black pig in Anhui south, country fair pig, Anqing six
The χ of white four groups of pig and Large White2Value is respectively less than 5.991, is in Hardy-Weinberg equilibrium state (P ＞ 0.05).
Genetic polymorphism and χ of 6 sites pig TLR4 gene C 1027A of table in group2It examines
Note：With 5 note of table.
The polymorphic detection that this experiment carries out the exon 3 sequence of pig TLR4 genes, finds 1 single base mutation, 1 altogether
To chain base mutation.The wherein mutational sites C1027A detect in four strains, black pig and the black swinerys of Huo Shou in Anhui south
It is protogene that each genotype frequency trend, which is CA ＞ AA ＞ CC, A allele, in body, and is tied in Large White, country fair pig groups
Fruit is on the contrary, genotype frequency CC ＞ CA ＞ AA, C allele is protogene.And the sites C318A/G417A the black pig in Anhui south,
Preponderant genotype is EE genotype in country fair pig, six white pig of Anqing and Large White group, frequency is respectively 79.05%,
71.00%, 81.08 and 76.76%, E allele is protogene.
Four groups detect base mutation in the sites TLR4 gene C 1027A and C318A/G417A, meet heredity
Label standard will be with certain generality if being used as genetic marker from now on.The sites C318A and the sites G417A are synonymous prominent
Become, does not cause the change of amino acid.Nevertheless, still the meaning of same sense mutation can not be ignored.When same sense mutation is in
The efficiency that may result in same amino acid translation degenerate codon when gene coding region is different, and then influences its transcriptional level,
Change protein expression level, influences the normal physiological function of body.Whether this linked mutation has resistance/neurological susceptibility of disease
It influences, can need further to research and analyse as the label of a disease resistance trait.Amino acid caused by the sites C1027
Variation is missense mutation Gln343Lys, which causes amino acid nature to change, it is made to be changed into band just by uncharged Gln
The Lys of charge, the quantity of electric charge increase, and may cause the variation of structure, to influence protein function.And external pig kind is compared, I
State's local pig breed all has the characteristics that disease-resistant, resistance is strong, and TLR4 gene C 1027A loci polymorphisms are distributed in China and foreign countries' pig inter-species
Huge difference, if it is related with variety resistance, be worth further research.
Two, the correlation analysis of TLR4 gene pleiomorphisms and weanling pig sero-immunity index
Test material
Experimental animal
100 country fair pigs pick up from the safe and sound agricultural development Co., Ltd in Guangde Anhui in experimental animal, and the black pig in 105 Anhui south is picked up from
The plump eco-agricultural development Co., Ltd in Jixi Anhui, 92 six white pigs of Anqing pick up from Wangjiang Anhui Hao Yu animal husbandry Co., Ltd,
132 Large Whites pick up from the safe and sound agricultural development Co., Ltd in Feidong Anhui, and all sampling pigs are 35 age in days weanling pigs.In ＜
Specific implementation mode ＞ (one) parts adopt ear tissue simultaneously, and the whole blood of acquisition is stood ice by every pig empty stomach vena cava anterior blood sampling
Overnight, then 1000-3000rpm is centrifuged 10 minutes 4 DEG C of case, and serum is taken to be saved backup in -20 DEG C.
Testing equipment and equipment
(1) microplate reader (450nm Detection wavelength optical filters, 570nm or 630nm tuning wavelengths optical filter).
(2) board-washing machine (adjustable reservoir quantity ensures not spilling over per 350 μ l washing lotions of hole).
(3) superclean bench, Biohazard Safety Equipment, vent cabinet.
(4) high-precision single-channel charger (range 0.5-10ul, 2-20 μ l, 20-200 μ l, 200-1000 μ l).
(5) high-precision multiple tracks charger (8 Dao Huo 12, range are 50-300 μ l)
(6) 37 DEG C of insulating boxs.
(7) refrigerated centrifuge.
(8) refrigerator (4 DEG C, -20 DEG C, -86 DEG C)
(9) assay balance.
(10) scissors, tweezers, pliers etc..
(11) whirlpool mixed instrument, low-frequency oscillator etc..
Test consumable and reagent
(1) pig IFN γ, IL-10, IL-6, IL-12, TNF α enzyme-linked immunoassay kit.
(2) centrifuge tube (capacity 1.5mL, 5mL etc.).
(3) disposable tip (range is 0.5-10 μ L, 2-20 μ L, 20-200 μ L, 200-1000 μ L)
(4) pure water or distilled water.
(5) graph paper.
(6) blotting paper.
(7) EDTA, sodium citrate, heparin.
Test method
Pig vena cava anterior is taken a blood sample, conventional to detach serum, immune indexes is measured using pig ELISA kit, in microplate reader
Following mass concentration is surveyed at 450nm：1000,500,250,125,62.5, the 31.25, standard items of 15.6pg/mL totally 7 concentration
Absorbance (OD values), draw out standard curve, measure the concentration of first sample sheet.
Test principle
This experiment uses double antibody sandwich ELISA.The ELISA Kit provided are that typical sandwich method enzyme linked immunological is inhaled
Attached assay kit (ELISA).Advance coated antibody is anti-pig monoclonal antibody.Detection phase antibody is polyclonal antibody, warp
Biotin (biotin) marks.After the reaction of ELISA Plate hole is successively added in sample and biotin labelled antibodies, PBS or TBS washings.With
The Avidin reaction of peroxidase labelling is added afterwards；With substrate TMB colour developings after the thorough washing of PBS or TBS.TMB exists
Au bleu is converted under the catalysis of peroxidase, and is converted to final yellow under the action of an acid.The depth and sample of color
In the factor to be measured be proportionate.
Operating procedure (by taking IL-6 as an example, IFN γ, IL-10, IL-12, the same IL-6 of TNF α detection operating procedure)
(a) from lath needed for taking-up experiment has been balanced into the hermetic bag of room temperature, unused lath and drier please be put back to
2-8 DEG C is sealed in aluminium foil bag.
(b) reserve blank well (if using dual wavelength read plate, blank well can not be set).
(c) corresponding aperture is added in sample or various concentration pig IL-6 standard items (holes 0pg/mL add standard dilutions) respectively
In (100 holes μ L/), seal reacting hole with sealing plate gummed paper, 37 DEG C of incubators are incubated 90 minutes.
(d) shift to an earlier date 30 minutes and prepare biotinylation pig IL-6 antibody working solutions.
(e) board-washing 3 times.
(f) biotinylation pig IL-6 antibody working solution (100 holes μ l/) is added.Reacting hole is sealed with sealing plate gummed paper, 37 DEG C incubate
Case is incubated 60 minutes.
(h) shift to an earlier date 30 minutes and prepare enzyme conjugates working solution.Room temperature avoid light place.
(i) board-washing 3 times.
(j) in addition to blank well, enzyme conjugates working solution (100 holes μ l/) is added.Reacting hole is sealed with sealing plate gummed paper, 37 DEG C
Incubator is protected from light incubation 30 minutes.
(k) board-washing 5 times.
(1) TMB colour developings working solution (including blank well) 100 holes μ L/ are added, 37 DEG C of incubators are protected from light incubation, work as standard curve
The color of high concentration is deeper, when having apparent color gradient, you can terminates.It please don't be more than 30 minutes to test chromogenic reaction.
(m) terminate liquid (including blank well) 100 holes μ L/ are added, OD (450nm) value is measured at once (10 minutes after mixing
It is interior).
Result judgement and calculating
Make abscissa with standard concentration, OD values make ordinate, and the coordinate points of each standard items are connected with sweep.Pass through sample
This OD values can find its concentration on standard curve.If sample OD values are higher than the standard curve upper limit, resurveyed after should suitably diluting,
It should be multiplied by extension rate when calculating concentration.
Mathematical analysis model
Between kind the difference analysis of sero-immunity index using SPSS19.0 statistical softwares One-way ANOVA programs into
Row variance analysis and significance test, experiment the data obtained are indicated with " average ± SD ".
The mathematical model that different genotype uses the effect variance analysis of each immune indexes for：Yij=μ+Bi+Gj+eij；
Wherein, YijFor immune indexes phenotypic number；μ is group's mean value；BiIndicate the wean batch effect of weanling pig；GjIndicate jth kind
Genotype effects；eijIndicate random error；Become using the heredity detected to this experiment of SPSS19.0 statistical softwares GLM programs
The different relevance with sero-immunity index is analyzed.
As a result with analysis：
Each pig interspecies immunity index error specific analysis
The variance analysis of immune indexes level is shown (table 7) between 4 pig kinds：Blood serum IL-6 content, six white pig of Anqing are extremely aobvious
It writes and is higher than country fair pig and Large White (P ＜ 0.01), the black pig pole in Anhui south is significantly higher than Large White (P ＜ 0.01)；And -10 content of serum IL
Exactly the opposite, six white pig pole of Anqing is substantially less than country fair pig, the black pig in Anhui south and Large White (P ＜ 0.01)；- 12 content of serum IL, country fair
Pig pole is substantially less than the black pigs of Huo Shou, the black pig in Anhui south and Large White (P ＜ 0.01)；Difference of the blood IFN-γ content in 4 pig inter-species
Different is extremely significantly；At the same time, blood TNF-α content is also extremely significantly in the difference of 4 pig inter-species.In addition, serum IL-
6 contents highest in six white pig of Anqing, serum IL -10, IL-12 contents highest in Large White；Serum I FN- γ, TNF-α contain
Amount highest in country fair pig.
Each immune indexes level difference unit between 7 kind of table：pg/mL
Note：Same column indicates significant difference (P ＜ 0.05) with different lowercase shoulder marks, carries different capitalization shoulder marks
Indicate difference extremely significantly (P ＜ 0.01).
The correlation analysis of TLR4 gene pleiomorphisms and weanling pig sero-immunity index
The association analysis of pig TLR4 gene C 1027A loci polymorphisms and immune indexes
The association analysis of C1027A loci polymorphisms and immune indexes in six white pig of Anqing
As shown in Table 8, in six white pig groups of this experiment Anqing the sites TLR4 gene C 1027A AA genotype individuals serum
Immune indexes content is individual higher than CA, CC type, wherein in serum TNF α, IL-12 content's index, the difference of AA types and CC type individuals
Different to reach the pole level of signifiance (P ＜ 0.01), AA types and the difference of CA type individuals reach the level of signifiance (P ＜ 0.05)；Serum
IFN γ content reaches the level of signifiance (P ＜ 0.05) between AA and CC genotype individuals.
The association analysis unit in 8 Anqing of table, six sites white pig TLR4 gene C 1027A：pg/mL
Note：Colleague indicates significant difference (P ＜ 0.05) with different lowercase shoulder marks, carries different capitalization shoulder marks
Indicate difference extremely significantly (P ＜ 0.01).
The association analysis of C1027A loci polymorphisms and immune indexes in country fair pig
As shown in Table 9, the sero-immunity of the sites TLR4 gene C 1027A AA genotype individuals refers in this experiment country fair pig groups
Content is marked higher than CA, CC type individual, wherein in serum I FN γ, IL-6 content's index, AA types and the difference of CA type individuals reach
The level of signifiance (P ＜ 0.05), AA types and the difference of CC type individuals reach the pole level of signifiance (P ＜ 0.01)；Serum TNF alpha content refers to
It is marked between AA and CC genotype individuals and reaches the level of signifiance (P ＜ 0.05), it is not notable with CA genotype individuals differences.
The association analysis unit in 9 sites country fair pig TLR4 gene C 1027A of table：pg/mL
Note：Colleague indicates significant difference (P ＜ 0.05) with different lowercase shoulder marks, carries different capitalization shoulder marks
Indicate difference extremely significantly (P ＜ 0.01).
The association analysis of C1027A loci polymorphisms and immune indexes in the black pig in Anhui south
As shown in Table 10, the serum of the sites TLR4 gene C 1027A AA genotype individuals is exempted from the black pig groups in this experiment Anhui south
Epidemic disease index content is individual higher than CA, CC type, wherein in serum TNF α, IL-12 content's index, the difference of AA types and CC type individuals
Reach the level of signifiance (P ＜ 0.05)；- 10 content of serum IL reaches the pole level of signifiance (P ＜ between AA and CC genotype individuals
0.01), reach the level of signifiance (P ＜ 0.05) between AA and CA genotype individuals.
The association analysis unit in 10 Anhui of the table south sites black pig TLR4 gene C 1027A：pg/mL
Note：Colleague indicates significant difference (P ＜ 0.05) with different lowercase shoulder marks, carries different capitalization shoulder marks
Indicate difference extremely significantly (P ＜ 0.01).
The association analysis of C1027A loci polymorphisms and immune indexes in Large White
As shown in Table 11, in this experiment Large White group the sites TLR4 gene C 1027A AA genotype individuals sero-immunity
Index content is higher than CA, CC type individual, wherein in serum I FN γ, IL-6 content's index, AA types and the difference of CC type individuals reach
To the pole level of signifiance (P ＜ 0.01), blood serum IL-6 content reaches the level of signifiance (P ＜ 0.05) between AA and CA genotype individuals,
Serum I FN γ contents reach the pole level of signifiance (P ＜ 0.01) between AA, CA and CC genotype individuals；- 10 content's index of serum IL
Reach the level of signifiance (P ＜ 0.05) between AA and CC genotype individuals.
The association analysis unit in 11 sites Large White TLR4 gene C 1027A of table：pg/mL
Note：Colleague indicates significant difference (P ＜ 0.05) with different lowercase shoulder marks, carries different capitalization shoulder marks
Indicate difference extremely significantly (P ＜ 0.01).
The association analysis of pig TLR4 gene C 318A/G417A loci polymorphisms and immune indexes
The association analysis of C318A/G417A loci polymorphisms and immune indexes in six white pig of Anqing
As shown in Table 12, the sites TLR4 gene C 318A/G417A EE genotype individuals in six white pig groups of this experiment Anqing
Sero-immunity index content higher than EF types individual, wherein in serum IL -10, IL-12 content's index, EE types and EF type individuals
Difference reach the pole level of signifiance (P ＜ 0.01)；Serum TNF alpha content reaches the level of signifiance between EE and EF genotype individuals
(P ＜ 0.05).
The association analysis unit in 12 Anqing of table, six sites white pig TLR4 gene C 318A/G417A：pg/ml
Note：Same column indicates significant difference (P ＜ 0.05) with different lowercase shoulder marks, carries different capitalization shoulder marks
Indicate difference extremely significantly (P ＜ 0.01).
The association analysis of C318A/G417A loci polymorphisms and immune indexes in country fair pig
As shown in Table 13, in this experiment country fair pig groups the sites TLR4 gene C 318A/G417A EE genotype individuals serum
Immune indexes content is individual higher than EF types, wherein in serum I FN γ, TNF α content's index, the difference of EE types and EF type individuals
Reach the level of signifiance（P ＜ 0.05).
The association analysis unit in 12 sites country fair pig TLR4 gene C 318A/G417A of table：pg/ml
Note：Same column indicates significant difference (P ＜ 0.05) with different lowercase shoulder marks, carries different capitalization shoulder marks
Indicate difference extremely significantly (P ＜ 0.01).
The black pig C318A/G417A polymorphic sites in Anhui south and each immune indexes significance analysis
As shown in Table 14, the sites TLR4 gene C 318A/G417A EE genotype individuals in the black pig groups in this experiment Anhui south
Sero-immunity index content is individual higher than EF types, wherein in serum IL -12, IL-10 content's index, EE types and EF type individuals
Difference reaches the level of signifiance (P ＜ 0.05)；Serum TNF alpha content reaches the pole level of signifiance (P between EE and EF genotype individuals
＜ 0.01).
The association analysis unit in 14 Anhui of the table south sites black pig TLR4 gene C 318A/G417A：pg/ml
Note：Same column indicates significant difference (P ＜ 0.05) with different lowercase shoulder marks, carries different capitalization shoulder marks
Indicate difference extremely significantly (P ＜ 0.01).
Large White C318A/G417A polymorphic sites and each immune indexes significance analysis
As shown in Table 15, in this experiment Large White group the sites TLR4 gene C 318A/G417A EE genotype individuals blood
Clear immune indexes content is individual higher than EF types, wherein in serum I FN γ, IL-10 content's index, the difference of EE types and EF type individuals
It is different to reach the level of signifiance (P ＜ 0.05)；Blood serum IL-6 content's index reaches the pole level of signifiance (P between EE and EF genotype individuals
＜ 0.01).
The association analysis unit in 13 sites Large White TLR4 gene C 318A/G417A of table：pg/ml
Note：Same column indicates significant difference (P ＜ 0.05) with different lowercase shoulder marks, carries different capitalization shoulder marks
Indicate difference extremely significantly (P ＜ 0.01).
Five immune indexes that this research is chosen have very important importance in immunity of organism.IL-6 is as a kind of
Multi-functional inflammatory cytokine can balance the damage effect of some cell factors, to play a protective role.Leucocyte is situated between
- 10 (IL-10) of element are a kind of cell factors that can play anti-inflammatory effect, can promote the differentiation of B cell and promote antibody
Generation, while inhibiting macrophage, and Th1 cells is inhibited to secrete cytokine profiles.Helper T lymphocyte (helper T
Cell, Th) include two subgroups of Th1 and Th2, wherein the former participates in cellular immunity, mainly generates IL-2, IFN γ etc., and the latter
Main to generate IL-10 etc., Help B Cells generate antibody, and participate in humoral immunity.The effect of IL-12 is that independent induction early stage is auxiliary
Helping property T mother cell differentiating into T h1 cells, and promote development and the proliferation of Th1 cells, it is to mediate Th1 cellullar immunologic responses
Key cytokines.IL-12 can stimulate NK cells and T cell to generate IFN γ.IFN γ can stimulating expression of macrophage generate it is big
The nitric oxide of amount, directly kills pathogen.TNF-α can mediate the activation of NF-kB, and NF-kB is transcribed as a kind of fast response
The factor can regulate and control proinflammatory cytokine, chemotactic factor (CF) and the isogenic activation of adhesion factor and expression.IL-10 is by Th2 cells
It generates, IL-10 can almost inhibit the synthesis and release of all inflammatory immune indexes of such as IL-6, wherein IL-12 to promote Th1 thin
Born of the same parents break up and the ability of proliferation can also be inhibited by IL-10.But meanwhile the reaction of Th2 cells and the secretion capacity of cell factor
Also can be inhibited in turn by the key cytokines IL-12 as startup and maintenance Th1 cells.Therefore, IL-10 and IL-
12 dynamic equilibrium can be directly related to resistance of the body to disease.It can be seen that influence each other between each immune indexes, phase
Mutually cooperation, to play a significant role in immunity of organism.
This tests every sero-immunity index value detected by each pig population in range of normal value.In different swinerys
In, the mean differences of each immune indexes is substantially all as extremely significantly, this may be due to the genetic background of each swinery and its right
The premunition of disease it is different and caused by.
The correlation analysis result of each group's different genotype in the sites TLR4 gene C 1027A is shown：Country fair pig, Large White, Anhui
Between the black pig in south, six Bai Zhuge groups different genotype of Anqing the variation tendency of immune indexes is consistent, i.e. AA genotype individuals
Sero-immunity index content is higher than CA, CC type individual；Wherein in country fair Swine serum IFN γ, IL-6, TNF α content's index, AA types
Reach extremely notable or the level of signifiance with the difference of CA types, CC type individuals；In Large White serum I FN γ, IL-6, IL-10 content
In index, the difference of AA types and CA, CC type individual reaches the extremely notable or level of signifiance；Black Swine serum TNF α, IL- in Anhui south
12, in IL-10 content's index, the difference of AA types and CA, CC type individual reaches the extremely notable or level of signifiance；In the black pig bloods of Huo Shou
Clear TNF α, IL-12, in IFN γ content's index, the difference of AA types and CA, CC type individual reaches the extremely notable or level of signifiance；Therefore
Speculate in four groups, the disease resistance of the sites TLR4 gene C 1027A AA types individual may exist with CA, CC type individual
Difference, A allele may have certain advantage in resistance of the pig to disease.
The correlation analysis result of each group's different genotype in the sites TLR4 gene C 318A/G417A is shown：Country fair pig, great Bai
The variation tendency of immune indexes is consistent between pig, the black pig in Anhui south, six Bai Zhuge groups different genotype of Anqing, i.e. EE genotype
The sero-immunity index content of body is higher than EF type individuals；Wherein in country fair Swine serum IFN γ, TNF α content's index, EE types and EF
The difference of type individual reaches the level of signifiance；In Large White serum I FN γ, IL-6, IL-10 content's index, EE types and EF types
The difference of body reaches the extremely notable or level of signifiance；In the black Swine serum TNF α in Anhui south, IL-12, IL-10 content's index, EE types
Reach extremely notable or the level of signifiance with the difference of EF type individuals；In Anqing, six white Swine serum TNF αs, IL-12, IL-10 content refer to
It puts on, the difference of EE types and EF type individuals reaches the extremely notable or level of signifiance；Therefore speculate in four groups, TLR4 genes
The disease resistance of the sites C318A/G417A EE types individual may have differences with EF type individuals, and E allele may be in pig to disease
There is certain advantage in the resistance of disease.
Result of study prompts, and TLR4 gene Cs 1027A, C318A/G417A site can be used as candidate target site and be applied to peace
The breeding for disease resistance of emblem local pig breed country fair pig, the black pig in Anhui south, six white pig of Anqing and adventive Large White.
Embodiment described above is only that the preferred embodiment of the present invention is described, not to the model of the present invention
It encloses and is defined, under the premise of not departing from design spirit of the present invention, technical side of the those of ordinary skill in the art to the present invention
The various modifications and improvement that case is made should all be fallen into the protection domain of claims of the present invention determination.
Claims (8)
1. a kind of screening technique of Anhui's local pig kind weanling pig premunition candidate genetic label, which is characterized in that including such as
Lower step：
(1) selection of control-animal model：Select Anhui Native pig kind country fair pig, the black pig in Anhui south, six white pig of Anqing and external pig
The piglet of kind 28 age in days of Large White wean is the control-animal model for studying immune indexes breed difference；
(2) sample collection：When 35 age in days of piglet, acquisition ear sample tissue is simultaneously taken a blood sample on an empty stomach；
(3) ear sample tissue DNA extracts；
(4) PCR amplification Toll-like receptor 4 is TLR4 gene target fragments；
(5) target fragment single-strand conformation polymorphism, that is, sscp analysis；
(6) pcr amplification product bidirectional sequencing is compared with NCBI accession number for AY753179, and there are following mutation：C318A、
G417A and C1027A；
(7) C318A, G417A and C1027A described in step (6) are directed to and carry out Population Genetics specificity analysis；
(8) measurement of piglet sero-immunity index；
(9) relevance of the hereditary variation and sero-immunity index of the TLR4 genes detected by mathematics model analysis is used, and
The otherness of sero-immunity index between analysis kind；
According to the correlation analysis of TLR4 gene mononucleotide polymorphisms SNP and immune indexes, detected SNP site is inquired into
Whether have an impact to different swinery weanling pig Resistance indexes, to which can the detected SNP site of judgement be used as piglet
The candidate significant notation site of breeding for disease resistance；
The process of PCR amplification target fragment is in the step (4)：The ear tissue sample DNA obtained using step (3) is template, root
According to 3 sequence of GenBank pig TLR4 gene extrons, accession number AY753179 is set using Primer Premier5.0 softwares
2 pairs of primers are counted, carry out the clone of 3 partial sequence of TLR4 gene extrons, and using the detection clone's production of 2% agarose gel electrophoresis
Whether object is purpose segment；Wherein SEQ ID NO in the nucleotide sequence of primer TLR4 exon3-1 such as sequence table:1 and SEQ
ID NO:Shown in 2, SEQ ID NO in the nucleotide sequence of primer TLR4 exon3-2 such as sequence table:3 and SEQ ID NO:4 institutes
Show, annealing temperature and target fragment length are shown in Table 1；
1 pig TLR4 gene primer sequences of table
The continuous mode of piglet sero-immunity index is in the step (8)：35 age in days weanling pig empty stomach vena cava anteriors are taken a blood sample,
Conventional separation serum, measures immune indexes, mass metering concentration is respectively at microplate reader 450nm using pig ELISA kit
1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.25pg/ml, 15.6pg/ml totally 7 concentration
Standard items absorbance, draw out standard curve, measure the concentration of first sample sheet；And then according to the mark of corresponding standard items
Directrix curve calculates IL-6, IL-10, IL-12, IFN-γ, the concentration of TNF-α in serum successively.
2. the screening technique of Anhui's local pig kind weanling pig premunition candidate genetic label according to claim 1, special
Sign is that control-animal model is selected as described in the step (1)：Country fair pig, the black pig in Anhui south, the six white pig of Anqing of 35 ages in days
And 28 age in days weanling pig of Large White selects 100,105,92,132 respectively, wherein country fair pig picks up from the safe and sound agricultural in Guangde Anhui and opens
Co., Ltd is sent out, the black pig in Anhui south picks up from the plump eco-agricultural development Co., Ltd in Jixi Anhui, and six white pig of Anqing picks up from Wangjiang peace
Emblem modern times breeding cultivates Co., Ltd, and Large White picks up from the safe and sound agricultural development Co., Ltd in Feidong Anhui.
3. the screening technique of Anhui's local pig kind weanling pig premunition candidate genetic label according to claim 2, special
Sign is that step (2) the middle ear sample tissue sampling process is：In advance 70% volume is injected in the Eppendorf pipes of 1.5mL
The alcohol of fraction concentration, then every pig adopt ear tissue block 1.5g, be fitted into the Eppendorf pipes, it is standby in -20 DEG C of preservations
With.
4. the screening technique of Anhui's local pig kind weanling pig premunition candidate genetic label according to claim 3, special
Sign is that step (3) the middle ear sample tissue DNA extraction process is：1. with eye scissors clip 0.2g ear tissues, its surface is removed
Hair and alcohol are fitted into 1.5mL Eppendorf pipes, and as far as possible shred ear tissue；2. the extraction of DNA is complete using Beijing
The tissue DNA extracts kit of the bio tech ltd Shi Jin is extracted；3. by obtained DNA tune final concentration to 100ng/ μ L,
It is placed on 2-8 degrees Celsius of preservation；4. DNA purity detectings：1 μ L DNA are taken to measure the ratio of OD260/OD280 on SMA 1000, when
OD260/OD280 ratios illustrate that carried DNA purity is higher between 1.8-2.0；5. quality testing：By 1.5% fine jades of carried DNA
Sepharose electrophoresis detection, observation extract the quality of product to carry out subsequent experimental.
5. the screening technique of Anhui's local pig kind weanling pig premunition candidate genetic label according to claim 4, special
Sign is that the process of target fragment single-strand conformation polymorphism is in the step (5)：First by described TLR4-exon3-1, TLR4-
The pcr amplification product of exon3-2 primer pairs carries out native polyacrylamide gel electrophoresis respectively, then carries out silver nitrate respectively
After dyeing the weanling pig TAP1 gene single-strand conformation polymorphisms are obtained with gel imaging system observation electrophoresis result；
Wherein, the native polyacrylamide gel electrophoresis specifically comprises the following steps：
(A) glass plate used in cleaning glue, clip, tightrope, sample comb, dry；
(B) making sheet：Upward by clean and dry bottom plate flour milling, the round end on ground glass side seals glass plate downward, with tightrope
Three faces, clamp；
(C) 10% polyacrylamide gel 72mL, distilled water 42mL, 30% PAGE 24mL, 10 × TBE 6mL are prepared,
50 330 μ L of μ L, AP of TEMED, quick encapsulating after mixing；
(D) glue is filled to the edge of glass plate, is inserted into comb, pay attention to checking in glue whether there is bubble at this time, at room temperature
It places, is used after glue polymerization；
(E) after gel polymerisation is good, 1 × TBE is added into electrophoresis tank, removes lower clip, tightrope and comb, glass plate is fixed on electricity
It swims on slot, irrigation with syringe well is used in combination；
(F) prerunning 30min under 300V voltages；
(G) it takes in 3 μ LPCR amplified productions to 96 hole PCR plates, 7 μ L sample-loading buffers is added, after blowing and beating mixing with rifle, use is transparent
Tape seal；
(H) it is denaturalized 10min for 98 DEG C, then rapid ice bath 10min, then point sample；
(I) 240V electrophoresis 1h, 150V electrophoresis 12h；
Wherein, the cma staining specifically comprises the following steps：
(a) after the completion of electrophoresis, glass plate is removed, it is careful to take out gel and mark, it is rinsed 2 times with distilled water；
(b) fixer is poured into, the fixer is that the distilled water containing 10% volume fraction ethyl alcohol and 0.5% volume fraction acetic acid is molten
Liquid, slight oscillatory 5min recycle fixer；
(c) silver nitrate solution for pouring into 2% mass fraction is protected from light slight oscillatory 15min, recycles silver nitrate solution；
(d) after being rinsed 2 times with distilled water, developing solution is poured into, the developing solution is containing 3% volume fraction NaOH and 0.1% volume
The double steaming solution of score HCHO, oscillation carries out chromogenic reaction, until band is clear；
(e) distilled water rinses, and electrophoresis result is observed in gel imaging system.
6. the screening technique of Anhui's local pig kind weanling pig premunition candidate genetic label according to claim 5, special
Sign is that the process of pcr amplification product bidirectional sequencing is in the step (6)：According to single-strand conformation polymorphism as a result, selecting not
With the sample of banding pattern, each banding pattern selects 3 samples, and 50 μ L systems of PCR amplification are sent two-way to the progress of Shanghai Sheng Gong Co., Ltds
Sequencing, sequencing result DNAStar or DNAMan, the analysis of Chromas softwares.
7. the screening technique of Anhui's local pig kind weanling pig premunition candidate genetic label according to claim 6, special
Sign is that Population Genetics specificity analysis process is in the step (7)：Step (5), (6) are detected using popgene softwares
The sites SNPs arrived carry out Population Genetics analysis, including gene frequency, genotype frequency, homozygosity, heterozygosity, effectively
The calculating of number of alleles, polymorphism information content judges whether the distribution of genotype in group is in Chi-square Test
Hardy-Weinberg balances carry out 2 comptibility tests of χ calculating using popgene softwares.
8. the screening technique of Anhui's local pig kind weanling pig premunition candidate genetic label according to claim 7, special
Sign is, the mathematical analysis model used in the step (9) for：1. the difference analysis of sero-immunity index uses between kind
SPSS19.0 statistical softwares One-way ANOVA programs carry out variance analysis and significance test, and experiment the data obtained is with " flat
Mean ± SD " is indicated；2. the mathematical model that different genotype uses the effect variance analysis of each immune indexes for：Yij=μ+Bi
+Gj+eij；Wherein, YijFor immune indexes phenotypic number；μ is group's mean value；BiIndicate the wean batch effect of weanling pig；GjTable
Show jth kind genotype effects；eijIndicate random error；Using SPSS19.0 statistical softwares GLM programs to detected by this experiment
Hereditary variation and the relevance of sero-immunity index analyzed.
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