CN101503688A - CD163 gene fragment clone related to pig immune trait and use thereof as molecular marker - Google Patents

Abstract

The invention belongs to the technical field of preparing livestock molecular markers, and in particular relates to preparation and application of a molecular marker applied as porcine marker for assisted selection and relevant to porcine immunity. The molecular marker is obtained by cloning a porcine CD163 gene, and a mRNA sequence of the molecular marker is shown in a sequence table SEQ ID NO:1. A 2439bp locus of the sequence table SEQ ID NO:1 has an A-to-G base mutation; a 2454bp locus of the sequence has a G-to-C base mutation; a 2537bp locus of the sequence has a G-to-A base mutation; and a 2685bp locus of the sequence has an A-to-C base mutation. The invention also discloses a primer for amplifying partial coding regions of the CD163 gene and a method for detecting a 2537bp G-to-A molecular marker and a 2685bp A-to-C molecular marker. The invention provides two novel molecular markers for porcine marker-assisted selection.

Description

The CD163 gene fragment clone relevant and as the application of molecule marker with pig immune trait

Technical field

The invention belongs to domestic animal molecule marker (genetic marker) preparing technical field, relate in particular to a kind of clone and application of the molecule marker of using as the pig marker assisted selection relevant with pig immune trait.

Background technology

Porcine reproductive and respiratory syndrome PRRS (Porcine Reproductive and Respiratory Syndrome, claim " pig blue-ear disease " again because of suffering from cyanosis change indigo plants such as pig ear) cause by porcine reproductive and respiratory syndrome virus (PRRSV) infection, be one of most important disease of puzzlement Swine Production, in worldwide, caused serious economy loss.In recent years, owing to seriously break out in the world wides such as PRRS, bird flu and foot and mouth disease, residual animal-derived food product safety and the environment of influencing of medicine and objectionable impurities thereof, animal disease resistant genetic breeding more and more come into one's own, and have become the important goal of improvement of breed.And, make and utilize molecule breeding for disease resistance technology effectively to cultivate disease-resistant varieties fast to become possibility, open up a new way for improving animal disease resistant power along with the development of molecular biology and information biology.Molecular marker assisted selection is at present effective breeding for disease resistance technology, and transgenic technology is cultivated disease-resistant varieties and will be with a wide range of applications.

Great mass of data has shown that the heredity difference of pig is relevant with the existence of pathogenic agent, and as atrophic rhinitis, respiratory system disease and intestinal tract disease etc., breed difference is in the news with the related of sickness rate with other inheritable variations.Research at present think the host the infection of PRRS virus and PRRS have important effect in the groove, the pig of different varieties (being) shows notable difference to PRRSV immune response, opposing or tolerance.Mainly concentrate on the screening of resistance marker for the research of host's disease resistance, the research of reaction, effect path and the relevant cell factor of innate immunity behind virus infection.

Isolation identification has gone out Suleparoid (Hs, Heparan sulfate) from the PAM of the main target cell of PRRSV, (Sn is sialoadhesin) with three PRRSV acceptor genes of CD163 for sialoadhesin.CD163 may work to assist undress shell and geneome RNA is discharged in the tenuigenin of endocytosis, virus, is the principal recipient of PRRSV.Found for the first time the CD163 molecule in 1987, but people to it understanding but seldom, found just up in recent years that it was a kind of special scavenger receptor protein (scavenger receptor (SR) protein), the identification that scavenger receptor protein participates in various parts by a large amount of cell surface parts and soluble glycoprotein, comprise protein, polynucleotide, polysaccharide and lipid.CD163 is rich in halfcystine, belongs to scavenger receptor family (SRCR, scavenger receptor cysteine-rich).With the position and the number of cysteine residues, the molecule that comprises the SRCR zone is divided two classes: category-A contains 6 cysteine residues, and category-B contains 8 cysteine residues.CD163 is an I strand transmembrane glycoprotein molecule, is made up of outer 9 the category-B SRCR structural domains of born of the same parents, a transmembrane segment and short and small cytoplasmic tail three parts.CD163 regulate to remove oxyphorase by identification Hb-Hp complex body (haptoglobin-haemoglobin complexes), thereby prevents the inflammatory reaction that caused by excessive free protoheme.The CD163 molecule also can pass to by signal in the direct inducing cell on the other hand, regulates the expression-secretion of cell anti-inflammatory factors.Studies show that in a large number the CD163 molecule plays important effect in the immune response of mononuclear macrophage group mediation.CD163 is found the 3rd acceptor as PRRSV, be because in the non-sensitive cell of PRRSV, have only the expression of preceding two acceptor molecules, can mediate virus absorption and endocytosis, infect but can't finish the virus particle shelling and produce effectively, play an important role so CD163 enters cell and causes in the course of infection at PRRSV.

Have now and studies show that the proterties related SNP mark overwhelming majority is in the functional gene dna sequence dna, some researchs filter out more disease resistances relevant with the pig hereditary basis successively better related candidate gene, helps to screen new molecule marker and cultivates disease-resistant variety.

But the domestic and international at present research to the CD163 gene mainly concentrates on the function in the anti-inflammatory response, and pig CD163 gene pleiomorphism is not also reported, does not study the pig CD163 gene molecule marker relevant with pig immunity up to now.Therefore, the applicant has cloned the portion C DS of relevant CD163 gene with pig immunity, utilizes this sequence to carry out researchs such as polymorphism and association analysis, and is new for the relevant molecule marker of pig immunity in the hope of obtaining.

Summary of the invention

The objective of the invention is to clone with the association analysis of pig immunity correlated character use molecule marker (or claim genetic marker, down with), utilize this molecule marker as the application in the pig marker-assisted breeding.

The present invention is achieved through the following technical solutions:

The applicant clone obtains a kind of molecule marker relevant with pig immunity, and it is the nucleotide sequence shown in the sequence table SEQ ID NO:1.There is 1 base mutation by A2439-G2439 at 2439bp place at sequence table SEQ ID NO:1; There is 1 base mutation by G2454-C2454 at 2454bp place at sequence table SEQ ID NO:1; There is 1 base mutation by G2537-A2537 at 2537bp place at sequence table SEQ ID NO:1, introduces the mutational site, and the sudden change of above-mentioned three base pairs causes the HpaII-RFLP polymorphism; There is 1 base mutation by A2685-C2685 at 2685bp place at sequence table SEQ ID NO:1, introduces the mutational site, causes the BsiYI-RFLP polymorphism

Wherein:

The right dna sequence dna of the segmental primer of clone pig CD163 Gene Partial is as follows:

Forward primer: 5 '-TGGTCAACTTCGCCTGGT-3 ', reverse primer: 5 '-AGCACGTCACAGCAGCAT-3 '.

It is as follows to detect the right dna sequence dna of the forward and reverse primer of base mutation of G2537-A2537:

Forward primer: 5 '-GAGCTTGGGGCAGCGTTGGCC-3 ' reverse primer: 5 '-ACACTGAACATTGTCCACCCAC-3 '.

It is as follows to detect the right dna sequence dna of the forward and reverse primer of base mutation of A2685-C2685:

Forward primer: 5 '-GGAAGTTTTTTACAACGGAGC-3 ' reverse primer: 5 '-GGAGATGATGGGCCCTGCCATAG-3 '.

A kind of preparation method of molecule marker is characterized in that according to following steps:

Extract total RNA with pig variety PBMC genome, with pig CD163 gene cDNA sequence is template design primer, pcr amplification, PCR product purification and cloning and sequencing, the nucleotide sequence of acquisition shown in sequence table SEQ ID NO:1 used the polymorphism that the CRS-PCR-RFLP method detects pig CD163 gene.

More detailed technical scheme is referring to " embodiment ".

Description of drawings

Sequence table SEQ ID NO:1 is the pig CD163 Gene Partial CDS sequence that the present invention clones.

Fig. 1: be techniqueflow chart of the present invention.

Fig. 2: the present invention clone's pig CD163 Gene Partial CDS sequence, among the figure: " square frame " is labeled as the mutational site of base, from left to right be successively: A2439-G2439, G2454-C2454, G2537-A2537 and A2685-C2685, " shadow zone " of the head of nucleotide sequence and tail is that primer is right.

Fig. 3: be the electrophoretogram of pig CD163 gene C DS partial sequence amplification among the present invention, clip size is 938bp (agarose gel concentration is 1.5%).Among the figure: the Marker swimming lane is a dna molecular amount standard.

Fig. 4: be the partial dna sequence that pig CD163 gene amplification contains the G2537-A2537 mutational site among the present invention, " shadow zone " of sequence head and the tail is right for the introducing restriction enzyme site primer of design, square frame in the upstream primer " C " is the mutational site, creates a restriction enzyme site HpaII.

Fig. 5: be the partial dna sequence that pig CD163 gene amplification contains the A2685-C2685 mutational site among the present invention, sequence head and the tail " shadow zone is right for the introducing restriction enzyme site primer of design; in the downstream primer in the square frame " A " be the mutational site, create a restriction enzyme site BsiYI.

Fig. 6: be the electrophoretogram of the dna fragmentation that contains G2537-A2537 and A2685-C2685 mutational site of pig CD163 gene amplification among the present invention, clip size is 152bp and 210bp (agarose gel concentration is 1.5%) respectively.Among the figure: the Marker swimming lane is a dna molecular amount standard.

Fig. 7: be 4 mutational sites finding after the pig CD163 gene sequencing, from left to right be successively: A2439-G2439, G2454-C2454, G2537-A2537 and A2685-C2685.

Fig. 8: three kinds of genotype (GG, AA and the AG) electrophoretogram that is pig CD163 gene PCR-HpaII-RFLP among the present invention.

Fig. 9: three kinds of genotype (AA, CC and the AC) electrophoretogram that is pig CD163 gene PCR-BsiYI-RFLP among the present invention.

Embodiment

Embodiment 1:

One, the screening in segmental clone of CD163 Gene Partial and mutational site

1. design of primers

Utilize primer-design software Primer 5.0, (Genebank accession number NM_213976.1) is the amplimer that stencil design goes out to comprise the part coding region with pig CD163 gene mRNA sequence.It is synthetic that this primer is given birth to worker's biotechnology company limited by Shanghai, and the right dna sequence dna of this primer is as follows:

Forward primer: 5 '-TGGTCAACTTCGCCTGGT-3 ',

Reverse primer: 5 '-AGCACGTCACAGCAGCAT-3 '.

2.PCR the purifying of product and order-checking

(1) pcr amplification

Pcr amplification reaction system 20ul comprises the cDNA of 50ng, 1 * PCR buffer, 2.5mM MgCl, 0.2mM dNTP, 10pmol ofeach primer and 1 U Ampli Taq DNA polymerase (worker's biotechnology company limited is given birth in Shanghai).The PCR reaction conditions is: the response procedures of PCR is: 95 ℃ of 5min; 94 ℃ of 30s, 59 ℃ of 30s, 72 ℃ of 30-60s, 35 circulations; 72 ℃ are extended 5min.Amplified production carries out the electrophoresis detection (see figure 3) with 1.5% sepharose.

(2) purifying of PCR product and order-checking:

CDNA with 5 product boars is a template, and random choose 1 each and every one body is used for pcr amplification in each kind, reclaims 5 kind pcr amplification products, mixes order-checking.The miniprep dna fragment purification test kit that utilizes sky, Beijing root biotech company to produce carries out purifying and reclaims, and concrete grammar is with reference to the process specifications of this test kit.Sequencing is given birth to worker's biotechnology company limited by Shanghai and is finished.

(3) the dna sequence dna homology search is identified

By NCBI-BLAST (Basic Local Alignment Search Tool) instrument, the reference sequences of announcing in the sequence that order-checking back is obtained and the GenBank database is compared, and the result shows successfully to clone and obtains the fragment (see figure 2) that pig CD163 gene contains the regional 938bp of portion C DS.

(4) screening in mutational site in the dna sequence dna

By NCBI-BLAST (Basic Local Alignment Search Tool) instrument, the sequence of announcing in back sequence that obtains of order-checking and the GenBank database is compared, the mixing of 5 pig varieties order-checking peak figure is imported SeqMan software to compare, the result is presented at pig CD163 gene coding region has 4 sites appearance significantly bimodal, and this illustrates that this gene in these 4 site the base mutation (see figure 7) has taken place.These 4 sites are respectively: 2439A-G, 2454 G-C, 2537 G-A and 2685 A-C.

Two, the foundation of CRS-PCR-RFLP genotype detection method

1. introduce the restriction enzyme site design of primers

Design introducing restriction enzyme site primer respectively at polymorphic site 2537 G-A and 2685 A-C, be used for genotype detection, its dna sequence dna is as follows respectively:

Forward primer: 5 '-GAGCTTGGGGCAGCGTTGGCC-3 ' reverse primer: 5 '-ACACTGAACATTGTCCACCCAC-3 '

Forward primer: 5 '-GGAAGTTTTTTACAACGGAGC-3 ' reverse primer: 5 '-GGAGATGATGGGCCCTGCCATAG-3 '

2.PCR amplification

Add dna profiling 1 μ L in the reaction system of 15uL, distilled water 11.3 μ L, 10 * PCR buffer, 1.5 μ L, dNTP 0.45 μ L, each 0.3 μ L of 10mM primer, Taq enzyme 1U.The PCR reaction conditions is: behind 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 30s, 64 ℃ of annealing 30s, 72 ℃ of extension 25s, 35 circulations, last 72 ℃ are extended 5min.3 μ L PCR products detect (see figure 6) through 1.5% agarose gel electrophoresis, and positive is used for next step enzyme and cuts detection.

3.RFLP detect

(1) HpaII-RFLP detection molecules mark 2537 G-A

With PCR product 4 μ L, 10 * Buffer, 1 μ L, restriction enzyme HpaII is 3U, 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 10% polyacrylamide gel electrophoresis, and silver dyes and takes pictures.

Molecular marker gene type analysis: as shown in Figure 8, represent the different genotype of the SNP3 of 3537R place kind (genotype is represented with its mutating alkali yl) AA, GG, AG respectively, electrophoresis result demonstration AA genotype (has only a band, length is 152bp), GG genotype (two bands, length is 132bp and 20bp) and AG genotype (3 bands, length are 152bp/132bp/20bp).

(2) BsiYI-RFLP detection molecules mark 2685 A-C

With PCR product 4 μ L, 10 * Buffer, 1 μ L, restriction enzyme BsiYI is 3U, adds distilled water and mends to 10 μ L, with centrifugal behind the sample mixing, places 12h in 55 ℃ of water-baths, enzyme is cut product and is cut the result with 10% polyacrylamide gel electrophoresis detection enzyme.

Molecular marker gene type analysis: as shown in Figure 9, the primer amplification sequence contains the restriction enzyme site of two restriction enzyme BsiYI, and (one of them is for introducing identification 2685 A-C mutational sites behind the base mutation, the recognition site that another contains for sequence itself), 3 kinds of banding patterns appear after BsiYI digestion, represent 3 kinds of different genotype (genotype is represented with its mutating alkali yl) AA respectively, CC, AC, electrophoresis result demonstration AA genotype (has only two bands, length is 147bp and 63bp), CC genotype (3 bands, length is 130bp/63bp/17bp) and AC genotype (4 bands, length are 147bp/130bp/63bp/17bp).

(3) electrophoresis detection of the preparation of polyacrylamide gel and RFLP

A.10% the preparation of polyacrylamide gel

Distilled water 16.1ml

30% acrylamide (29:1) 11.7ml

5 * tbe buffer liquid 7ml

10%AP solution 0.23ml

TEMED 0.02ml

Cumulative volume 30ml

B. encapsulating

Slowly pour into the acrylamide glue along glass plate edge, note avoiding bubble to produce, stop encapsulating when irritating to from sheet glass upper edge 0.1cm the time, insert comb, the inclination offset plate became 30 to spend angles with the plane, in polymerized at room temperature 1 hour.

C. go up sample, electrophoresis

Pull out comb after the gel polymerisation, draw 1 * tbe buffer liquid flushing well with syringe.Assemble electrophoresis equipment, the agarose gel with 0.8% has sealed the slit between sheet glass and the electrophoresis chamber, prevents the electrophoretic buffer seepage.Pour 1 * TBE electrophoretic buffer of capacity up and down in the dashpot into.With the microsyringe of 50 μ l enzyme is cut product and slowly inject the comb hole.Open the electrophoresis apparatus power supply after last sample finishes, electrophoresis is 4 hours under the 100V constant voltage.

D. gel-colored

1. electrophoresis finishes, and takes off sheet glass, carefully gel is stripped down from sheet glass, puts into the distilled water rinsing once;

2. immerse fixedly 10min of 10% ethanolic soln.Fixedly finish, with the of short duration detergent gel of distilled water (changing water twice, each 30s);

3. with 1% nitric acid decolouring 3min, then once with the rapid rinsing of distilled water;

4. immerse 0.2% Silver Nitrate, lucifuge is placed on and dyes 15～25min on the shaking table;

5. deionization washing glue is three times, and each 1min adds 3%Na ₂CO ₃Na is outwelled in colour developing when colour developing is suitable ₂CO ₃, add 3% Glacial acetic acid rapidly and stop colour developing;

6. gel is placed in the gel imaging system camera bellows, scan and preserve electrophoretogram.

Embodiment 2:

The application of CD163 molecule marker classifying method in the immune character association analysis that the present invention sets up

(1) with the related immune character of CD163 gene 2537 G-A (HpaII-RFLP) molecule marker

(1) uses CRS-PCR-HpaII-RFLP detection molecules mark 2537 G-A genotype and carry out association analysis, the result shows: this molecule marker meets the Hardy-Weinberg equilibrium law in detecting colony, pig breeding and breathing syndrome virus (reproductive and respiratory syndrome virus) antibody test value to 0 age in days, MPW, volume of platelets, big thrombocyte ratio, thrombocyte and white corpuscle influence are significantly.Concrete outcome sees Table 1:

The be associated least square average of immune character of table 1 CD163 gene 2537G-2537A different genotype

P＜0.05, the expression significant difference; P＜0.01, expression difference is extremely remarkable

(2) with the related immune character of CD163 gene 2685A-C (BsiYI-RFLP) molecule marker

Use CRS-PCR-BsiYI-RFLP detection molecules mark 2685A-C genotype and carry out association analysis, the result shows: this molecule marker does not meet the Hardy-Weinberg equilibrium law in detecting colony, heavy to weaned piglet, the pig breeding of 0 age in days, 17 ages in days is remarkable with the hog cholera antibody blocking-up rate influence of breathing syndrome virus (reproductive and respiratory syndrome virus) antibody test value and 17 ages in days.Concrete outcome sees Table 2:

The be associated least square average of immune character of table 2 CD163 gene 2685A-2685C different genotype

P＜0.05, the expression significant difference; P＜0.01, expression difference is extremely remarkable

Sequence table

＜110〉Hua Zhong Agriculture University

＜120〉the CD163 gene fragment clone relevant and as the application of molecule marker with pig immune trait

<130>

<141>2009-03-17

<160>5

<170>PatentIn?version?3.1

<210>1

<211>3400

<212>DNA

＜213〉pig (Sus scrofa)

<220>

<221>CDS

<222>(1)..(3399)

<223>

<220>

<221>mutation

<222>(2685)..(2685)

<223>

<220>

<221>mutation

<222>(2537)..(2537)

<223>

<220>

<221>mutation

<222>(2454)..(2454)

<223>

<220>

<221>mutation

<222>(2439)..(2439)

<223>

<400>1

<210>2

<211>1110

<212>PRT

＜213〉pig (Sus scrofa)

<400>2

<210>3

<211>6

<212>PRT

＜213〉pig (Sus scrofa)

<400>3

<210>4

<211>6

<212>PRT

＜213〉pig (Sus scrofa)

<400>4

<210>5

<211>8

<212>PRT

＜213〉pig (Sus scrofa)

<400>5

Claims (8)

1, a kind of CD163 gene fragment relevant with pig immunity as molecule marker, it is the nucleotide sequence shown in the sequence table SEQ ID NO:1.

2, a kind of CD163 gene fragment relevant with pig immunity as molecule marker is characterized in that there is 1 base mutation by G2537-A2537 at the 2537bp place of sequence table SEQ ID NO:1, causes the HpaII-RFLP polymorphism.

3, a kind of CD163 gene fragment relevant with pig immunity as molecule marker is characterized in that there is 1 base mutation by A2685-C2685 at the 2685bp place of sequence table SEQ ID NO:1, causes the BsiYI-RFLP polymorphism.

4, the described CD163 gene fragment relevant of claim 1 with pig immunity as molecule marker, wherein the right dna sequence dna of the forward and reverse primer of amplification screening mutational site sequence is as follows:

Forward primer: 5 '-TGGTCAACTTCGCCTGGT-3 ', reverse primer: 5 '-AGCACGTCACAGCAGCAT-3 '.

5, the described CD163 gene fragment relevant of claim 1 with pig immunity as molecule marker, it is as follows wherein to detect the right dna sequence dna of the forward and reverse primer of base mutation of G2537-A2537:

Forward primer: 5 '-GAGCTTGGGGCAGCGTTGGCC-3 ' reverse primer: 5 '-ACACTGAACATTGTCCACCCAC-3 '.

6, the described CD163 gene fragment relevant of claim 1 with pig immunity as molecule marker, it is as follows wherein to detect the right dna sequence dna of the forward and reverse primer of base mutation of A2685-C2685:

Forward primer: 5 '-GGAAGTTTTTTACAACGGAGC-3 ' reverse primer: 5 '-GGAGATGATGGGCCCTGCCATAG-3 '.

7, a kind of preparation method of the molecule marker relevant with pig immunity is characterized in that according to following steps:

Extract total RNA with pig variety PBMC genome, with pig CD163 gene cDNA sequence is template design primer, pcr amplification, PCR product purification and cloning and sequencing, the nucleotide sequence of acquisition shown in sequence table SEQ ID NO:1 used the polymorphism that the CRS-PCR-RFLP method detects pig CD163 gene.

8, each described gene fragment of claim 1-3 or the claim 4-6 primer described in each is to the application in the pig marker assisted selection.

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