KR101748774B1 - Method for polymorphism analysis of Swine Leukocyte Antigen(SLA)-MIC2 gene - Google Patents

Abstract

The present invention relates to polymorphism analysis of the porcine leukocyte antigen (SLA) -MIC2 gene, a method of analyzing the polymorphism of the MIC2 gene through PCR amplification and sequencing of a specific region in genomic DNA extracted from seven kinds of pig tissues, And the new allele of SLA-MIC2 analyzed through this.
According to the present invention, by typing the SLA-MIC2 gene having a polymorphism through a simple process, it is possible to provide useful information for studying the relationship between the SLA-MIC2 gene and the immune response of the pig and immunological diseases .

Description

(SLA) -MIC2 gene (polymorphism analysis of Swine Leukocyte Antigen (SLA) -MIC2 gene)

The present invention relates to polymorphism analysis of a porcine leukocyte antigen (SLA) -MIC2 gene, and more particularly, to a method for detecting polymorphism of a SLA-MIC2 gene by PCR amplification and sequencing of a specific site in genomic DNA extracted from seven different kinds of pig tissues Gt; polymorphism < / RTI >

Pigs are economically important animals, and pigs that are similar in size and structure to human organs among organ transplant animals are being actively studied. Major Histocompatibility Complex (MHC) is called Swine Leukocyte Antigen (SLA) in pigs and it plays an important role in immune rejection in heterologous organ transplantation. .

MHC is an essential component of the immune system in all vertebrates. One of the greatest hallmarks of MHC-related genes is the presence of extreme polymorphisms within the locus. This is because evolution requires a gene encoding a highly polymorphic MHC molecule to recognize almost unlimited antigens. Thus, in immunogenics it is very important to find and classify polymorphisms within the MHC gene. The MHC class I chain-related (MIC) gene was first described in primates and mammals (Bahram et al. 1994). One or more functional MIC genes have been studied in some species and the number of pseudogene has also been reported (Bahram 2000; Chardon et al. 2001).

Seven MIC genes were identified in the human genome. Among them, MICA and MICB are functional genes, and MICC to MICG are the above genes (Bahram & Spies 1996). Three complete MIC genes were analyzed in the region of bovine MHC class I through bovine genome analysis (Birch et al. 2008) and were temporarily named BoLa MIC1, BoLa MIC2, and BoLa MIC3.

MIC1 and MIC2 have been reported in pigs (Velten et al. 1999), whereas MIC1 is an incomplete gastric gene, whereas MIC2 is a functional gene (Chardon et al., Renard et al., 2006 ) Detailed characteristics of gene structure, expression pattern, and function have not yet been clarified.

Accordingly, the present inventors extracted genomic DNA from seven kinds of pigs and 145 pigs, amplified and sequenced specific exon regions capable of genetic polymorphism analysis, and analyzed the genetic polymorphism of SLA-MIC2 gene through a simple process The present invention has been completed.

Related prior arts include Korea Patent No. 10-0834563 (genotype analysis method using base mutation of the CFS non-gene existing in the region 3 of porcine main histocompatibility complex and primers used therein), Korean Patent Laid- -0051803 (identification of base mutations located in region 3 of porcine major histocompatibility complex).

An object of the present invention is to amplify a specific exon region capable of polymorphism analysis of the SLA-MIC2 gene based on genomic DNA extracted from a variety of pig tissues and sequencing a functional part (exon) of the amplified DNA product MIC2 gene by analyzing the nucleotide sequence of the SLA-MIC2 gene by typing the allele of SLA-MIC2.

In order to achieve the above object, the present invention provides a primer set for PCR consisting of SEQ ID NOS: 1 and 2, which amplifies the entire exon 2 to exon 4 regions of a porcine leukocyte antigen (SLA) -MIC2 gene.

In addition, the present invention provides a sequencing primer set consisting of SEQ ID NOS: 3 and 4 for sequencing exon 2 of the porcine leukocyte antigen (SLA) -MIC2 gene in forward and reverse directions.

In addition, the present invention provides a sequencing primer set consisting of SEQ ID NOS: 5 and 6 for sequencing exon 3 of the porcine leukocyte antigen (SLA) -MIC2 gene in forward and reverse directions.

The present invention also provides a primer set for sequencing consisting of SEQ ID NOS: 7 and 8 for sequencing exon 4 of the porcine leukocyte antigen (SLA) -MIC2 gene in forward and reverse directions.

Further, the present invention provides a method for producing a recombinant vector comprising: (1) separating genomic DNA from a tissue of a pig; (2) PCR-amplifying the separated genomic DNA using a primer capable of amplifying the entire exon 2 to exon 4 regions of a porcine leukocyte antigen (SLA) -MIC2 gene; And (3) sequencing the DNA fragment amplified in step (2) using a sequencing primer; (SLA) -MIC2 gene comprising a polynucleotide of the present invention.

The method further comprises performing a bi-directional sequencing of the clone obtained by cloning the DNA fragment amplified in the step (2) using exon 2, 3 and 4 specific sequencing primers.

In addition, the method further comprises separating RNA from the tissue of the pig, performing RT-PCR, and performing bidirectional sequencing of the obtained cDNA product.

According to the present invention, it is possible to amplify a specific exon region capable of analyzing the polymorphism of the SLA-MIC2 gene based on genomic DNA extracted from a variety of pig tissues, and to measure the functional portion (exon) of the amplified DNA product By sequencing and analyzing the nucleotide sequence, the SLA-MIC2 gene having a polymorphism can be typed by a simple procedure, and the information on the genetic polymorphism can be used for the relationship between the SLA-MIC2 and the immune response of the pig, And to use it as a useful resource for research.

1 is a flow chart for the structure and polymorphism analysis of the SLA-MIC2 gene.
Figure 2 shows the results of PCR amplification of 15 alleles of SLA-MIC2.
FIG. 3 is a graph showing the RT-PCR results and the relative density of SLA-MIC and GAPDH for confirming the mRNA expression level of SLA-MIC2 in 15 tissues of pigs.
Figure 4 compares the amino acid sequences of the MIC genes of human, pig and cattle.
FIG. 5 is a graph showing the homology of MIC genes of human, pig, and bovine.
6 is a phylogenetic map of the MIC gene.

Hereinafter, the present invention will be described in detail.

The MIC2 gene of the present invention is a locus-specific gene located in the red-labeled region in SLA class I, which is a main histocompatibility complex of pigs.

The present invention provides a primer set for PCR consisting of SEQ ID NOS: 1 and 2, which amplifies all of the exon 2 to exon 4 regions of a porcine leukocyte antigen (SLA) -MIC2 gene.

In addition, the present invention provides a sequencing primer set consisting of SEQ ID NOS: 3 and 4 for sequencing exon 2 of the porcine leukocyte antigen (SLA) -MIC2 gene in forward and reverse directions.

In addition, the present invention provides a sequencing primer set consisting of SEQ ID NOS: 5 and 6 for sequencing exon 3 of the porcine leukocyte antigen (SLA) -MIC2 gene in forward and reverse directions.

The present invention also provides a primer set for sequencing consisting of SEQ ID NOS: 7 and 8 for sequencing exon 4 of the porcine leukocyte antigen (SLA) -MIC2 gene in forward and reverse directions.

Further, the present invention provides a method for producing a recombinant vector comprising: (1) separating genomic DNA from a tissue of a pig; (2) PCR-amplifying the separated genomic DNA using a primer capable of amplifying the entire exon 2 to exon 4 regions of a porcine leukocyte antigen (SLA) -MIC2 gene; And (3) sequencing the DNA fragment amplified in step (2) using a sequencing primer; (SLA) -MIC2 gene comprising a polynucleotide of the present invention.

The method further comprises performing a bi-directional sequencing of the clone obtained by cloning the DNA fragment amplified in the step (2) using exon 2, 3 and 4 specific sequencing primers.

In addition, the method further comprises separating RNA from the tissue of the pig, performing RT-PCR, and performing bidirectional sequencing of the obtained cDNA product.

Prior to analyzing the polymorphism of the SLA-MIC2 gene, CLUSTALW was used to align the available genomic sequences of SLA-MIC2 (CT737281, AJ251914 and NM_001114274) and analyze the exon-intron structure. There was a gap between the exon-intron structure (between exon 6 at the end of exon 4) in AJ251914 (Chardon et al., 2001) and the other two sequences (CT737281, NM_001114274). In order to solve this inconsistency, DNA fragments from a part of the exon 3 to the 3 'UTR were amplified using a forward primer (MIC2-cDNA-F) and an oligo (dT) ₁₇ reverse primer of SEQ ID NO: After sequencing, the sequences of SLA-MIC2 gDNA were complemented.

We used human MIC gene information to determine DNA amplification sites for polymorphism analysis of genomic DNA-based SLA-MIC2 gene. Namely, regions including exons 2, 3 and 4, which are functionally important regions in human MICA and MICB, were also applied to SLA-MIC2 of pigs, and this region was selected as a target region for DNA amplification. (SLA-MIC2-gDNA-F1) of SEQ ID NO: 1 and the reverse primer (SLA-MIC2 (SEQ ID NO: 2) of SEQ ID NO: 2) were amplified using various primer sets for successful genomic DNA- -gDNA-R1), the genomic DNA-based PCR was performed and all the samples were successfully amplified. In the PCR amplification, a part of intron 1 (235 bp), exon 2 (255 bp), intron 2 (281 bp), exon 3 (285 bp), intron 3 (575 bp), exon 4 (279 bp), intron 4 (120 bp) and a portion of intron 5 (361 bp).

Next, the obtained DNA fragment was subjected to a bi-directional sequencing reaction using the sequencing primers of SEQ ID NOS: 3 to 8 by direct sequencing, and as a result, a novel SLA-MIC2 allele which was not previously reported was analyzed . The new allele appears in both haplotypes and is divided into haplotypes by cloning to verify the correct sequence. The clone obtained through the cloning process is subjected to bidirectional sequencing using exon-specific primers, Was determined.

The exon-specific primer used in the above procedure was a forward primer (SLA-MIC2-E2sF) of SEQ ID NO: 3 and a reverse primer (SLA-MIC2-E2sR) of SEQ ID NO: 4, sequencing of exon 3 (SLA-MIC2-E3sF) of SEQ ID NO: 5 and a reverse primer (SLA-MIC2-E3sR) of SEQ ID NO: 6, a primer set for sequencing of exon 4, a forward primer -MIC2-E4sF) and the reverse primer of SEQ ID NO: 8 (SLA-MIC2-E4sR), and when sequential bi-directional sequencing was performed, consistent sequence information was derived.

In addition, RT-PCR was performed to identify the pseudogene of the newly analyzed allele.

Alleles of 10 new SLA-MIC2 were obtained and deposited in GenBank (Accession numbers: KM541686, KM541688, KM541688, KM541690, KM541691, KM541692, KM541693, KM541694 and KM541695)

The primer sequences used in the present invention are summarized in Table 1 below.

[Table 1] PCR and sequencing primer sequences used in the present invention

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1. Preparation of seven kinds of experimental animals and genomic DNA isolation

Seven varieties of pigs used in the present invention were 22 SNU (Seoul National University) mini pig, 25 KNP (Korean traditional pig), 13 NIH (National Health Research Institute) mini pig, 22 DUR (duroc), 20 LAN (land race) , 19 YOR (Yorkshire) and 24 BER (Berkshire). The method for isolating genomic DNA from the pigs was performed using standard separation techniques (Sambrook J. et. Al., 2001) from 1 ml of peripheral blood containing 0.5 g of ear tissue or 6% EDTA.

Specifically, the ear tissues were lysed in a cell lysis buffer solution [10 mM Tris-HCl (pH 8.0) and 0.1 M (pH 7.0) containing 0.5% SDS and 20 μl of 20 mg / ml proteinase K (Promega, Madison, WI) EDTA]. Next, the supernatant was purified using the phenol / chloroform extraction method, and DNA pellets were obtained by alcohol precipitation. DNA from blood samples was obtained from previously reported Miller A. et. al. (1998).

Example 2. PCR primer design

CLUSTALW was used to align the available genomic sequences of SLA-MIC2 (CT737281, AJ251914 and NM_001114274) and to analyze the exon-intron structure. There was a gap between the exon-intron structure (between exon 6 at the end of exon 4) in AJ251914 (Chardon et al., 2001) and the other two sequences (CT737281, NM_001114274). To solve this inconsistency, DNA fragments from a part of exon 3 to 3 'UTR were amplified using a forward primer (SLA-MIC2-cDNA-F) and an oligo (dT) ₁₇ reverse primer of SEQ ID NO: After sequencing the intron sequence, the sequence of the SLA-MIC2 gDNA was complemented. Primers for amplifying the cDNA were designed using the PRIMER DESIGNER (Version 2.0, Scientific and Educational Software 1990, 1991) for the reference sequence (CT37281).

Example 3. Genomic PCR

Amplification using genomic DNA (including all exons 2 to 4 exon) was carried out using the forward primer (MIC2-gDNA-F1) of SEQ ID NO: 1 and the reverse primer (MIC2-gDNA-R1) of SEQ ID NO: 2 . The PCR profile was subjected to 35 cycles of denaturation at 94 DEG C for 35 seconds, annealing at 66 DEG C for 45 seconds, and extension at 72 DEG C for 2 minutes after the initial denaturation at 95 DEG C using a T-3000 thermocycler (Biometra, Germany) , And final incubation at 72 [deg.] C for 10 minutes.

The PCR products were identified by electrophoresis on a 1.5% agarose gel under 1X TAE buffer for about 25 minutes at 100 V. The gel was stained with EtBr and visualized under ultraviolet light.

Example 4. Genomic DNA sequencing

For the direct sequencing of the PCR product obtained in Example 3, 5 μl of the PCR product was incubated with 4 U of exonuclease I (Fermentas, St. Leon-Rot, Germany) and 0.8 U of shrimp alkaline phosphatase (USB Corporation, Cleveland, Ohio). Sequencing reactions were performed using primers of SEQ ID NOS: 3 to 8 and ABI PRISM BigDye ™ Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, Calif.) And analyzed using an automated DNA analyzer (ABI 3730XL, Applied Biosystems). All sequences were manually identified and edited for ambiguous sequences using BioEdit V7.0 (Hall 1999).

Example 5. Identification of new alleles of SLA-MIC2

As a result of the direct sequence analysis of Example 4, a new allele of the SLA-MIC2 gene was analyzed, and a new allele was identified in both haploids, and then cloned into a haplotypes for accurate sequencing to perform bidirectional sequencing And the exact sequence of the new allele was verified.

The PCR products obtained in Example 3 above were purified using QIAquick (TM) Gel Extraction Kit (QIAGEN, The Netherlands) and ligated to pGEM-T vector (Promega Corporation, USA). The PCR product ligation vector was inserted into E. coli DH-5a competent cells, and the transformed E. coli was treated with 40 mg / ml X-gal, 100 mM IPTG and 50 μg / ml ampicillin at 37 ° C On an agar plate. Thereafter, 5 white colonies were selected, and the inserted region was amplified using T7 and SP6 primers to confirm insertion of the PCR product into the vector. The clone obtained through the above cloning was used as the exon 2 of the SLA-MIC2 gene in the forward direction (SLA-MIC2-E3sF) and reverse (SLA-MIC2-E3sR) primer set consisting of SEQ ID NOS: 3 and 4 sequencing in the reverse direction (SLA-MIC2-E2sF) Sequence sequencing reaction using a primer set consisting of SEQ ID NOS: 5 and 6 and a primer set consisting of SEQ ID NOS: 7 and 8 sequencing exon 4 in the forward direction (SLA-MIC2-E4sF) and reverse direction (SLA-MIC2-E4sR) Bi-directional sequencing of the cloned products was performed.

After verifying the sequencing results by manual inspection of the sequencing chromatogram, the sequences were blasted to the NCBI database and our database. The complete sequence of the exons 2,3,4 of the SLA-MIC2 gene was aligned using CLUSTALW (Thompson et al., 1994).

Example 6. RNA Isolation and RT-PCR

RNA was isolated and RT-PCR was performed to confirm the pseudogene of the newly analyzed allele through the present invention. In more detail, R < 5 > in nine different tissues (liver, stomach, lung, small intestine, heart, skin, tongue, spleen, muscle, large intestine, testis, kidney, ovary, Total RNA was isolated according to the manufacturer's manual using the A-BLUE ^™ RNA Extraction Kit (Intron Biotechnology, Korea).

Oligo (dT) ₁₇ and SuperScript (TM) III reverse transcriptase (Invitrogen, USA) were added to reverse transcribe the separated RNA, and ₂₅ μl reaction was performed at 50 ° C for 50 minutes. ≪ / RTI > for 15 minutes. Next, the full length SLA-MIC2-cDNA (5 'to 3' UTRs) was inserted into the forward primer of SEQ ID NO: 9 (SLA-MIC2-cDNA-F) and the reverse primer of SEQ ID NO: 10 (SLA-MIC2- MIC2-cDNA of exons 2 to 3 'UTR was amplified by using SEQ ID NO: 11 (SLA-MIC2-cDNA-F1) in order to confirm once again whether other gene loci including pseudogene were amplified. (SEQ ID NO: 10) (SLA-MIC2-cDNA-R)) were subjected to reverse transcription PCR (RT-PCR) using a reverse primer.

The PCR mixture contained 50 ng of cDNA, 0.5 U of Super-Therm DNA polymerase (JMR Holdings, Kent, UK), 0.5 uM primer, 1 X PCR reaction It consists of a buffer (1.5 mM MgCl ₂₎ and a 0.1mM and 0.1mM dNTPs.

Amplification was repeated 35 times for a cycle of initial denaturation at 95 캜 for 5 minutes, denaturation at 95 캜 for 1 minute, annealing at 48 for 1 minute, and extension at 72 캜 for 1 minute.

The RT-PCR product was amplified using the SLA-MIC2-cDNA sequencing primer set of the forward primer (SLA-MIC2-cDNA-sF) of SEQ ID NO: 13 and the reverse primer (SLA-MIC2- Sequence analysis was performed.

Example 7. Statistical treatment

The frequency of alleles, the number of alleles, the number of alleles available, the expected heterozygosity and the Hardy-Weinberg equilibrium are calculated by POPGENE 1.32 (Yet. Et. Al., 1999) .

Experimental Example 1. Genetic polymorphism analysis of SLA-MIC2 gene

As a result of direct sequence analysis of genomic DNA isolated from porcine tissue, a new allele of SLA-MIC2 which has not been reported so far was found, and a PCR product was cloned to precisely identify a new allele sequence. , 3, and 4 (Example 5). The results are shown in Table 2.

[Table 2] Polymorphism analysis of exons 2, 3 and 4 of the SLA-MIC2 allele

In Table 2 above, SLA-MIC2- * 01, SLA-MIC2- * 03, SLA-MIC2- * 04, SLA-MIC2- * 05 and SLA-MIC2- * 07 are alleles of previously reported SLA-MIC2 , The remaining MIC- * knO3 (-2), MIC- * knO2 (-3), MIC- * knO4 (-2), MIC- * knO4 (-3) The 10 alleles of kn05 (-2), MIC- * kn07 (-1a), MIC- * kn07 (-1t), MIC- * kn07 (-2) It is a newly acquired allele through polymorphism analysis.

In this new allele, 'kn' is an abbreviation for the konkuk new allele, which is arbitrarily named to distinguish the newly analyzed allele from the known allele, and the name of the allele existing in the Genbank database and the newly analyzed allele Where 01, 03, 04, 05 and 07 are the local name marking the position, and the negative numbers in parentheses represent the nucleotide difference from the previously reported alleles most closely related. The alleles SLA-MIC2- * kn07 (-1 _A ) and SLA-MIC2- * kn07 (-1 _T ) differ from SLA-MIC2- * 07 by one nucleotide (-1) In the case of SLA-MIC2- * kn07 (-1 _A ), G changed to "A" and in SLA-MIC2- * kn07 (-1 _T ), C changed to "T". In Table 2, the reference sequence refers to the region corresponding to SLA-MIC2 of the BAC sequence of NCBI accession number CT737281.

Experimental Example 2. Measurement of frequency and heterozygosity of SLA-MIC2 alleles in 7 different breeds of pigs

The frequency and heterozygosity of 15 alleles were measured in the seven varieties of pigs (BER, KNP, NIH, SNU, YOR, DUR, LAN) used in the present invention. The results are shown in Tables 3 and 4.

[Table 3] Frequency of SLA-MIC2 alleles in 7 varieties of pigs

[Table 4] SLA-MIC2 heterozygosity of 7 varieties of pigs

As shown in the results of Table 3 and Table 4, the previously reported MIC- * 01, SLA-MIC2- * 05 and SLA-MIC2- * 07 were the most common alleles and the observed heterozygosity range was 7 And 0.260-0.728 in the cultivars. The number of alleles in the SLA-MIC2 gene of pigs was smaller than the number of alleles of MHC class I and II, but the level of heterozygosity was similar.

Observed heterozygosity measured from 145 pigs of 7 different types of breeds ranged from 26% to 72.8% and expected heterozygosity ranged from 23.6% to 79.7%. From this, the proportion of pigs observed as heterozygotes in whole pigs was 53%, and the proportion of pigs expected to be statistically estimated was 85%.

The results of this heterozygosity correspond to the results of other previously studied SLA class II genes (Park et al., 2010; Thong et al., 2011). As expected, animal varieties including NIH and SNU mini pigs in inbred or closed breeding systems showed lower heterozygosity than outbred populations.

Experimental Example 3. Confirmation of PCR amplification products of 15 alleles of SLA-MIC2

PCR amplification products of 15 alleles of SLA-MIC2 were electrophoresed on 1.5% agarose gel, stained with EtBr and visualized under ultraviolet light (UV). The results are shown in FIG.

Experimental example 4. Analysis of mRNA expression level of SLA-MIC2

In order to analyze the mRNA expression level of SLA-MIC2 in each tissue, primers for amplifying the SLA-MIC2 exon 2 to 6 region were designed using the RNA isolated in Example 6, and semi-quantitative RT-PCR (Example 6). The amplification product (1080 bp) was then electrophoresed on an agarose gel, stained with EtBr, and visualized under ultraviolet light (UV). (GAPDH) gene was used to control the experimental fluctuation and GAPDH and SLA-MIC2 were amplified using Image Studio Analysis Software Version 4.0 (LI-COR Biosciences, USA) Density were compared.

The results are shown in FIG. Figure 3 (a) shows the level of expression in the 15 tissues of the GAPDH gene used as a control, and Figure 3 (b) shows the expression level of the RT-PCR amplified product (1080 bp) of the SLA-MIC2 exon 2-6 region FIG. 3 (c) is a graph showing the relative density of the amplified SLA-MIC2 and GAPDH calculated. FIG.

These results indicate that the SLA-MIC2 gene is expressed only in the small intestine, lung, and heart. In humans, the MICA and MICB genes are expressed only in cancer cells including lung, breast, ovarian, prostate, and colon cancers (Groh et al., 1999; 1996). These results suggest that the expression patterns of MIC genes among different species are not the same and that expression patterns of MIC genes may vary during the immune response even in the same species. Differences in the expression pattern of the MIC gene between humans and pigs suggest that MIC can cause significant differences in the immune responses of the two species.

Experimental Example 5. Analysis of gene structure of SLA-MIC2

In order to analyze the SLA-MIC2 gene structure, a primer set for the SLA-MIC2 sequence was first designed from the BAC sequence (Genbank accession number, AJ251914) and according to the exon-intron information of the SLA-MIC2 gene et al., 2001) attempted to amplify regions between the intron 1 to 4 region or the full-length cDNA of the SLA-MIC2 gene, but no amplification products were obtained. As a result of a comparison between the AJ251914 and another BAC sequence containing the SLA-MIC2 gene (Accession No. CT737281) through an additional search, a difference in the base sequence from the end of the exon 4 of the SLA-MIC2 gene to the end of the exon 6 was found . Also, in the ensemble genome browser, the present annotation of the SLA-MIC2 gene (Accession No. NM_001114274) was found to be inconsistent with AJ251914.

MIC2 exon 3 specific forward primer (MIC2-cDNA-sF) and poly A tail specific oligodeptide (dT) ₁₇ reverse primer (MIC2-cDNA-sF) were used for the RNA isolated by Example 6 to solve the above- RT-PCR (Reverse Transcription PCR) was performed to obtain a cDNA product of 670 bp. As a result of the analysis of the exon-intron structure of the full-length SLA-MIC2, the nucleotide sequence and the position of the exon-intron boundary of exon 5 and exon 6 were found as compared with the sequence information already reported , Which enabled us to identify the correct exon-intron structure of the entire SLA-MIC2 cDNA. The exon-intron structure of SLA-MIC2 can be confirmed from FIG. 1, and the exon region sequence and amino acid sequence of SLA-MIC2 can be confirmed by the following Table 5.

[Table 5] SLA-MIC2 exon region sequence and amino acid sequence

According to Table 5, the SLA-MIC2 gene is composed of 374 amino acids. As a result of the comparison with the human MICA gene, the leader sequence (exon 1), three extracellular domains? 1,? 2,? 3 2, 3 and 4), a transmembrane domail (exon 5), and a cytoplasmic domain (exon 6).

On the other hand, glycosylation is important for protein stability and biological function. The glycosylation reaction depends on the position and number of N-linked oligosaccharides added to the protein chain. In the present invention, N-linked glycosylation sites (three sites in alpha 1, four sites in alpha 3) from the SLA-MIC2 gene And seven sites were identified. It has been reported that there are six potential N-linked glycosylation sites in the bovine MIC gene and eight potential N-linked glycosylation sites in the human MICA gene.

In addition, cystein is one of the amino acids abundant in organisms and is known to be present in functionally important protein sites. In the present invention, ten cysteine residues concentrated in the region encoding the extracellular domain of the SLA-MIC2 gene were identified. (Cys-207, -219, -270 and -76) in alpha 1, five sites (Cys-99, -123, 130, 158, and -176) in alpha 2, 291) exists. It is known that human MICA contains seven cysteine residues.

Experimental Example 6. Comparison of amino acid sequence and homology of MIC gene between pigs, human and cow

As shown in Experimental Example 5, the amino acid sequence of the porcine SLA-MIC2 gene was identified and compared with the amino acid sequence of the MIC gene of human and bovine. As a result, as shown in FIG. 4, the MICA gene in human is composed of 383 amino acids, and the BoLA MIC1 and 2 are composed of 384 amino acids. On the other hand, the MIC2 gene of pigs is composed of 374 amino acids as shown in Table 4 above.

As a result of comparing the intron-exon structures of the MIC genes of human, bovine and porcine animals, the exon regions were almost identical except for some differences in exon regions as shown in FIG.

In addition, as a result of comparing homology based on the amino acid sequence of the MIC gene of human, bovine and porcine animals, as shown in Fig. 5, it was confirmed that the high sequence homology in extracellular domain, i.e.,? 1,? 2, , Suggesting that structural conservation of extracellular domains is important. Sequence homology between pigs and cows is much higher than sequence homology between pigs and humans. The transmembrane domain has low homology when compared to other domains.

Next, patterns of amino acid sequence conservation of the MIC gene between human, bovine, and porcine were compared. As a result, three conserved N-linked glycosylation sites (Asn-8, -255, -283) were found between the species as shown in FIG. In addition, except for the human MICA gene, pig, bovine and human MICB genes were conserved in α2 (Cys-99), α3 (Cys-276) and transmembrane domains (Cys-323). Pigs and cows share three conserved cysteine sequences in the a2 domain.

Experimental Example 7. Phylogenetic Analysis

 Phylogenetic analysis of the SLA-MIC2 allele was performed using sequences of exons 2, 3 and 4 in a Neighbor-joining method involving 1000-times repeated bootstrap analysis, and the evolutionary distance between alleles Was calculated using the Kimura 2-parameter model (Kimura 1980) using MEGA 5.2 software (Tamura et al., 2001). MICA and MICB genes were used in humans, Patr-MICA / B was used as chimpanzee, Mamu-MIC1 and Mamu-MIC2 as rhesus macaque, and BoLA-MIC1, BoLA-MIC2 and BoLA-MIC3 genes were used.

The phylogenetic analysis results have a low phylogenetic resolution, especially in the case of MIC2, as shown in FIG. This suggests that there was a restriction on the genetic mutation and the change of the gene sequence in the MIC gene including the recent pig MIC2. The MIC2 allele is closely clustered with the BoLA-MIC gene, showing a close evolutionary relationship between them. Primates, humans, chimpanzees, and Rhesus monkeys are found to be in a distant relationship with MIC2.

Having described specific portions of the present invention in detail, it will be apparent to those skilled in the art that this specific description is only a preferred embodiment and that the scope of the present invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> University-Industry Cooperation Foundation, Konkuk University <120> Method for polymorphism analysis of Swine Leukocyte          Antigen (SLA) -MIC2 gene <130> P14-E685 <160> 16 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 tgtcctctgc ttgccgatct c 21 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 atccagaacc acctagatcc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ttctggcccc ttgtacacat 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tccatgctca gctcacagac 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ccttgactca gcagcacagg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ggactgacca gaagagcaag 20 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 tgcatgaagg ctcagccag 19 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 agcctggcct ctggatctc 19 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 gagcgagtgt cccatttggg a 21 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ggccagaaca gggagttgaa ttc 23 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ggtacaactt cacggtgatg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 ggagaagacg tgcgacatgg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 ggagaagacg tgcgacatgg 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 ctctgtgaag ctggtccagg 20 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 actcacggca aattcaacgg c 21 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 atcacaaaca tgggggcatc g 21

Claims (7)

A primer set for PCR consisting of SEQ ID NOS: 1 and 2 which amplifies all of the exon 2 to exon 4 regions of a porcine leukocyte antigen (SLA) -MIC2 gene.
SEQ ID NOS: 3 and 4 for sequencing forward and reverse exon 2 of the porcine leukocyte antigen (SLA) -MIC2 gene.
SEQ ID NOS: 5 and 6 for sequencing forward and reverse exon 3 of the porcine leukocyte antigen (SLA) -MIC2 gene.
SEQ ID NOs: 7 and 8 for sequencing forward and reverse exon 4 of the porcine leukocyte antigen (SLA) -MIC2 gene.
(1) separating the genomic DNA from the tissue of the pig;
(2) PCR-amplifying the separated genomic DNA using primers consisting of SEQ ID NOS: 1 and 2 capable of amplifying the entire exon 2 to exon 4 regions of a porcine leukocyte antigen (SLA) -MIC2 gene; And
(3) The DNA fragment amplified in (2) above is composed of SEQ ID NOs: 3 and 4 in the case of exon 2, SEQ ID NOs: 5 and 6 in the case of exon 3, and SEQ ID NOs: 7 and 8 Performing sequencing using a sequencing primer comprising: (SLA) -MIC2 &lt; / RTI &gt; gene comprising the polymorphism of a porcine leukocyte antigen (SLA) -MIC2 gene.
6. The method of claim 5,
Further comprising the step of performing a bi-directional sequencing of a clone obtained by cloning the DNA fragment amplified in the step (2) using exon 2, 3 and 4 specific sequencing primers. Method of polymorphism analysis of leukocyte antigen (SLA) -MIC2 gene.
6. The method of claim 5,
Wherein the method further comprises the step of performing RT-PCR by separating the RNA from the tissue of the pig, and performing a bidirectional sequencing of the cDNA product obtained thereby, wherein the polymorphism of the porcine leukocyte antigen (SLA) -MIC2 gene Way.

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