CN1778818A - Pig production and immune character related protein, its coding gene and use - Google Patents

Abstract

A pig production and immune related protein, its coding gene and use are disclosed. The protein is one of the amino acid residue sequences as following: 1) SEQ ID No:6 in sequence table; 2) Amino acid residue sequence of SEQ ID No:6 in sequence table is prepared by substituting, losing and adding the protein related to pig production and immune property, inspecting pig production and immune property, PCR enlarging for gene group DNA to be tested a pair of primers with nucleotide sequence of SEQ ID No:2 and SEQ ID No:3 in sequence table, mononucleotide polypeptide inspecting for the PCR enlarged product, determining No.146 bit base as T from the 5, end of SEQ ID No:1 in sequence table, slicing it by Hin6 I enzyme and inspecting the obtained size and amount of sliced fraction. It can be used for breeding.

Description

One boar production, immune character related protein and encoding gene and application

Technical field

The present invention relates to a kind of protein and encoding gene thereof and application, particularly relate to a kind of pig production character and immune character related protein and encoding gene thereof and utilize the single nucleotide polymorphism of this protein coding gene and the method that restrictive fragment length polymerphism detects pig production character and immune character.

Background technology

GENERALIZATION OF MODERN BREEDING TECHNIQUE is significantly improved the production performance of pig, yet along with the raising of living standards of the people, the requirement of meat quality is also improved thereupon.When the emphasis that herding is produced has extended to the growth efficiency that improves lean tissue, improvement meat, improve the resistance of product, thereby reduce the production cost of unit product, and reduce drug residue, promptly when guaranteeing livestock product quantity, improve the quality of livestock product.

Individual immunity power is to weigh an important indicator of animal health condition, it belongs to quantitative character, related genes involved is more, molecular mechanism also complicated (Knapp etc., Relationships between geneticschanges and infections disease in domestic livestock, (eds.Hill W G, BishopS C, McGuirk B.) brit.Soc Anim Sci., Edinburgh Occasional publication no.27,65-80.2000).Part Study shows that immunizing power and production performance are usually expressed as the phenotype negative correlativing relation, and this is big obstacle for breeding work is provided with, and promptly how improving the individual immunity ability on the basis of maintenance and raising production performance is the new research topic in herding breeding field.But it is two-way the relation that studies show that pig immunological competence and production performance on a small quantity also being arranged abroad, and the immunological competence that promptly strengthens pig under certain situation (standard state) can improve main production performance such as its speed of growth, feed efficiency.So far, the mutual relationship between pig immunological competence and production performance is still uncertain, and therefore very crucial from the mutual relationship between molecular level announcement production performance and immunizing power controlling gene, also the breeding for disease resistance work for domestic animal provides new thinking.

The healthy state of pig depends on and infects and the defensive enginery results of interaction, if defensive enginery just shows the nature disease resistance by force.Disease resistance can be divided into special disease resistance and general disease resistance by the hereditary basis difference.Special disease resistance is meant the resistance of pig to certain specified disease or pathogenic agent, and this resistance is controlled by mainly in a key-gene site, also can be subjected to other site (comprising regulator) and such environmental effects to some extent.Studies show that the inherent mechanism of special disease resistance is to exist in the host or lack certain molecule or its variant, this molecule has following effect: 1. determine allosome identification and specificity simplified reaction; 2. determine the special attachments power of pathogenic agent; 3. pathogenic agent is bred after entering in the body in vivo, and can decision cause the host to fall ill.General disease resistance is not limited to anti-a certain pathogenic agent, it is subjected to the combined influence of polygene and environmental factors, the antigenic specificity of pathogenic agent is minimum to general disease resistance influence, even basic not influence, and this disease resistance has embodied the whole defense function of body to disease.

Disease resistance major histocompatibility complex (Major Histocompatibility complex, MHC) be and disease resistance and the closely-related one group of gene group of immunne response, the MHC of pig (called after SLA) is positioned at (Warner on No. 7 karyomit(e)s of pig, Mapping of C2, Bf, and C4genes to the swine majorhistocompatibility complex.J.Immunology.1987,139:3388-3395), comprise I type and II type gene, wherein I type gene has extremely strong polymorphism.The research of proterties such as pig SLA gene haplotype and pig birth weight, the speed of growth, the thickness of backfat is engaged in the Rothschild laboratory for a long time, they think that there are correlationship (Rothschild MF in SLA gene haplotype and above-mentioned production performance, Identification of quantitativetrait loci and interesting candidate genes in the pig:progress and prospects.Proc 6th WCGALP 1998,26:403-409).Mallard etc. (1998) find that in high immunne response of selecting through 8 generations to form of pig (H system) and low immunne response (L system) strain H system reaches market weight than the Zao 10d of L system.The above-mentioned SLA that studies show that pig and the multiple production traits all have chain, and get in touch the relation of being proportionate between the SLA of pig and growth, back fat and reproductive trait more.(Draper，D.D.，Effects of divergentselection for leg weakness on bone and muscle cross-sectional areas in Durocswine.Am.J.Vet.Res.1991，52：164-168；Stalder，K.J.，Maternal swine traitgenetic parameter estimates measured on Landrace females with known porcinestress syndrome genotypes.J.Anim.Breed.Genet.1998，115：199-209)。

PSMB8 is one of moiety of proteasome.Proteasome (26S proteasome) is the necessary moiety of the proteolysis approach of the interior dependency ATP of cell in the eukaryote, participate in active degraded (the Cannon MJ of a large amount of intracellular protein, Pate JL.Expression and regulation of interferongamma-inducible proteasomal subunits LMP7and LMP10 in the bovine corpusluteum.Biol Reprod.2003,68 (4), 1447-54.).Proteasome has following hydrolytic activity: (1) side chain hydrogen base acid close preferendum activity (BrAAP); (2) Chymotrypsin sample activity; (3) trypsin-like; (4) peptide acyl paddy amine acylpetide hydrolase activity.Short-lived albumen in the proteasome selectivity degradation of cell, generation is suitable for MHCI quasi-molecule bonded antigen peptide, contacting directly body immunizing power (Peng Rui, Qin Jun river. proteasome 26S Proteasome Structure and Function progress. biological chemistry and biophysics progress .1998; 25 (3): 235-238).

Studies show that in the human cancer cell of kinds more than half, proteasome all can morph.The protein degradation that ubiquitin is regulated is of crucial importance in organism, thereby its pioneering research is acquired a special sense.At present, worldwide, research is constantly arranged and find the cell new function relevant with this protein degradation process.These researchs are to the further secret that discloses biology, and genesis mechanism and the treatment means thereof of exploring some diseases are significant.

The proteolytic activity of proteolytic enzyme complex body is finished by a plurality of subunits are collaborative, and change that subunit is formed and interpolation chemically modified all can change its activity.The subunit of the core 20S proteasome of PSMB8 genes encoding 26S proteasome, and can under the inducing of interferon-gamma, produce, another one proteasome subunit PSMB5 replaced.Therefore the functional study of PSMB8 gene is very important.

The existing report of the 26S Proteasome Structure and Function of people PSMB8 gene, Glenn etc. show that after deliberation there is obviously relevant (Glenn YD in the polymorphism of PSMB8 gene with insulin-dependent diabetes, Muir A, Maclaren NK, et al.Association of LMP2 and LMP7 genes within the major histocompatibilitycomplex with insulin-dependent diabetes mellitus:population and familystudies.Am J Hum Genet, 1995,56 (2): 528-534).People's PSMB8 gene and MHC I quasi-molecule close linkage on karyomit(e), be positioned at HAS6p21, form (Castro etc. by 6 exons and 5 introns, An antecedent of the MHC-linked genomic region inamphioxus.Immunogenetics.2004,11 (55): 782-784).So far, the functional study of PSMB8 gene also is not very thorough, the polymorphism of research mutational site in colony, and carry out the strong means that the proterties association analysis is the research gene function.

(single nucleotide polymorphism SNP), mainly is meant on genomic level by the caused dna sequence polymorphism of the variation of single Nucleotide single nucleotide polymorphism.The polymorphism that SNP showed only relates to the variation of single base, and this variation can be caused by the conversion (transition) or the transversion (transversion) of single base, also can be by due to the insertion or disappearance of base.But usually said SNP does not comprise back two kinds of situations.This variation may be that (C → T then is G → A) on its complementary strand, also may be transversion (C → A, G → T, C → G, A → T) in conversion.The incidence of conversion is always apparently higher than other several variations, and the SNP with conversion hysteria variation accounts for 2/3, and the occurrence probability of other several variations is similar.The SNP detection method often adopts some existing mature technologies, as dna sequencing, restriction fragment length polymorphism (RFLP), single strand conformation polymorphism (SSCP), allele specific oligonucleotide oligonucleotide hybridization (ASO) etc.No matter adopt any method, all at first must increase, just can carry out other detection then target sequence.

The PCR-RFLP full name is that (restrictionfragment length Polymorphism RFLP) analyzes polymerase chain reaction,PCR (PCR) restriction fragment length polymorphism.Mainly comprise three links: (1) target gene pcr amplification; (2) Kuo Zeng dna fragmentation restriction map (length polymorphism), this technology is used PCR target gene fragment is increased more than 1,000,000 times, even sample nucleic acid amount is less than the pg level, also can be amplified out, this has not only improved the sensitivity of genetic analysis greatly, and need not mark or radioactive probe crossover process .PCR-RFLP is mainly used in nucleic acid Variability Analysis and comparison, when DNA or RNA molecule since single base mutation or sequence reset, certain restriction enzyme site is increased, reduce or disappearance, cause restriction fragment length to change, just produced restriction fragment length polymorphism, because restriction enzyme has special restriction enzyme site on dna molecular, position and quantity according to its recognition sequence, can be size is similar and the variant DNA of sequence cuts into different fragments passes through nucleic acid electrophoresis, the DNA sheet that is uneven in length can be separated and show its length polymorphism (segmental size) .PCR-RFLP and be used for genetic analysis and have the resolution height, good reproducibility, advantages such as easy quicklook, be mainly used in the somatotype branch hypotype of hiving off of microorganism, oncogene is analyzed, the diagnosis of inherited disease, the analysis of HLA somatotype and lipophorin gene etc.

Summary of the invention

The purpose of this invention is to provide a boar production, immune character related protein and encoding gene thereof.

Pig provided by the present invention produces, immune character related protein, and name is called PSMB8, derives from pig, is the protein with one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 6;

2) with SEQ ID № in the sequence table: 6 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and the protein relevant with pig production character and immune character.

Wherein, the SEQ ID № in the sequence table: 6 are made up of 276 amino-acid residues.

Above-mentioned pig produces, the encoding gene (PSMB8) of immune character related protein also belongs to protection scope of the present invention.

Above-mentioned pig produces, the encoding gene of immune character related protein comprises that the cDNA gene of pig production, immune character related protein and pig produce, the genomic gene of immune character related protein.Its cDNA gene is one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 5 nucleotide sequence;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 6 protein sequences;

3) under the rigorous condition of height can with the SEQ ID № in the sequence table: the nucleotide sequence of the 5 dna sequence dnas hybridization that limit.

The rigorous condition of described height for hybridization back with contain 0.1 * SSPE (or 0.1 * SSC), the solution of 0.1%SDS washes film under 65 ℃.

SEQ ID № in the sequence table: 5 by 1104 based compositions, and the encoding sequence of this cDNA sequence is SEQ ID № in the sequence table: 5 from 5 ' end the 47th bit base to the 877 bit bases.

Its genomic gene is one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 4 nucleotide sequence;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 6 protein sequence;

3) under the rigorous condition of height can with the SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits.

The rigorous condition of described height for hybridization back with contain 0.1 * SSPE (or 0.1 * SSC), the solution of 0.1%SDS washes film under 65 ℃.

Sequence 4 in the sequence table is by 3102 based compositions, 1-193 bit base from 5 ' end is first exon of this genomic gene, 975-1122 bit base from 5 ' end is second exon of this genomic gene, 1293-1403 bit base from 5 ' end is the 3rd exon of this genomic gene, 1837-1966 bit base from 5 ' end is the 4th exon of this gene, 2337-2541 bit base from 5 ' end is the 5th exon of this gene, 2787-3102 bit base from 5 ' end is the 6th exon of this gene, from 5 ' the 47-49 bit base of end be the initiator codon ATG of this gene, from 5 ' the 2873-2875 bit base held is the terminator codon TAA of this genomic gene; 194-974 bit base from 5 ' end is first intron of this genomic gene, 1123-1292 bit base from 5 ' end is second intron of this genomic gene, 1404-1836 bit base from 5 ' end is the 3rd intron of this genomic gene, from 5 ' the 1967-2336 bit base of end be the 4th intron of this genomic gene, from 5 ' the 2542-2786 bit base held is the 5th intron of this genomic gene.

The carrier, clone and the host bacterium that contain pig production of the present invention, immune character related protein encoding gene all belong to protection scope of the present invention.

Second purpose of the present invention provides a kind of method that detects pig production, immune character.

Detection pig provided by the present invention produces, the method for immune character, be to use by SEQ ID № in the sequence table: 2 and SEQ ID №: a pair of primer that 3 nucleotide sequence is formed carries out pcr amplification to the genomic dna of pig to be measured, then pcr amplification product is carried out following at least a detection:

1) described pcr amplification product is carried out single nucleotide polymorphism and detect, determine that from 5 of SEQ ID NO:1 ' end the 146th bit base be T;

2) cut described pcr amplification product with the Hin6I enzyme, detect in the endonuclease bamhi that obtains whether contain a 441bp band and/or a 143bp band and a 298bp band.

If the single nucleotide polymorphism detected result of pcr amplification product is from 5 of SEQ ID NO:1 ' end the 146th bit base, promptly in sequence table 5 of sequence 4 ' when being T, its homozygotic genotype is TT to end the 2533rd bit base (5 of sequence 5 ' end the 780th bit base in sequence table); From 5 of SEQ ID NO:1 ' end the 146th bit base, promptly in sequence table 5 of sequence 4 ' end the 2533rd bit base (5 of sequence 5 ' end the 780th bit base in sequence table) when being C, its homozygotic genotype is CC; Their heterozygote genotype is TC.

The Hin6I enzyme is cut described pcr amplification product, if the endonuclease bamhi that obtains is the band of a 441bp, determines that then the genotype of pig to be measured is TT; If the endonuclease bamhi that obtains is two bands of a 143bp and a 298bp, determine that then the genotype of pig to be measured is CC; If the endonuclease bamhi that obtains is the band of a 441bp, three bands of a 143bp and a 298bp determine that then the genotype of pig to be measured is TC.

Wherein, the immune character of TC genotype individuality (content of hemoglobin, pcv amount, mean corpuscular volume (MCV)) is better than the genotypic individuality of TT; The dressing percentage of TT genotype individuality, estimation eye muscle area (cm ²) and leg stern ratio proterties all be significantly higher than the TC genotype, nascent market weight (90KG) age in days that reaches of TC genotype individuality, these 3 proterties of leaf fat rate and intramuscular fat all are significantly higher than the TT genotype, show that the intramuscular fat of TC genotype individuality and leaf fat rate are than higher, the speed of growth of TT genotype individuality is than very fast, and it is all better to produce meat and lean meat ratio.

Described single nucleotide polymorphism detects and can adopt dna sequencing, restriction fragment length polymorphism (RFLP), single strand conformation polymorphism (SSCP), the hybridization of allele specific oligonucleotide oligonucleotide (ASO), the little sequencing according to the DNA array, dynamic allele specific oligonucleotide hybridization, special connection, DNA chip and the TaqMan system etc. of oligonucleotide to detect.

Pig production of the present invention, immune character related protein and encoding gene thereof can be used for detecting immune characters such as PINPROL content, pcv, mean corpuscular volume (MCV), and be nascent to the body weight of going on the market (90KG) age in days, dressing percentage, estimation eye muscle area (cm ²) production traits such as leaf fat rate, leg stern ratio, meat, intramuscular fat, for the molecular breeding of pig provides a new genetic marker, will in the breeding of pig, play a significant role.

Description of drawings

Fig. 1 is for detecting the schema of pig PSMB8 partial dna sequence polymorphism

Fig. 2 is the amplification of pig PSMB8 partial dna sequence

Fig. 3 detects the agarose gel electrophoresis detected result of pig PSMB8 Gene Partial dna sequence polymorphism for the PCR-RFLP method

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment.

The acquisition of embodiment 1, pig PSMB8 gene

1, the acquisition of the genomic gene of pig PSMB8

CDNA sequence (the GenBank number of including: NM_004159) be the information probe of personnel selection PSMB8, utilize the BLAST instrument of NCBI in GenBank pig est database, to do the homologous sequence retrieval, obtain a series of homologys and reach ESTs (fragment length is greater than 100bp) more than 90%, corresponding sequence is inquired about in the number of including of these ESTs in the ENTREZ of NCBI (http://www.ncbi.nlm.nih.gov/Web/Search/index.htmL), use the EST contig of the Seqman program construction pig among the software DNAStar then, at last according to 6 pairs of EST splicing sequences Design amplimers:

1PL：5’-ACTTCCTCCTGCGAGAGAGC-3’

1PR：5’-GTTCCTTTCTCCATCCCCAC-3’

2PL：5’-ACGTACAGATTGAGATGGCT-3’

2PR：5’-AGACGCTCCCAATACTGACA-3’

3PL：5’-CATTAAGAGTGAACAAGGTG-3’

3PR：5’-CATCATATTGGAGAGCAGTT-3’

4PL：5’-TATCTCAGTGTCCGCAGCCT-3’

4PR：5’-TGACAACGCCTCCAGAATAG-3’

5PL：5’-ATGTTCTCCACTGGTAGCGG-3’

5PR：5’-TACTCTCCACCTTCACCCAC-3’

6PL：5’-TACCACATGAAGGAAGATGG-3’

6PR：5’-ATTGTGCTTAGTGAGATGCC-3’

In the genomic dna of pig, increase, obtain 6 specific bands, obtain the sequence of a 3.1kb with the splicing of the Seqman program among the software DNAStar, nucleotide sequence with sequence 4 in the sequence table, this sequence is the genome sequence of pig PSMB8, the amino acid residue sequence (pig PSMB8) that coding has sequence 6 in the sequence table.1-193 bit base from 5 ' end is first exon of this genomic gene, 975-1122 bit base from 5 ' end is second exon of this genomic gene, 1293-1403 bit base from 5 ' end is the 3rd exon of this genomic gene, 1837-1966 bit base from 5 ' end is the 4th exon of this gene, 2337-2541 bit base from 5 ' end is the 5th exon of this gene, 2787-3102 bit base from 5 ' end is the 6th exon of this gene, from 5 ' the 47-49 bit base of end be the initiator codon ATG of this gene, from 5 ' the 2873-2875 bit base held is the terminator codon TAA of this genomic gene; 194-974 bit base from 5 ' end is first intron of this genomic gene, 1123-1292 bit base from 5 ' end is second intron of this genomic gene, 1404-1836 bit base from 5 ' end is the 3rd intron of this genomic gene, from 5 ' the 1967-2336 bit base of end be the 4th intron of this genomic gene, from 5 ' the 2542-2786 bit base held is the 5th intron of this genomic gene.

2, the acquisition of the cDNA gene of pig PSMB8

6 dna fragmentations obtaining and pig EST splicing sequence and people's dna sequence dna are compared, confirm the exon part, exon partly is stitched together, just obtain the cDNA sequence of pig PSMB8.

3, the location of pig PSMB8 gene

With pig * rodents somatic cell hybrid clone panel RH clone plate (INRA-Minnesota porcineradiation hybrid panel, ImpRH, 118 hybrid cell systems) DNA in is (available from French Academy of Agricultural Sciences, Laboratoire de G é n é tique Cellulaire, INRA) be template, with primer 1 (forward): 5 '-ATGTTCTCCACTGGTAGCGG-3 ' and primer 2 (oppositely): 5 '-TACTCTCCACCTTCACCCAC-3 ' is that primer carries out pcr amplification.Amplification condition is: 94 ℃ of sex change 4min, and 94 ℃ of sex change 20s, 59 ℃ of annealing 20s, 72 ℃ are extended 20s, 30 circulations.Extend 5min at 72 ℃ at last.Reaction system wherein consists of: PCR reaction cumulative volume is 10 μ l, and wherein the about 25ng of RH clone plate DNA contains 1 * damping fluid (Promega), 2mmol/L MgCl ₂, the dNTP final concentration is 150 μ mol/L, the primer final concentration is 0.2 μ mol/L, 1U TaqDNA polysaccharase (Promega).The PCR reaction product is carried out the pcr amplified fragment somatotype with 1.5% agarose gel electrophoresis, wherein the 1st, 6, and 10,18,19,20,23,25,27,29,34,35,40,41,46,52,54,55,59,60,61,65,67,75,79,80,83,85,88,93,96,99,100,105,110,111,113,114, obtain positive amplification among the DNA during 115, No. 116 hybrid cell is.PCR somatotype data are submitted to HybWeb (http://imprh.toulouse.inra.fr/) carry out statistical study to obtain zone location information, the data analysis result shows that the genomic gene of pig PSMB8 is positioned on No. 7 karyomit(e)s of pig, with microsatellite marker SSC2B02 close linkage, the LOD value is 7.72.

The single nucleotide polymorphism of the partial dna sequence of embodiment 2, pig PSMB8 and RFLP polymorphism detect

The flow process that the single nucleotide polymorphism of the partial dna sequence of pig PSMB8 and RFLP polymorphism detect as shown in Figure 1, concrete steps are as follows:

One, the detection of the single nucleotide polymorphism of the partial dna sequence of pig PSMB8

Detailed process comprises the steps:

1, design of primers

CDNA sequence (the GenBank number of including: NM_004159) be the information probe of personnel selection PSMB8, utilize the BLAST instrument of NCBI in GenBank pig est database, to do the homologous sequence retrieval, obtain a series of homologys and reach ESTs (fragment length is greater than 100bp) more than 90%, corresponding sequence is inquired about in the number of including of these ESTs in the ENTREZ of NCBI (http://www.ncbi.nlm.nih.gov/Web/Search/index.htmL), use the EST contig of the ASSEMBLY program construction pig among the software GeneTool then, according to EST splicing sequences Design amplimer, primer sequence is as follows at last:

Primer 1 (forward): 5 '-ATGTTCTCCACTGGTAGCGG-3 ' (sequence 2)

Primer 2 (oppositely): 5 '-TACTCTCCACCTTCACCCAC-3 ' (sequence 3)

2, the purifying of pcr amplification and amplified production thereof, clone and order-checking

Respectively to greatly enhance 22 of logical combinations (Da Bai * length in vain * Tongcheng pig), the total DNA of poba gene group of 30 of logical combination (long white * Da Bai * Tongcheng pig) 25 and Tongcheng pigs of growing up is a template, under the guiding of primer 1 and primer 2, pcr amplification pig PSMB8 partial dna sequence.PCR reaction cumulative volume is 20 μ l, wherein, and the about 100ng of pig genomic dna, 2 μ l, 10 * PCR damping fluids (Promega), MgCl ₂Final concentration is 1.5mmol/L, and the dNTP final concentration is 150 μ mol/L, and primer 1 and primer 2 final concentration respectively are 0.2 μ mol/L, 2U Taq archaeal dna polymerase.The PCR response procedures is: 94 ℃ of 4min of elder generation; 94 ℃ of 30s then, 58 ℃ of 30s, 72 ℃ of 20s, 30 circulations; Last 72 ℃ are extended 5min.The PCR reaction product is carried out 1.5% agarose gel electrophoresis, and electrophoresis result shows that the PCR product size of acquisition is about 441bp as shown in Figure 2.Among Fig. 2,2 swimming lanes are pcr amplification product, and the M swimming lane is dna molecular amount mark (100-1000bp ladder).Carry out purifying, clone and the order-checking of PCR product then as follows:

(1) purifying of PCR product: under ultraviolet lamp, contain the segmental gel of purpose from the sepharose cutting-out, put into 1.5ml Ependorff pipe, bathe to gel in 70 ℃ of temperature and to melt fully, use PCR product purification test kit (Promega) purified pcr product then, according to the operation of test kit specification sheets, concrete steps are to add 1ml resin (Resin), mixing 20s in the gel that per 300 μ l melt, with the Resin/DNA mixture syringe of packing into, make slurries pass through microtrabeculae (Minicolumn) and extrude.In syringe, add 80% Virahol 2ml again, touch piston Virahol is extruded by Minicolumn, take off Minicolumn and pack in the 1.5ml Ependorff pipe 10 into, the centrifugal 2min of 000g is with dry Resin, Minicolumn is packed in another clean 1.5ml Ependorff pipe, add 30-50 μ l sterilized water, leave standstill 1min, 10, the centrifugal 20s of 000g abandons supernatant, and eluted dna is stored in the Ependorff pipe.

(2) ligation: the PCR product and the pGEM-T carrier (Promega) of step (1) purifying are used T ₄Dna ligase connects, and obtains recombinant vectors.Linked system is: 2.5 μ L2 * damping fluids, 0.5 μ L pGEM-T carrier, the PCR product of 0.5 μ L purifying, 0.5 μ L T ₄Dna ligase, 1 μ L aqua sterilisa, 16 ℃ of water-bath 12-24h.

(3) preparation of competent cell: with bacillus coli DH 5 alpha (TaKaRa) on the LB solid medium 37 ℃ cultivate 16-20h after, picking list colony inoculation is in 2mL LB liquid nutrient medium, 37 ℃ of shaking culture 3h, getting 1mL bacterium liquid then transfers in containing the shaking in the bottle of 30mL LB liquid nutrient medium, continue the about 4h of shaking culture at 37 ℃, to shake bottle when the value for the treatment of bacterium liquid OD600 reaches 0.3-0.4 takes out from shaking table, ice bath 10-15min makes it cooling, then bacterium liquid is changed in the centrifuge tube in 4 ℃, 4, the centrifugal 10min of 000g collects somatic cells, centrifuge tube is inverted to abandon clean nutrient solution, is iced the CaCl of the 0.1mol/L of precooling with 10mL ₂The resuspended precipitation of solution, i.e. somatic cells, behind the ice bath 30min, 4 ℃ 4, the centrifugal 10min of 000g is again with the CaCl of the 0.1mol/L of 4mL ice precooling ₂The resuspended precipitation of solution, it is standby to put 4 ℃ of preservations.

(4) transform: the competent cell of getting 100-120 μ l step (3) preparation under the sterile state is in the 1.5mLEpendorff pipe, add the connection product that 5 μ L steps (2) obtain again, mixing, ice bath 30min, 42 ℃ of heat shock 90s (not shaking the Ependorff pipe in the above-mentioned conversion process), take out back ice bath 3-4min, add 400 μ l again and do not contain antibiotic LB liquid nutrient medium, at 37 ℃ of shaking culture 45min, get 100 μ L bacterium liquid at last and coat the 200mg/mLIPTG (Isopropylthio-β-D-galactoside that contains 4 μ L, isopropylthio-) and on the LB flat board of the 20mg/mL X-gal of 40 μ L, is inverted under the same conditions behind 37 ℃ of pre-1h of cultivation earlier and cultivates 12-24h;

(5) a small amount of of plasmid preparation: the single colony inoculation that grows on the picking agar plate is in 2-3mL LB liquid nutrient medium, and 37 ℃ of 300r/min cultivate 12-24h, collects thalline with the centrifugal several seconds of 1.5mL EP pipe 12000r/min again.Every pipe adds the ice-cold solution I of 100 μ l (50mM glucose, 25mM Tris-HCl (pH8.0), 10mM EDTA (pH8.0)), and vortex vibrates to thalline and fully suspends.Add new preparation solution II (0.2M NaOH, 1%SDS) 200 μ l put upside down mixing fast, ice bath 5min adds solution III (5M potassium acetate, glacial acetic acid 11.5ml, the H of precooling then ₂O 28.5ml) 150 μ l, ice bath 5min behind the mixing, the centrifugal 5min of 12000r/min, supernatant is gone in another EP pipe, add phenol: chloroform: primary isoamyl alcohol 500 μ l, vortex vibration, the careful upper strata water of drawing in centrifugal back, the dehydrated alcohol that adds 2 times of volumes,-20 ℃ of precipitation 30min, the centrifugal 5min of 12000r/min, precipitation is with 70% washing with alcohol 2 times, drain, add the TE 20 μ l that contain the RNA enzyme.

(6) enzyme of recombinant plasmid is cut evaluation: get 3 μ l plasmid DNA and distilled water mixing, making its cumulative volume is 15 μ l, add restricted endoenzyme EcoRI of 2-3U and the corresponding 10 * restriction enzyme reaction damping fluid of 2 μ l, flick tube wall mixing and centrifugal, put 37 ℃ of water-bath 1-2 hours, get 2-3 μ l reaction solution and detect, obtain enzyme and cut the result and estimate identical purpose recombinant plasmid in agarose gel electrophoresis.

(7) order-checking: recombinant plasmid adopts the terminal cessation method of two deoxidations to check order on automatic dna sequencer, and sequencing is finished by Shanghai Bo Ya Bioisystech Co., Ltd.Sequencing result shows that the length of this PCR product is 441bp, has the nucleotide sequence in the sequence table, wherein, from 5 ' the 1-154 bit base of end is the partial sequence of the 5th exon of the genome sequence of PSMB8, i.e. 5 of sequence 4 ' end 2388-2541 bit base (5 of sequence 5 ' end 635-788 bit base in sequence table) in sequence table; 155-399 bit base from 5 ' end is genomic the 5th the intron complete sequence of this gene, i.e. 5 of sequence 4 ' end 2542-2786 bit base in sequence table; From 5 ' the 400-441 bit base of end is i.e. 5 of the sequence 4 ' end 2787-2828 bit base (5 of sequence 5 ' end 789-830 bit base in sequence table) in sequence table of partial sequence of genomic the 6th exon of PSMB8.From 5 of SEQ ID NO:1 ' end the 146th bit base, promptly 5 of sequence 4 ' end the 2533rd bit base (5 of sequence 5 ' end the 780th bit base in sequence table) locates to exist two allelotrope of T, C in sequence table, is pleomorphism site.As from 5 of SEQ ID NO:1 ' end the 146th bit base, promptly in sequence table 5 of sequence 4 ' end the 2533rd bit base (5 of sequence 5 ' end the 780th bit base in sequence table) when being T, its homozygotic genotype is TT; From 5 of SEQ ID NO:1 ' end the 146th bit base, promptly in sequence table 5 of sequence 4 ' end the 2533rd bit base (5 of sequence 5 ' end the 780th bit base in sequence table) when being C, its homozygotic genotype is CC; Their heterozygote genotype is TC.

3, the dna sequence dna homology search is identified

By the American National biotechnology (NCBI of information center, National Center for BiotechnologyInformation, http://www.ncbi.nlm.nih.gov) BLAST of website (Basic LocalAlignment Search Tool) software, the known physiological function gene of announcing in the dna sequence dna (SEQ ID NO:1) that order-checking back is obtained and the GenBank database carries out sequence homology relatively, to identify and to obtain the function information of this dna sequence dna, the result shows that pig and people's sequence similarity is 88%, the proteasome subunit PSMB8 of this sequence encoding pig.

Two, the detection of the RFLP polymorphism of the partial dna sequence of pig PSMB8

Respectively to greatly enhance 22 of logical combinations (Da Bai * length in vain * Tongcheng pig), the total DNA of poba gene group of 30 of logical combination (long white * Da Bai * Tongcheng pig) 25 and Tongcheng pigs of growing up is a template, carries out pcr amplification under the guiding of primer 1:5 '-ATGTTCTCCACTGGTAGCGG-3 ' and primer 2: 5 '-TACTCTCCACCTTCACCCAC-3 '.PCR reaction cumulative volume is 20 μ l, wherein, and the about 100ng of pig genomic dna, 2 μ l, 10 * PCR damping fluids (Promega), MgCl ₂Final concentration is 1.5mmol/L, and the dNTP final concentration is 150 μ mol/L, and primer 1 and primer 2 final concentration respectively are 0.2 μ mol/L, 2U Taq archaeal dna polymerase.The PCR reaction conditions is: 94 ℃ of 4min of elder generation; 94 ℃ of 30s then, 58 ℃ of 30s, 72 ℃ of 20s, 35 circulations; Last 72 ℃ are extended 5min.Then the PCR reaction product is detected with 1.5% agarose gel electrophoresis, the result has obtained the electrophoretic band of 441bp size.With PCR product Hind6I digestion with restriction enzyme, endonuclease reaction system (10 μ l): 1 * enzyme cutting buffering liquid, 1 μ l, PCR product 3-5 μ l, restriction enzyme 0.5 μ l (5U) uses H ₂O supplies 10 μ l.The enzyme tangent condition is: 37 ℃ of water-bath 4h.Detecting enzyme with 2% agarose gel electrophoresis cuts the result and writes down genotype, the result is as shown in Figure 3 (among Fig. 3, swimming lane M is Marker, swimming lane TT is that genotype is the homozygote of TT, swimming lane CC is that genotype is the homozygote of CC, swimming lane TC is that genotype is the heterozygote of TC), show in the TT genotype individuality, have to the band of a 441bp; In the TC genotype individuality, obtain three bands: a 441bp, a 298bp and a 143bp band; In the CC genotype individuality, obtain two bands: a 298bp and a 143bp band.When allelotrope is C, recognition site (the G ↓ CGC) that occurs restriction enzyme Hin6I from 5 of SEQ ID NO:1 ' end 143-146 bit base, if cut this fragment with the Hin6I enzyme, then fragment of 441bp only appears in T allelotrope, 143bp and two fragments of 298bp then can appear in C allelotrope, owing to this locus is controlled by above-mentioned two allelotrope, thereby can form TT, TC, three kinds of genotype of CC.

Embodiment 3, detection pig immune trait and the production traits

With 15 of Large Whites, 15 of landraces, 15 of durocs, 37 of Tongcheng pigs totally 82 individualities are experimental subjects, detect the single nucleotide polymorphism and the RFLP polymorphism of 82 individualities according to the method for embodiment 2, determine their genotype, its result is as shown in table 1, shows all not detect the CC genotype in the individuality of 4 pig varieties that detected, only in the pig of Tongcheng, detect the TC genotype, illustrate that T allelotrope has comparative advantage.

The distribution of each genotype of table 1 in 4 pig varieties

Kind	Number of individuals	Genotype			Gene frequency
							CC	CT	TT	C	T
		Large White landrace duroc Tongcheng pig	15 15 15 37	0 0 0 0	0 0 0 11	15 15 15 26						0 0 0 0.1486	1 1 1 0.8514

Method according to embodiment 2 detects following 80 individualities: greatly enhance 21 pigs of logical combination (Da Bai * (in vain long * Tongcheng)), 25 pigs of logical combination (in vain long * (Da Bai * Tongcheng)) grow up, single nucleotide polymorphism and the RFLP polymorphism of 34 pigs of Tongcheng pure breeding group, determine their genotype, and carry out genotype and production, immune character association analysis.

With following least square model analysis immune character (content of hemoglobin, pcv, mean corpuscular volume (MCV), blood IgG antibody content, NCHC, average content of hemoglobin, total white blood cells and red corpuscle sum) and the production traits (nascent end body weight (90KG) age in days that reaches, dressing percentage, estimation eye muscle area (cm ²), leg stern ratio, leaf fat rate, intramuscular fat, shearing rate, pH value, average daily gain, the thickness of backfat, yellowish pink, marble grain, percentage of water loss and drip loss):

y _ijk＝μ+GENOTYPE _i+SEX _j+COMBINATION _k+ε _ijk，

Wherein, y _IjkBe the character observation value, μ is a population mean, GENOTYPE _iBe genotype effect, SEX _jBe sex effect, COMBINATION _kBe the effect of Different Cross Combinations, ε _IjkBe random error, suppose obey N (0, σ ²) distribute.

The result of the proterties significant difference of proterties between the different genotype (least square mean and standard error analysis) is shown in table 2 and table 3, and other proterties does not have significant difference between different genotype.

The association analysis of table 2 different genotype and part producing proterties

Genotype	Nascent reaching finished the body weight age in days	Dressing percentage	Estimation eye muscle area (cm 2)	The leaf fat rate	Leg stern ratio	Intramuscular fat (%)
							TT	197.094±2.332	75.509±0.268	28.949±0.948	3.9820.183	29.4060.231	3.154±0.172
TC	215.185±6.219	72.976±0.714	21.078±2.529	5.3160.488	28.0060.615	4.827±0.460
							TT/TC	0.008**	0.001**	0.005**	0.012*	0.036*	0.001**

Annotate: * * represents that difference is extremely remarkable, and * represents significant difference.

As can be seen from Table 2, the genotypic dressing percentage of TT, estimation eye muscle area (cm ²) and leg stern ratio character value all the utmost point be significantly higher than the genotypic respective value of TC; Reach market weight (90KG) age in days for nascent, these 3 proterties of leaf fat rate and intramuscular fat, the TC genotype respective value all utmost point is significantly higher than TT genotype respective value.The intramuscular fat of TC genotype individuality and leaf fat rate are than higher; The speed of growth of TT genotype individuality is than very fast, and it is all relatively good to produce meat and lean meat ratio.

The association analysis of table 3 different genotype and partial immunity proterties

Genotype	Hb H GB content (g/L)	Pcv HCT (%)	Mean corpuscular volume (MCV) MCV (fL)
				TT	98.362±2.058	36.357±0.850	58.397±0.569
TC	110.000±5.224	42.000±2.158	62.222±1.444

TT/CT

0.042*

0.018*

0.016*

Annotate: * * represents that difference is extremely remarkable, and * represents significant difference.

As shown in Table 3, at HGB (g/L), in these 3 immune characters of HCT (%) and MCV (fL), the genotypic respective value of TC all is significantly higher than the genotypic respective value of TT, shows that the immune character of TC genotype individuality is better.

Sequence table

<160>6

<210>1

<211>441

<212>DNA

＜213〉wild boar be born in the year of pig (Sus scrofa domestica Brisson)

<220>

<221>misc-feature

<222>(146)

＜223〉n=t or c

<400>1

atgttctcca ctggtagcgg gaacacctac gcttatgggg tcatggacag tggccatcgg 60

tacgatctta gcattgaaga ggcctatgac ctgggccgga gggctattgt tcatgccact 120

catcgagaca gctattctgg aggcgntgtc aatagtaaga gactaaggcc ccccgccccc 180

atcctggcgg gtggggggag ggggtttgag attgggagac gtggaggagg cagaggggca 240

gggagcaggt gttcagagcc agaacagggg catcggaagt cccagcatga aaggctttga 300

cattgcgtgt tgctgttgcc cccgtcaaca gaacatcacg tataatagga actcaccgaa 360

ggggaaagtc acgccctgtt ctttctgcct tgcttgcagt gtaccacatg aaggaagatg 420

ggtgggtgaa ggtggagagt a 441

<210>2

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>2

atgttctcca ctggtagcgg 20

<210>3

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>3

tactctccac cttcacccac 20

<210>4

<211>3102

<212>DNA

＜213〉wild boar be born in the year of pig (Sus scrofa domestica Brisson)

<220>

<221>misc-feature

<222>(2533)

＜223〉n=t or c

<400>4

acttcctcct gcgagagagc ggacgcatct ccggtgctag gcgatcatgg cgctgctgga 60

tgtttgcgga gccccccgag cgcagaagga agactgggct ttcccagccg cggaaagccg 120

gcagcgctcg gaccctggtc actacagttt ctctatgcga tctccggaac tcgccctccc 180

ccggggaatg caggtcaggg ctgtaagaaa atcctctggg aggtgggcag gagggcgcag 240

agtaatggag ggtctgcctt gagattgtct aaaaggcggg gaaatgcgga ccggaccacc 300

ctttagcccg gtgagagaca catcctgggt ccaggacctg agccaggaca gaggccacgt 360

gggacttggg gactggcagc ctgagggtta ggaagtgcag agtagggcag agagttagag 420

gtgagatggg ggctcccatc cggagacaag aaagcttttt caccacacac aaatcacgca 480

gcctttctct tgggactatc ttaggataca agggctgtgt attgtcttag aaacctatgc 540

gtttcagcgg tcagcctcgc ctcagccact cactgtggtg tggcttggat caagtccctg 600

tctaagtctt gggcttctct tccataaaat caggatacta ttaataaaat gtgttccctg 660

agggttaaag gacatataat gaaaaaaaaa aaacaaaaca aaaaaccatt atagtggccc 720

aaagttttta gcctacagta acattttctt tctttttgac tgtgctggaa aatgaaaaag 780

gttggtgtca tggcctcttc tttcccttct ctcaaaccat ttccttgttg agatggattg 840

attcttagcc gtttcactta ttgattgttg attctgtgta tctctgcact cccacagtct 900

ctgaaatcct ttcctggcgc ttccagcttc ggaagtttcc agtgcccctt tctctgcctt 960

ttctgtctca ctagcccact gaattcctcc ggtccctggg tggggatgga gaaaggaacg 1020

tacagattga gatggctcat ggcactacca cgctggcctt caagttccag cacggagtga 1080

ttgtggcggt ggattctcgg gcctcggccg ggagttacat tggtgagtgt gtgttccagc 1140

tggcagcgtc tggacatccg ggccctcctc tgagcccggg ttcgacatca gcaccaaagt 1200

ggaagaagat gctgatttag gacatacagg gagagctgtg tgtgtgtgtg tggaggtcat 1260

tgctcctttc tcaaaactcc atcttcttcc agccacatta agagtgaaca aggtgattga 1320

gattaaccct tacctgcttg gcaccatgtc tggctctgcg gcggactgtc agtattggga 1380

gcgtctgctg gccaaggagg caggtaggtg aggcctctga cctctcttcc aagcctaggt 1440

ttcaatctcg gattctgggt cttccagttt gctgctcctc attcacttcc cctgtgctaa 1500

cgtagcattt tagcgctaac gaagtagttc aaaaaacagc cttttggtga ttgaaaatcc 1560

tctcccaaat ctcagcctgt gctcctgccc ataagccaga caatttgtea gtcatttagc 1620

actgacacca tcggtgctgg gtgggatctt atggttcact gggatctctc gatgtggctt 1680

agatacttta cattccaaat ctttccctga tccttagagc ccacaaaacc cttcctttca 1740

aatacttagc tcttcagtca ttgtctgcac ccccagctag ccttctcctg ccacttcgaa 1800

gtcccctccc aaaccgaacc gccctgtcct ccccaggttg tactatctgc ggaatgggga 1860

ccgtatctca gtgtccgcag cctccaaact gctctccaat atgatgtacc agtaccgggg 1920

catgggcctc tccatgggca gtatgatctg tggctgggac aagaaggtga gtctcccact 1980

gtggatgaga gatcagtttc cccatcctac actttcactc cctcccctcc gaggtatgtg 2040

gcacgtggac tccttggtta cagaactgtt ccagcacgtc cactggcaat ttctgggtct 2100

ggctgaggac ccactgtttc ataaacttgt tccttttgac aaaatagctg attccaagtt 2160

tgtccccccc ccccaaagtt ctatgattgt taccaaagtg ctatgttacc caaagttcaa 2220

atgtttctgt ggtcaccctt attctttcgt ccacttgtcc tggctcacac ttgaatctgt 2280

ccgttaccaa gagcttcctc tccagtcccc acctggaacc ttcttacatc ctatagggtc 2340

ctggactcta ctatgtggat gaaaatggga ctcggctctc gggaaatatg ttctccactg 2400

gtagcgggaa cacctacgct tatggggtca tggacagtgg ccatcggtac gatcttagca 2460

ttgaagaggc ctatgacctg ggccggaggg ctattgttca tgccactcat cgagacagct 2520

attctggagg cgntgtcaat agtaagagac taaggccccc cgcccccatc ctggcgggtg 2580

gggggagggg gtttgagatt gggagacgtg gaggaggcag aggggcaggg agcaggtgtt 2640

cagagccaga acaggggcat cggaagtccc agcatgaaag gctttgacat tgcgtgttgc 2700

tgttgccccc gtcaacagaa catcacgtat aataggaact caccgaaggg gaaagtcacg 2760

ccctgttctt tctgccttgc ttgcagtgta ccacatgaag gaagatgggt gggtgaaggt 2820

ggagagtacg gacgtcagtg acctgatgca ccagtaccgg gaggccagtc tgtaagggtg 2880

gcggcggcag ctgggccggc ctctcctgag gaagccgggc ctactcaggg acctggacca 2940

gttcaggccc cagaagaaag aggggcctaa cgtgacccag agggaatgaa ccagtggtcc 3000

ctgggaggca gggcggagtt tatccttttg tgtagtctct gcttcaatgg agcctccccc 3060

cctccccaac cccaaccttc tgggcatctc actaagcaca at 3102

<210>5

<211>1104

<212>cDNA

＜213〉wild boar be born in the year of pig (Sus scrofa domestica Brisson)

<220>

<221>misc-feature

<222>(780)

＜223〉n=t or c

<400>5

acttcctcct gcgagagagc ggacgcatct ccggtgctag gcgatcatgg cgctgctgga 60

tgtttgcgga gccccccgag cgcagaagga agactgggct ttcccagccg cggaaagccg 120

gcagcgctcg gaccctggtc actacagttt ctctatgcga tctccggaac tcgccctccc 180

ccggggaatg cagcccactg aattcctccg gtccctgggt ggggatggag aaaggaacgt 240

acagattgag atggctcatg gcactaccac gctggccttc aagttccagc acggagtgat 300

tgtggcggtg gattctcggg cctcggccgg gagttacatt gccacattaa gagtgaacaa 360

ggtgattgag attaaccctt acctgcttgg caccatgtct ggctctgcgg cggactgtca 420

gtattgggag cgtctgctgg ccaaggagtg caggttgtac tatctgcgga atggggaccg 480

tatctcagtg tccgcagcct ccaaactgct ctccaatatg atgtaccagt accggggcat 540

gggcctctcc atgggcagta tgatctgtgg ctgggacaag aagggtcctg gactctacta 600

tgtggatgaa aatgggactc ggctctcggg aaatatgttc tccactggta gcgggaacac 660

ctacgcttat ggggtcatgg acagtggcca tcggtacgat cttagcattg aagaggccta 720

tgacctgggc cggagggcta ttgttcatgc cactcatcga gacagctatt ctggaggcgn 780

tgtcaatatg taccacatga aggaagatgg gtgggtgaag gtggagagta cggacgtcag 840

tgacctgatg caccagtacc gggaggccag tctgtaaggg tggcggcggc agctgggccg 900

gcctctcctg aggaagccgg gcctactcag ggacctggac cagttcaggc cccagaagaa 960

agaggggcct aacgtgaccc agagggaatg aaccagtggt ccctgggagg cagggcggag 1020

tttatccttt tgtgtagtct ctgcttcaat ggagcctccc cccctcccca accccaacct 1080

tctgggcatc tcactaagca caat 1104

<210>6

<211>276

<212>PRT

＜213〉wild boar be born in the year of pig (Sus scrofa domestica Brisson)

<400>6

Met Ala Leu Leu Asp Val Cys Gly Ala Pro Arg Ala Gln Lys Glu Asp

1 5 10 15

Trp Ala Phe Pro Ala Ala Glu Ser Arg Gln Arg Ser Asp Pro Gly His

20 25 30

Tyr Ser Phe Ser Met Arg Ser Pro Glu Leu Ala Leu Pro Arg Gly Met

35 40 45

Gln Pro Thr Glu Phe Leu Arg Ser Leu Gly Gly Asp Gly Glu Arg Asn

50 55 60

Val Gln Ile Glu Met Ala His Gly Thr Thr Thr Leu Ala Phe Lys Phe

65 70 75 80

Gln His Gly Val Ile Val Ala Val Asp Ser Arg Ala Ser Ala Gly Ser

85 90 95

Tyr Ile Ala Thr Leu Arg Val Asn Lys Val Ile Glu Ile Asn Pro Tyr

100 105 110

Leu Leu Gly Thr Met Ser Gly Ser Ala Ala Asp Cys Gln Tyr Trp Glu

115 120 125

Arg Leu Leu Ala Lys Glu Cys Arg Leu Tyr Tyr Leu Arg Asn Gly Asp

130 135 140

Arg Ile Ser Val Ser Ala Ala Ser Lys Leu Leu Ser Asn Met Met Tyr

145 150 155 160

Gln Tyr Arg Gly Met Gly Leu Ser Met Gly Ser Met Ile Cys Gly Trp

165 170 175

Asp Lys Lys Gly Pro Gly Leu Tyr Tyr Val Asp Glu Asn Gly Thr Arg

180 185 190

Leu Ser Gly Asn Met Phe Ser Thr Gly Ser Gly Asn Thr Tyr Ala Tyr

195 200 205

Gly Val Met Asp Ser Gly His Arg Tyr Asp Leu Ser Ile Glu Glu Ala

210 215 220

Tyr Asp Leu Gly Arg Arg Ala Ile Val His Ala Thr His Arg Asp Ser

225 230 235 240

Tyr Ser Gly Gly Val Val Asn Met Tyr His Met Lys Glu Asp Gly Trp

245 250 255

Val Lys Val Glu Ser Thr Asp Val Ser Asp Leu Met His Gln Tyr Arg

260 265 270

Glu Ala Ser Leu

275

Claims (8)

1, boar production, immune character related protein are the protein with one of following amino acid residue sequences:

1) the SEQ ID No:6 in the sequence table;

2) with the amino acid residue sequence of SEQ ID No:6 in the sequence table through replacement, disappearance or the interpolation of one to ten amino-acid residue and the protein relevant with pig production character and immune character.

The encoding gene of 2, the described pig production of claim 1, immune character related protein.

3, encoding gene according to claim 2 is characterized in that: described encoding gene has one of following nucleotide sequence:

1) polynucleotide of SEQ ID No:5 in the sequence table;

2) polynucleotide of SEQ ID No:6 protein sequence in the code sequence tabulation;

3) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with the SEQ ID No:5 in the sequence table.

4, encoding gene according to claim 2 is characterized in that: described encoding gene has one of following nucleotide sequence:

1) polynucleotide of SEQ ID No:4 in the sequence table;

2) SEQ ID No:6 protein sequence in the code sequence tabulation;

3) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with the SEQ ID No:1 in the sequence table.

5, the carrier that contains claim 2 or 3 or 4 described encoding genes.

6, the clone that contains claim 2 or 3 or 4 described encoding genes.

7, the host bacterium that contains claim 2 or 3 or 4 described encoding genes.

8, a kind of method that detects pig production, immune character, be to use a pair of primer formed by the nucleotide sequence of SEQ ID No:2 in the sequence table and SEQ IDNo:3 that the genomic dna of pig to be measured is carried out pcr amplification, then pcr amplification product carried out following at least a detection:

1) described pcr amplification product is carried out single nucleotide polymorphism and detect, determine that from 5 of SEQ ID NO:1 ' end the 146th bit base be T;

2) cut described pcr amplification product with the Hin6I enzyme, detect in the endonuclease bamhi that obtains whether contain a 441bp band and/or a 143bp band and a 298bp band.

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