CN104046620B - SNP marker that in leukocyte differentiation antigen CD34 gene, reproductive and respiratory syndrome anti-to pig is relevant and application thereof - Google Patents
SNP marker that in leukocyte differentiation antigen CD34 gene, reproductive and respiratory syndrome anti-to pig is relevant and application thereof Download PDFInfo
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- CN104046620B CN104046620B CN201310081967.2A CN201310081967A CN104046620B CN 104046620 B CN104046620 B CN 104046620B CN 201310081967 A CN201310081967 A CN 201310081967A CN 104046620 B CN104046620 B CN 104046620B
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Abstract
The present invention relates to SNP marker and application thereof that in leukocyte differentiation antigen CD34 gene, reproductive and respiratory syndrome anti-to pig is relevant, the SNP marker that described reproductive and respiratory syndrome anti-to pig is relevant is positioned on the leukocyte differentiation antigen CD34 gene of pig, the nucleotide sequence of described SNP marker is as shown in SEQ ID NO.1, wherein, at 256bp and 260bp two of this sequence base be the pig to be measured of A be susceptible reproductive and respiratory syndrome pig variety, and this at two base be the pig to be measured of G be anti-reproductive and respiratory syndrome pig variety.The molecular genetic marker that the present invention provides is not limited by age of pig, sex etc., can be used for the early stage selection-breeding of anti-reproductive and respiratory syndrome pig variety, even just can screen exactly when pig is just born, can greatly speed up the breeding process of anti-reproductive and respiratory syndrome pig；Screening technique is accurate, simple to operate, low cost, and efficiency is high.
Description
Technical field
The present invention relates to genetic engineering and biology field, specifically, relate to leukocyte
SNP marker that in differentiation antigen CD34 gene, reproductive and respiratory syndrome anti-to pig is relevant and application thereof.
Background technology
Porcine reproductive and respiratory syndrome (PRRS), being commonly called as " reproductive and respiratory syndrome " is to be bred by pig and exhale
Inhaling what syndrome virus (PRRSV) caused, this virus is nido virales Arteriviridae
A member, the virus belonged to together also has equine arteritis virus, molluscum contagiosum acidohydrogenase enzymophathy poison and
SHF virus.This disease mainly causes breeding and the respiratory symptom of pig, shows as chromic fibrous lung
Inflammation and Sow abortion mummy tire etc., cause huge economy to global pig industry every year
Loss.Within 2007, having broken out a hyperpyrexia disease in China, this disease spreads in the whole country rapidly,
Infected more than 2,000,000 pigs, 400,000 pig death, this disease finally confirms it is by a kind of high cause
The reproductive and respiratory syndrome poison of characteristic of disease causes.The pulmonary alveolar macrophage of PRRSV main infection pig, grinds now
Study carefully and show that in innate immunity, PRRSV infection can not cause effective I type after infecting PRRSV
Interferon response, humoral immunization can not produce effective neutralizing antibody in time, and can be formed anti-
The viral enhancement effect that body relies on, ultimately results in immunosuppressant and persistent infection, due to this virus
Mutation rate high, traditional vaccine method can not effectively control this disease, and a lot of researchs are all endeavoured
In finding RNAi, the new method such as interferon is come for this virus, is used for making up traditional method
Deficiency.
Due to different pig varieties and individual genetic background difference, to the resistance of reproductive and respiratory syndrome the most not
With, research shows in clinical symptoms and physiological and biochemical index, and Tongcheng pig, prunus mume (sieb.) sieb.et zucc. mountain pig etc. are domestic
Pig variety has stronger after infecting highly pathogenic PRRSV compared to external Landrace, Large White etc.
Resistance.By the method for high-flux sequence, to infecting Landrace and Tongcheng pig before and after PRRSV
Tissue do differential genes expression analysis, some Ia gene expression amount before infection after six
Significant difference is had between individual kind.Additionally, there are some interracial SNP site, these bases
Because serving critically important effect in the immunne response of host, and these SNP are positioned at gene
Exon in, by finding out the gene expression difference that PRRSV infection between different cultivars causes,
It is possible not only to study the infection mechanism of PRRSV, it is also possible to find out resistance or susceptible relevant base
Cause, by interracial SNP compares analysis, thus filters out the pig of better resistance.
Summary of the invention
It is an object of the invention to provide reproductive and respiratory syndrome anti-with pig in leukocyte differentiation antigen CD34 gene
Relevant SNP marker and application thereof.
CD34 molecule is a kind of cluster of differentiation, and adhesion molecule rises in hematopoiesis in early days
Important function, plays an important role in T cell enters adenoid transition process, but sometimes
Stop the adhesion of mastocyte.By the analysis of bioinformatics, result shows two differences
The pig variety of resistance: there are 6 SNP between Landrace (susceptible pig), Tongcheng pig (disease-resistant pig)
Site, in susceptible pig, 6 SNP bases are respectively as follows: A, A, C, A, G, A and A,
And 6 bases in resistance pig are respectively as follows: G, G, T, C, A, T and G, the present invention
People have chosen 2 SNP site, detects the base in these 2 sites by conventional molecular biological method
Type, can be as a kind of method detecting disease-resistant and susceptible pig.
In order to realize the object of the invention, the present invention provides the SNP that a kind of reproductive and respiratory syndrome anti-to pig is relevant
Labelling, it is positioned on the leukocyte differentiation antigen CD34 gene of pig, the core of described SNP marker
Nucleotide sequence is as shown in SEQ ID NO.1, wherein, and alkali at 256bp and 260bp two of this sequence
Base be the pig to be measured of A be susceptible reproductive and respiratory syndrome pig variety, and this at two base be that the pig to be measured of G is
Anti-reproductive and respiratory syndrome pig variety.
The present invention also provides for the primer pair for detecting the relevant SNP marker of reproductive and respiratory syndrome anti-to pig,
It includes forward primer F:5 '-ACAGCTATCACCGGGTCAAC-3 ' and downstream primer
R:5'-AGCTCTGGTGGCTCCTAACA-3'.
The present invention also provides for the relevant SNP marker of reproductive and respiratory syndrome anti-to pig and is identifying anti-reproductive and respiratory syndrome pig
Application in kind, it includes step: 1) extract the total serum IgE in pig sample tissue to be measured,
Reverse transcription becomes cDNA；2) with the cDNA in step 1) as template, F and R is primer, enters
Row RT-PCR reacts；3) PCR primer is analyzed, if 256bp and 260bp in amplified production sequence
At two, base is G, and pig the most to be measured is anti-reproductive and respiratory syndrome pig variety.
The step of reverse transcriptional PCR is:
I) RNA secondary structure is opened
The little centrifuge tube that a nuclease free is polluted adds:
RNA 2μg
oligodT 1μg
Nuclease free water polishing is to 15 μ l.
Heating centrifuge tube to 70 DEG C, 5 minutes.
Ii) reverse transcriptional PCR
Add in above-mentioned centrifuge tube:
The reaction condition of reverse transcriptional PCR is: 42 DEG C, 60min.
The amplification system that Standard PCR uses is calculated as with 15 μ l:
The reaction condition of Standard PCR is:: 95 DEG C of 5min；With 95 DEG C of 30s, 50 DEG C-70 DEG C,
30s-2min；72 DEG C of 30s-2min do 35 circulations；72 DEG C of 10min, in 4 DEG C of insulations.
The present invention also provides for the reagent for detecting anti-reproductive and respiratory syndrome pig variety containing primers F and R
Box.Described test kit also includes dNTPs, Taq archaeal dna polymerase, Mg2+, PCR reaction slow
Rush one or more in liquid.Preferably, described test kit also includes standard positive template.
The present invention further provides reproductive and respiratory syndrome anti-to the pig relevant SNP marker molecule mark pig
Application in note assistant breeding.
The invention provides a kind of based on blue ear anti-with pig in leukocyte differentiation antigen CD34 gene
The method that sick relevant SNP marker screens anti-reproductive and respiratory syndrome pig, the advantage of the method is: (one)
The molecular genetic marker that the present invention provides is not limited by age of pig, sex etc., can be used for anti-indigo plant
The early stage selection-breeding of otopathy pig variety, even just can screen exactly when pig is just born, can
Greatly speed up the breeding process of anti-reproductive and respiratory syndrome pig；(2) screening technique is accurate, simple to operate,
Low cost, efficiency is high.
Accompanying drawing explanation
Fig. 1 carries out, by the embodiment of the present invention 1, the amplification program that Standard PCR is used.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.If not
Specializing, embodiment is all according to conventional laboratory conditions, and such as Sambrook equimolecular, clone is real
Test handbook (New York:Gold Spring Harbor Laboratory Press, 1989), or press
Condition according to manufacturer's description suggestion.
The SNP that in embodiment 1 leukocyte differentiation antigen CD34 gene, reproductive and respiratory syndrome anti-to pig is relevant
The method of label screening anti-reproductive and respiratory syndrome pig
Previous experiments is by (Landrace, Large White, duroc, peaceful to 6 kind pigs
Pig, prunus mume (sieb.) sieb.et zucc. mountain pig, Tongcheng pig) infect high-pathogenicity blue ear disease poison (PRRSV) carry out disease-resistant pig
Screening, by clinical symptoms record: feed intake, body temperature, Physiology and biochemistry, cytokine with
And the detection of time-to-live etc., finally obtain six product bigger to reproductive and respiratory syndrome Resistant Difference
Kind: Landrace (susceptible pig), Tongcheng pig (disease-resistant pig).In order to study high-pathogenicity blue ear disease
Infection mechanism and screening resistant gene, later experiments have chosen the 35 disease-resistant pig of age in days and susceptible pigs
Each 16 carry out challenge test, carry out the Pulmonis Sus domestica transcript profile of different time points after infecting PRRSV
High flux solexa order-checking, by the method for molecular bioinformatics, finds to be correlated with in immunity
Leukocyte differentiation antigen CD34 gene (GenBank accession number is NM_214086.1) show outward
Son exists 2 interracial SNP site, the monokaryon glycosides in these 2 sites in resistance pig variety
Acid is guanine (G) respectively, and in susceptible pig, the mononucleotide in these 2 sites is respectively
Adenine (A), by designing primer amplification purpose fragment near these 2 SNP site, surveys
Sequence detection SNP base type, available same result, the primer sequence of amplified fragments is as follows:
Forward primer F:5 '-ACAGCTATCACCGGGTCAAC-3 ' and downstream primer R:
5'-AGCTCTGGTGGCTCCTAACA-3'.With this, primer is detected pig to be measured these six
The base type of SNP site, i.e. can be used to screen disease-resistant pig.
One, the extraction of total serum IgE
1. animal tissue: take fresh or-70 DEG C of frozen animal tissues, every 30-50mg tissue adds
Entering 1ml Trizol, Syrup-homogenizing instrument carries out homogenized.Sample volume does not typically exceed Trizol body
Long-pending 10%.
2. above sample is placed 5 minutes at 15-30 DEG C so that nucleic acid-protein complex divides completely
From.
3. optional step: 4 DEG C 12, centrifugal 10 minutes of 000rpm (about 13,400g), take supernatant.
Note: if containing relatively polyprotein, fat, polysaccharide or muscle, plant tuberosity in sample
Parts etc., can centrifugal segregation.
4. in above solution, add chloroform, often use 1ml Trizol to add 0.2ml chloroform, lid
Good lid, acutely vibration 15 seconds, room temperature is placed 3 minutes.
5.4 DEG C 12, centrifugal 10-15 minute of 000rpm (about 13,400g), now sample is divided into three layers:
The aqueous phase that the organic facies of yellow, intermediate layer and upper strata are colourless, RNA is mainly in aqueous phase, water
Phase (about 500 μ l) is transferred in a new RNase-Free centrifuge tube.
6. adding equal-volume isopropanol, mixing in the aqueous phase solution obtained, room temperature places 20-30
Minute.
7.4 DEG C 12, centrifugal 10 minutes of 000rpm (about 13,400g), abandons supernatant.
8. add 75% ethanol (preparing with RNase-free water) washing precipitation.Often use 1ml
Precipitation is washed by Trizol 1ml75% ethanol.
9.4 DEG C of 5,000rpm (about 2,300g) are centrifuged 3 minutes.By rifle head careful sucking-off upper liquid
Body, retains precipitation.
Note: not sucking-off precipitation, remaining a small amount of liquid can of short duration be centrifuged, and then uses rifle head
Sucking-off, is careful not to suction and abandons precipitation.
10. room temperature is placed 2-3 minute, dries, and adds 30-100 μ l RNase-free water, fully
Dissolve RNA.Obtained RNA puts-70 DEG C of preservations.
Two, reverse transcriptional PCR
1. open RNA secondary structure
The little centrifuge tube that a nuclease free is polluted adds:
RNA 2μg
oligodT 1μg
Nuclease free water polishing is to 15 μ l.
Heating centrifuge tube to 70 DEG C, 5 minutes.
2. reverse transcriptional PCR
Add in above-mentioned centrifuge tube:
Hatch 60 minutes at 42 DEG C and react.
Three, Standard PCR reaction
1. it is loaded by following reaction system (15 μ L system)
2. carry out PCR amplification by reaction condition as described in Figure 1
Four, clone and check order
1. with E.Z.N.A Gel Extraction Kit glue, PCR primer is reclaimed test kit to reclaim
2. the product reclaimed is connected pMD-19T carrier (Takara), convert escherichia coli, obtain
Obtain positive colony, shake bacterium, order-checking (the raw work in Shanghai).Enzyme action, connection, conversion operation are detailed
Step is shown in " molecular cloning " third edition.
Five, interpretation of result
Sequencing result is as shown in SEQ ID NO.1, wherein, 256bp and 260bp two of this sequence
Place's base be the pig to be measured of A be susceptible reproductive and respiratory syndrome pig variety, and this at two base be the to be measured of G
Pig is anti-reproductive and respiratory syndrome pig variety.
Although, the most with a general description of the specific embodiments the present invention has been made in detail
Most description, but on the basis of the present invention, it can be made some modifications or improvements, this is to this
It is apparent from for skilled person.Therefore, on the basis without departing from spirit of the present invention
Upper these modifications or improvements, belong to the scope of protection of present invention.
Claims (5)
1. the SNP marker that a reproductive and respiratory syndrome anti-to pig is relevant, it is characterised in that it is positioned at pig
Leukocyte differentiation antigen CD34 on, the nucleotide sequence of described SNP marker such as SEQ ID
Shown in NO.1, wherein, at 256bp and 260bp two of this sequence base be the pig to be measured of A be easy
Sense reproductive and respiratory syndrome pig variety, and this at two base be the pig to be measured of G be anti-reproductive and respiratory syndrome pig variety.
2. it is used for the primer of the SNP marker that test right requires that reproductive and respiratory syndrome anti-to pig described in 1 is relevant
Right, it is characterised in that to include forward primer F:
5 '-ACAGCTATCACCGGGTCAAC-3 ' and downstream primer R:
5'-AGCTCTGGTGGCTCCTAACA-3'。
3. contain the reagent to being used for detecting anti-reproductive and respiratory syndrome pig variety of the primer described in claim 2
Box.
Test kit the most according to claim 3, it is characterised in that described test kit also wraps
Include dNTPs, Taq archaeal dna polymerase, Mg2+, one or more in PCR reaction buffer.
5. according to the test kit described in claim 3 or 4, it is characterised in that described test kit
Also include standard positive template.
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| CN201310081967.2A CN104046620B (en) | 2013-03-14 | 2013-03-14 | SNP marker that in leukocyte differentiation antigen CD34 gene, reproductive and respiratory syndrome anti-to pig is relevant and application thereof |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2010088633A2 (en) * | 2009-02-02 | 2010-08-05 | Chromocell Corporation | Novel cell lines and methods |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010088633A2 (en) * | 2009-02-02 | 2010-08-05 | Chromocell Corporation | Novel cell lines and methods |
Non-Patent Citations (2)
| Title |
|---|
| PRRS 病毒感染对猪细胞因子IL-2、IL-4 和IL-10mRNA 转录的影响;郑洁等;《畜牧兽医学报》;20031231;483-489 * |
| 重组合细胞介素一提高PRRS抗体阳性猪的猪瘟疫苗免度效果试验;李祥瑞等;《畜牧与兽医》;20021231;9-11 * |
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