CN102296074B - Pig immune trait associated CD169 gene and preparation method and use thereof - Google Patents
Pig immune trait associated CD169 gene and preparation method and use thereof Download PDFInfo
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Abstract
The invention discloses a pig immune trait associated CD169 gene and a preparation method and use thereof. The preparation method comprises: extracting total DNA from the blood of a pig; designing premiers (a forward primer: 5'-GGGCATTCAAAGCCAAACTC-3', and a backward primer: 5'-CCTCGGGTGTAGCCCTCAAA-3') by using pig CD169 sequence (GeneBank registry number: N_010459.2) issued by GenBank as a template; performing polymerase chain reaction (PCR) amplification; purifying the product of PCR amplification, cloning the product of PCR amplification and sequencing the product of PCR amplification; and thus, obtaining the nucleotide sequence represented by SEQ ID No.1, wherein the sequence comprises a mutation locus C305-A305 that can be identified by HinfI endonucleases and therefore the polymorphism of the pig CD169 gene can be detected by a created restriction site-polymerase chain reaction- restriction fragment length polymorphism (CRS-PCR-RFLP) method. The gene is mainly used to mediate the adhesion and endocytosis of porcine reproductive and respiratory syndrome virus (PRRSV). After invading into a body, the PRRSV first bonds with receptor proteins (for example CD169) on the membrane of the cell and then moves into the cell under action of endocytosis with assistance from the CD169. The invention provides a new molecular marker for the marker-assisted selection of pigs.
Description
Technical field
The invention belongs to the domestic animal gene engineering technology field; Be specifically related to the relevant CD169 gene of a kind of pig immune trait; The preparation method who also relates to the relevant CD169 gene of a kind of pig immune trait simultaneously also relates to the purposes of the relevant CD169 gene of a kind of pig immune trait.
Background technology
Since the pig breeding came to light with breathing syndrome virus (PRRSV), most of areas was popular in the whole world for it, had brought enormous economic loss for global pig industry.For the pathogeny of in depth understanding pig breeding and respiratory syndrome and control this disease, investigators need the biological property of research PRRSV acceptor, specifically comprise the acceptor of participating in viral absorption, internalization, dismounting nucleocapsid, the genome dispose procedure.But traditional treatment has a lot of limitation, can't deal with problems effectively, and medicine and objectionable impurities is residual influences animal-derived food product safety and environment, so Animal Genetics and molecule marker ancillary technique more and more comes into one's own.
(Sialoadhesin Sn), has another name called sialoadhesin to CD169, is a kind of I type transmembrane glycoprotein.Be accredited as a kind of sialyl binding domain-immunoglobulin appearance agglutinin receptor; Complex construction with 17 zones; Molecular structure comprises the cell outskirt, strides film district and intracellular region; Wherein extracellular region contains Tegeline V district and C district spline structure (.Sialoadhesin such as Crocker PR; A macrophage sialic acid binding receptor for haemopoietic cells with 17 immunoglobulin-like domains.The EMBO Journal, 1994,13 (19): 4490-4503.).
CD169 has intensive scavenger cell taxis, mainly on tissue macrophages, expresses.After PRRSV invades porcine alveolar macrophage; CD169 mainly plays a part to adhere to and endocytosis; This process need is by sialyl (the .Porcine arterivirus infection of alveolar macrophages is mediated by sialic acid on the virus.Journal of Virology such as Delputte PL of virus surface; 2004,78 (15): 8094-8101.).After Van Breedam etc. have proved that through experiment sialyl and the pSn on the porcine alveolar macrophage (PAM) on the virus envelope reacts, the endocytosis of challenge virus then.And make up the lysate generation immunoprecipitation of soluble pSn and PRRSV, the result shows it is the M/GP5 dimer and the pSn interaction of virus.
Though PRRSV has strict cell tropism, behind the pig sialoadhesin of mammiferous nonpermissive cell surface expression reorganization, PRRSV can adsorb these reconstitution cells effectively.The plasmid transfection that for example will comprise Sn is to nonpermissive cell PK-15; Make Sn express back inoculation PRRSV; Though in reconstitution cell, do not find duplicating of virus nucleocapsid dismounting and nuclear virus particle; But in the positive cell slurry, found a large amount of virus, shown that pSn is the acceptor of mediation PRRSV absorption and endocytosis.Peace is celebrated the experimental result that waits together and is shown, the N end 17-150 amino acid of pSn maybe be relevant with the absorption of virus, and C end cytoplasmic tail possibly be the factor that mediates viral endocytosis.Recently; There is experiment to show; Virus can be adsorbed one and only striden fragment (the .Porcine reproductive and respiratory syndrome virus attachment is mediated by the N-terminal domain of the sialoadhesin receptor.Vet Microbiol such as An TQ that film district and C lack cytoplasmic tail by the direct coupling of N end regions of pSn; 2010,143 (2/4): 371-378.).The sialyl agglutination activity has been brought into play important effect in the combining of PRRSV and acceptor CD169, utilize the R of the method for point mutation with the N end
116Amino acid converts E to
116Behind the amino acid; Though whole proteinic structure does not significantly change; But pSn has lost sialyl agglutinative ability; Cause virus can't adsorb and endocytosis (.Porcine arterivirus attachment to the macrophage-specific receptor sialoadhesin is dependent on the sialic acid-binding activity of the N-terminal immunoglobulin domain of sialoadhesin.J Virol such as Delputte PL; 2007,81 (17): 9546-9550.), show R
116Be CD169 and the viral important site of M/GP5 dimer bonded.In addition, the R of the sialyl calmodulin binding domain CaM of CD169
97Not only can form salt bridge, and can form hydrophobic the contact, work as R with sialic glycerine side chain with sialic glucide
97Be replaced by after the Methionin, influence the hydrophobicity effect between CD169 and the sialyl, hinder the formation of salt bridge, cause significantly reducing with the binding ability of sialyllactose.Similarly, with R
97Convert the ability that L-Ala also can cause CD169 forfeiture bound sialic acid to.
After using the specific antibody MAb 41D3 processing PAM of heparinase and pSn simultaneously; Blocked the adsorption of PRRSV fully to PAM; Explain that Suleparoid and pSn have mediated the adsorption of virus to acceptor; And possibly there is not other the acceptor of participating in absorption PRRSV process in the PAM surface, to such an extent as to or exist but quantity seldom can't detect.
In view of the CD169 gene infects the vital role in the PAM process at PRRSV; Therefore be target gene with acceptor gene CD169; Regulate and control its function, thereby reach the purpose of breeding of effective control pig and respiratory syndrome, and by molecular marker assisted selection; Screening CD169 gene potential and immunologic function related molecular marker are for breeding for disease resistance provides reference.
Summary of the invention
First purpose of the present invention is the CD169 gene that has been to provide a kind of pig immune trait relevant, and this gene significant feature is adhesion and the endocytosis of mediation PRRSV.After PRRSV invades body, at first with cytolemma on receptor protein (for example CD169) combine, under the assistance of CD169, gulp down effect then and get in the cell through bag.
Second purpose of the present invention is the preparation method who has been to provide the relevant CD169 gene of a kind of pig immune trait; This method is through the primer of designs specificity; Amplification comprises the part fragment of the CD169 gene in a mutational site, and confirms the different gene type according to the different endonuclease bamhis that the HinfI-RFLP method obtains.
The 3rd purpose of the present invention is the application of CD169 gene in the pig molecule mark assisted Selection that has been to provide a kind of pig immune trait relevant.
In order to realize above-mentioned purpose, the present invention adopts following technical scheme:
The preparation method of the CD169 gene that a kind of pig immune trait is relevant the steps include:
1. design of primers:
Utilize primer-design software Primer 5.0 (Premier company; Canada); With pig CD169 gene (GeneBank accession number: NC_010459.2) be the amplimer that stencil design goes out to comprise partial sequence; Primer is synthetic by Shanghai biotechnology Services Co., Ltd, and the right dna sequence dna of primer is following:
Forward primer: 5 '-GGGCATTCAAAGCCAAACTC-3 ' (SEQ ID NO:2);
Reverse primer: 5 '-CCTCGGGTGTAGCCCTCAAA-3 ' (SEQ IDNO:3).
2.PCR the purifying of product and order-checking:
Pcr amplification reaction system 20ul comprises the DNA of 500ng, 2.0ul 10 * PCR buffer, 0.6ul 10mM dNTP, 0.8ul of each primer (10uM) and 1 U Ampli Taq DNA polymerase (worker's biotechnology company is given birth in Shanghai).The PCR reaction conditions is: the response procedures of PCR is: 95 ℃ of 5min; 94 ℃ of 40s, 58 ℃ of 30s, 72 ℃ of 30s, 34 circulations; 72 ℃ are extended 10min.Amplified production carries out the electrophoresis detection (see figure 2) with 1.5% sepharose.
Reclaim the PCR product of 5 pig varieties (length is white, and Da Bai is peaceful, Jinhua, Xiao Mei mountain), each kind is selected 3 samples, mixes order-checking.Order-checking product and pig CD169 gene (GeneBank accession number: NC_010459.2) compare, successfully obtain the fragment (seeing sequence table SEQ ID NO:1) of pig CD169 Gene Partial sequence 615bp.Sequencer map occurs obviously bimodal, and the mutational site is C305-A305 (seeing sequence table SEQ ID NO:1).
3.CRS-PCR-RFLP method detection molecules mark C305-A305:
With primer sequence (seeing sequence table SEQ ID NO:2 and SEQ IDNO:3) amplification CD169 gene, obtain the fragment (seeing sequence table SEQ ID NO:1) of 615bp, differentiate CC, CA, three kinds of genotype of AA with restriction enzyme HinfI.
On the NCBI website, find 615bp fragment (seeing sequence table SEQ ID NO:1) corresponding amino acid sequence with CD169; With online software analysis (http://smart.embl-heidelberg.de/); This sequence belongs to the proteic Igc2 of CD169 zone; Comprise fibroblast growth factor acceptor, vascular endothelial growth factor receptor, interleukin 6 acceptor and nerve cell adhesion molecule; And these cytokine binding sites play an important role in immunoregulation effect, show that this zone and immunologic function are closely related.
The application of CD169 gene in the pig molecule mark assisted Selection that a kind of pig immune trait is relevant; DNA with pig is a template; Utilize the part fragment of primer (seeing sequence table SEQ IDNO:2 and SEQ IDNO:3) amplification CD169, obtain the PCR product (SEQID NO:1) of 615bp, detect the polymorphum of C305-A305 with the PCR-HinfI-RFLP method; Identify CC, CA, three kinds of genotype of AA; Related through three kinds of genotypic genotype frequencies of statistical study and morbidity relative risk filters out the AA genotype and possibly be and Ia molecule marker, for the pig molecule mark assisted Selection provides reference.
The present invention compared with prior art has the following advantages and effect:
1. molecule marker is to be the basis with the dna polymorphism, and performance is stable, and directly with the dna form performance, inorganization organ, developmental stage specificity are not influenced by envrionment conditions, interaction of genes polymorphum;
2. allelic variation is present in occurring in nature, need not the special artificial particular inheritance material of creating, and this has created condition for sample collection;
3. marker assisted selection can shorten the generation interval, reduces the required time of cultivating improved seeds;
4. simple to operate, required plant and instrument is less, and cost is low, and common laboratory all can be carried out;
5. sequencer map is directly perceived, can screen the mutational site exactly;
6. agarose gel electrophoresis gets final product the sldh gene type, simple and fast, and picture is directly perceived, accurate.
Description of drawings
Fig. 1 is preparing method's schema of the relevant CD169 gene of a kind of pig immune trait.
Fig. 2 is the electrophoretogram of a boar CD169 Gene Partial sequence amplification.
Clip size is 615bp (agarose gel concentration is 1.5%).Among the figure: the Marker swimming lane is a dna molecular amount standard.
Fig. 3 is the C305-A305 mutational site of finding after the boar CD169 gene sequencing.
Fig. 4 for a kind of be three kinds of genotype (CC, CA and AA) electrophoretogram of pig CD169 gene PCR-HinfI-RFLP among the present invention.
Embodiment
Embodiment 1:
The preparation method of the CD169 gene that a kind of pig immune trait is relevant the steps include:
According to pig CD169 gene (the GeneBank accession number: NC_010459.2) sequences Design primer, from the blood of pig, extract DNA as template, amplification PCR reclaims, purifying, order-checking.Search the mutational site,, and analyze 3 kinds of genotypic morbidity relative risks, screening and Ia molecule marker (Fig. 1) with the method sldh gene type of CRS-PCR-RFLP.
1. design of primers
Utilize primer-design software Primer 5.0 (Premier company; Canada); With pig CD169 gene (GeneBank accession number: NC_010459.2) be the amplimer that stencil design goes out to comprise partial sequence; Be used for the partial sequence of amplification " DLY " (by external kind " Du Luoke ", " long white " and " Da Bai " three mixing breeds obtain, and are the commercial pigs that a kind of China generally applies) CD169 gene.Primer is synthetic by Shanghai biotechnology Services Co., Ltd, and the right dna sequence dna of primer is following:
Forward primer: 5 '-GGGCATTCAAAGCCAAACTC-3 ',
Reverse primer: 5 '-CCTCGGGTGTAGCCCTCAAA-3 '.
2.PCR the purifying of product and order-checking:
(1) pcr amplification:
From pig variety blood, extract total DNA, with this DNA as template.Pcr amplification reaction system 20ul comprises the DNA of 500ng, 2.0ul10 * PCR buffer, 0.6ul 10mM dNTP, 0.8ul of each primer (10uM) and 1 U Ampli Taq DNA polymerase (worker's biotechnology company is given birth in Shanghai).The PCR reaction conditions is: the response procedures of PCR is: 95 ℃ of 5min; 94 ℃ of 40s, 58 ℃ of 30s, 72 ℃ of 30s, 34 circulations; 72 ℃ are extended 10min.Amplified production carries out the electrophoresis detection (see figure 2) with 1.5% sepharose.
(2) purifying of PCR product and order-checking:
DNA with 5 pig varieties (length is white, and Da Bai is peaceful, Jinhua, Xiao Mei mountain) is a template, and random choose 3 individuals are used for pcr amplification in each kind, reclaim 5 kind pcr amplification products of pig, mix and check order.The miniprep dna fragment purification test kit that utilizes sky, Beijing root biotech company to produce carries out purifying and recovering, and concrete grammar is with reference to the process specifications of this test kit.Sequencing is given birth to worker's biotechnology company by Shanghai and is accomplished.
(3) the dna sequence dna homology search is identified:
Through NCBI-BLAST (Basic Local Alignment Search Tool) instrument; With the reference sequences (accession number of announcing in sequence that obtains after the order-checking and the GenBank DB: NC_010459.2) compare; Its sequence is the nucleotide sequence shown in the SEQ ID NO:1, and the result shows that successfully the clone obtains the fragment (seeing sequence table SEQ ID NO:1) of pig CD169 Gene Partial sequence 615bp.
(4) screening in mutational site in the dna sequence dna:
Through NCBI-BLAST (Basic Local Alignment Search Tool) instrument; (accession number: NC_010459.2) compare, (length is white, Da Bai with 5 pig varieties with the sequence of announcing in sequence that obtains after the order-checking and the GenBank DB; Peaceful; Jinhua, Xiao Mei mountain) mixing order-checking peak figure imports SeqMan software (DNASTAR company, the U.S.) and compares; It is significantly bimodal that the result is presented at 1 site appearance of pig CD169 Gene Partial sequence, and this this gene of explanation in this site the base mutation (see figure 3) has taken place.This mutational site is: C305-A305 (seeing sequence table SEQ ID NO:1).
The foundation of CRS-PCR-RFLP genotype detection method:
1. design of primers:
Designed a pair of primer to polymorphic site C305-A305 point, be used for genotype detection, its nucleotide sequence sequence is (identical with the sequence shown in sequence table SEQ ID NO:2 and the SEQ ID NO:3) as follows respectively:
Forward primer: 5 '-GGGCATTCAAAGCCAAACTC-3 ' (seeing sequence table SEQ ID NO:2)
Reverse primer: 5 '-CCTCGGGTGTAGCCCTCAAA-3 ' (seeing sequence table SEQ ID NO:3)
2.PCR amplification:
Add dna profiling 1 μ L in the reaction system of 20uL, distilled water 14.3 μ L, 10 * PCR buffer, 2.0 μ L, 10mMdNTP 0.6 μ L, each 0.8 μ L of 10uM primer, Taq enzyme 1U.The PCR reaction conditions is: behind 94 ℃ of preparatory sex change 5min, and 94 ℃ of sex change 40s, 58 ℃ of annealing 30s, 72 ℃ of extension 30s, 34 circulations, last 72 ℃ are extended 10min.3 μ L PCR products detect (see figure 3) through 1.5% agarose gel electrophoresis, and positive is used for next step enzyme and cuts detection.
3.RFLP detect:
HinfI-RFLP detection molecules mark C305-A305
With PCR product 6 μ L, 10 * Buffer, 1 μ L, restriction enzyme HinfI is 4U, adds distilled water and mends to 10 μ L, and with centrifugal behind the sample mixing, 37 ℃ of incubators are placed 12h, and enzyme is cut product and is detected somatotype with 1.5% agarose gel electrophoresis.
Molecular marker gene type analysis: as shown in Figure 4; Represent 3 kinds of different gene types of C305-A305 place SNP (SNP) (genotype is represented CC, AA, CA with its mutating alkali yl) respectively; Electrophoresis result shows that the CC genotype (has 4 bands; Length is 471bp, 92bp, 36bp, 17bp), CA genotype (6 bands are arranged, and length is 471bp, 268bp, 202bp, 92bp, 36bp and 17bp) and AA genotype (5 bands are arranged, and length is 268bp/202bp/92bp/36bp/17bp); Wherein 36bp and 17bp can't show with agarose gel because fragment is too little.
Embodiment 2:
The application of CD169 molecule marker classifying method in the immune character association analysis:
Test has detected 256 polymorphums that " DLY " pig is individual altogether, comprises 215 of morbidity pigs, 41 of health pig.Confirm its genotype with the CRS-PCR-RFLP method, and carry out the association analysis of gene mutation site and porcine reproductive and respiratory syndrome.Use the Logistic analysis of regression model method in SAS software (match bodyguard software (Beijing) ltd, Beijing), (full name of OR value is odd ratio to set a genotypic OR value; Claim odds ratio again; For the very low disease of sickness rate, its OR value promptly is the accurate estimated value of relative risk) be reference value (OR=1), calculate other 2 kinds of genotypic OR values; If the OR value is greater than 1; And the P value is less than 0.05, and then this genotype possibly be a risk factor, promptly carries this easy infected pigs of genotypic individuality reproductive and respiratory syndrome in the swinery; Otherwise, if the OR value less than 1, and the P value is less than 0.05, then this genotype possibly be the protection factor, promptly carrying this genotypic individuality in the swinery has resistibility to porcine reproductive and respiratory syndrome.95%CI representes 95% fiducial interval (the Andrew M.Smith MD etc. of OR value; Environmental Tobacco Smoke and Interleukin 4 Polymorphism (C-589T) Gene:Environment Interaction Increases Risk of Wheezing in African-American Infants.The Journal of Pediatrics; 2008,152 (5): 709-715; Yen-Li Lo etc., ATM polymorphisms and risk of lung cancer among never smokers.LungCancer, 2010,69:148-154.).
(1) the genotype detection result is illustrated in 256 individuals, and the CC genotype has 85 individuals, and the CA genotype has 127 individuals, and the AA genotype has 44 individuals.Allele C and the distribution situation of allelotrope A in each colony are seen table 1, can find out that through observing C allelotrope is advantage allelotrope in " DLY ".
The SNP of table 1CD169 gene is genotype frequency and gene frequency distribution in colony
(2) with CD169 gene C 305-A305 (HinfI-RFLP) variant sites genotype frequency and gene frequency and related with the morbidity relative risk
Use CRS-PCR-HinfI-RFLP detection molecules mark C305-A305 genotype and adopt three kinds of genotype of Logistic regression analysis analyzing gene CD169 the morbidity relative risk in back health pig, two swinerys of morbidity pig to take place in disease.The genotypic morbidity relative risk of AA possibly be genotypic 3.167 times of CC (OR=3.167, P=0.0183), significant difference.Concrete outcome is seen table 2.The result shows, the AA genotype possibly be and an Ia molecule marker, for the molecular marker assisted selection of pig provides a reference.
Table 2 pig CD169 gene HinfI variant sites genotype frequency and gene frequency and with morbidity relative risk association analysis
Annotate: P＜0.05 expression significant difference.
SEQUENCE?LISTING
?
<110>Hua Zhong Agriculture University
<120>CD169 gene and preparation method and application that a kind of pig immune trait is relevant
<130>CD169 gene and preparation method and application that a kind of pig immune trait is relevant
<160> 3
<170> PatentIn?version?3.1
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<211> 615
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gggcattcaa?agccaaactc?tcctgatcat?tgagagattc?gcagagctga?tgtaatgaag 60
ctcagacctc?cctacaattg?tatggggaga?ggggaaccct?ggcgggcttc?ctgcagtggg 120
gggcatggaa?ccgcggatgt?gtgggggagc?tgagcctccc?tctctccctc?tcctgccagc 180
agcccgcctc?ctcatcagcc?cagcagcaga?ggtggtggaa?gggcaggcgg?tgacactgag 240
ctgcaggagc?agcctgagcc?tgatgcctga?cacccgtttt?tcctggtacc?ggaacggggc 300
cctgcttctc?gagggcccag?cagcagcctc?ctgctcccag?cagcctccag?cacagatgcc 360
ggctcatacc?actgccgggc?ccagagcagc?cacagcgcca?gtgggccctc?ctcacctgct 420
gttctcaccg?tgctctgtga?gtagccggcc?tgccagccgt?ggctgctagg?gggagggtca 480
gctcccacct?cctccaggcc?ctcctgactc?tgccctgtct?gggagtcttg?ataaggaagg 540
cagcccaggc?cacccagagc?tgaaatttgt?tttgttttgt?tttgttttgc?ttttttttga 600
gggctacacc?cgagg 615
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Claims (2)
1. molecule marker relevant with pig immunity, it is characterized in that: molecule marker C305-A305 is a template with pig CD169 gene, and primer SEQ ID NO:2 and SEQ ID NO:3 amplification obtain, and its sequence is shown in the SEQ ID NO.1, and size is 615bp.
2. the application of the described molecule marker of claim 1 in the pig molecule mark assisted Selection.
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| CN103320426A (en) * | 2012-03-19 | 2013-09-25 | 华中农业大学 | Angiogenin gene as molecular marker of swine immune trait |
| CN106399531B (en) * | 2016-10-17 | 2019-07-09 | 江苏省农业科学院 | One influences molecular labeling, detection method and its application of pig growth and PRRSV resistance |
| CN110452928A (en) * | 2019-08-13 | 2019-11-15 | 成都天邦生物制品有限公司 | The building and application of one plant of PK-15 stable cell line |
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| CN101235419A (en) * | 2008-01-25 | 2008-08-06 | 华中农业大学 | Clone and application of swine mark auxiliary selection molecule mark RNASE6 correlated with immunity |
| CN101503688A (en) * | 2009-03-18 | 2009-08-12 | 华中农业大学 | CD163 gene fragment clone related to pig immune trait and use thereof as molecular marker |
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| CN101235419A (en) * | 2008-01-25 | 2008-08-06 | 华中农业大学 | Clone and application of swine mark auxiliary selection molecule mark RNASE6 correlated with immunity |
| CN101503688A (en) * | 2009-03-18 | 2009-08-12 | 华中农业大学 | CD163 gene fragment clone related to pig immune trait and use thereof as molecular marker |
Non-Patent Citations (3)
| Title |
|---|
| 兰旅涛等.分子标记技术在现代猪育种中的应用现状与前景.《江西农业学报》.2004,第16卷(第2期),46-50. |
| 分子标记技术在现代猪育种中的应用现状与前景;兰旅涛等;《江西农业学报》;20041231;第16卷(第2期);46-50 * |
| 庞卫军等.猪H-FABP基因PCR-RFLP分子标记研究.《西北农林科技大学学报(自然科学版)》.2004,第32卷(第7期),11-15. * |
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