JP2012044965A - Screening method based on gene information of chicken having resistance against virus infection - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of evaluating resistance against a virus infection, especially an RSV infection, in a chicken.SOLUTION: There is provided the method of evaluating the resistance against the virus infection, especially the RSV infection, in the chicken. The method includes detecting and evaluating a polymorphism of a microsatellite marker LEI0258 in a MHC-B region located at least on the 16th chromosome.

Description

  The present invention relates to a method for evaluating resistance to viruses in chickens, particularly RSV infection, and a method for selecting chickens having the resistance using the method.

  Avian leukosis virus (hereinafter referred to as “ALV”) is a kind of avian leukemia-sarcoma virus (ALSV), which infects beef chickens and develops myelosphere (myeloid). It is known to develop leukocyte juveniles (myelocytic myeloid leucosis), renal tumors (nephromas) and other tumors (tumors). If chicken leukemia occurs at the production site for beef chickens, the titer of chickens in all poultry houses will be determined by measuring the antibody titer of all chicken houses and checking whether ALV infection has occurred. After disposing of the separately infected chickens, disinfection operations are performed to remove the contamination of the poultry house, and there is a problem in terms of time and economical burdens such as not using the contaminated poultry house for a long time.

  In order to solve such problems, breeding business related to resistance to viral diseases in chickens has been carried out for about 10 years from around 1980, centered on the Livestock Improvement Center (formerly the Shirakawa breeding farm of the Ministry of Agriculture, Forestry and Fisheries).

  In the breeding business, in order to evaluate resistance (susceptibility) to ALV infection, an infection test using Rous Sarcoma Virus (hereinafter referred to as “RSV”) is performed instead of ALV. It was.

  RSV is known to be a pseudotype of ALV. The gene structure of ALV and RSV consists of gag (making a polyprotein that is cleaved to become capsid protein), pol (making an enzyme involved in integrating reverse transcriptase and viral genes into the host genome), env (envelope glycoprotein) The RSV contains the src (oncogene) portion in addition to these. RSV can develop tumors in as little as 5 days after chicken infection. RSV is normally used instead of ALV when investigating the resistance (susceptibility) of chickens to ALV infection due to these properties (Non-patent Document 1).

  There are two types of chicken resistance to viral infection.

  One is “resistance” by not accepting the virus into the cell, ie, insensitivity. This is caused by a decrease in the affinity between the receptor protein and the viral protein due to the mutation of the viral receptor on the surface of the host cell, or lack of an enzyme suitable for cleaving the viral capsid composed of the protein.

  The other is “resistance” by infection of the virus but suppression of increase in virus by the action of cellular immunity and / or humoral immunity. The “resistance” can be assessed by tumor regression caused by viral infection.

  From the results of the infection test using RSV conducted by the Livestock Improvement Center, 10 types of white legphones (WL01, WL02, WL04, WL05, WL06, WL11, WL12, WL14, WL15) Among the strains and the WL21 strain), tumor regression was observed at a high rate in the WL11 strain, and it was revealed that the WL11 strain is resistant to virus infection (Non-patent Document 2).

  However, tumor regression was not observed in all of the chickens belonging to the WL11 strain, and tumor regression was observed only in about 50% of the individuals, and tumor regression was not observed in the remaining individuals. Therefore, a method for selecting individuals having resistance to viral infection has been eagerly desired in the art.

  We analyzed which chromosomes are lodged with genes associated with resistance to viral infection with tumor regression caused by RSV (especially RSV-A) infection. More specifically, an experimental family was established by crossing the WL11 strain with a Rhode Island Red strain that does not show tumor regression (hereinafter referred to as “RYRYS”), and an RSV infection test was performed. A linkage analysis using a macro satellite was performed.

  As a result, the chromosomal region related to the resistance was found in chromosomes 1, 2, 4 and 16, and it was found and reported that the significance of chromosome 16 was particularly highest (Non-patent Document 3).

  From the above results by the present inventors, it was understood that resistance to viral infection depends on chromosome 16 with particularly high significance, but this information alone is resistant to viral infection. In this field, a new method for selecting an individual having resistance has been demanded.

Breeding for leukemia anti-disease in chickens Yukio Yamada Livestock Technology (1969) 7: p217-221 Data on anti-disease breeding business of chicken (1990) Shirakawa breeding farm Y. Uemoto et al. (2009) Asian-Aust. J. et al. Anim. Sci. 22 (10): 1359-1365

  The present invention provides a method for evaluating resistance to viruses in chickens, particularly RSV infection, and a method for selecting chickens having the resistance using the methods.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have determined that at least a polymorphism of microsatellite LEI0258 (hereinafter referred to as “LEI0258”) in the MHC-B region located on the chromosome 16. By detecting and evaluating, it was found that chickens having resistance to viruses, particularly RSV infection, can be selected. Furthermore, in addition to the above-mentioned LEI0258 polymorphism, a TAPBP gene polymorphism in the MHC-B region on chromosome 16 can also be detected, so that a chicken having resistance to RSV infection can be obtained with high accuracy. The inventors have found that they can be selected and have completed the present invention.

That is, the present invention has the following features.
[1] A method for evaluating resistance to viral infection in chickens, comprising detecting and evaluating a polysatellite LEI0258 polymorphism in the MHC-B region located on chromosome 16.
[2] The method of [1], wherein the virus is Rous sarcoma virus (RSV).
[3] The method of [2], wherein the virus is avian RSV-J.
[4] The method according to any one of [1] to [3], wherein when the microsatellite LEI0258 has a base length of 443 bp, it is judged to have resistance to viral infection.
[5] Further, one or more genes selected from the group consisting of a Blec2 gene, a TAPBP (TAP Binding protein) gene, a BLB2 gene, a DMB1 gene and a TAP2 gene in the MHC-B region located on chromosome 16 The method in any one of [1]-[4] including detecting and evaluating the gene polymorphism of.
[6] The method according to [5], wherein a gene polymorphism of a TAPBP (TAP Binding protein) gene is detected and evaluated.
[7] The method according to [6], wherein when the 944th nucleotide in the amino acid coding sequence of a TAPBP (TAP Binding protein) gene is adenine, it is judged to have resistance to viral infection.
[8] A method for selecting a chicken having resistance to viral infection, using any one of the methods [1] to [7].
[9] A reagent kit for evaluating resistance to viral infection in chickens, comprising a primer for amplifying microsatellite LEI0258 by PCR reaction in any one of [1] to [8].
[10] The reagent kit according to [9], further comprising a primer or an oligonucleotide for detecting a gene polymorphism of the TAPBP gene.

  By using the present invention, it becomes possible to evaluate resistance to virus in chickens, in particular RSV infection, and it becomes possible to select chickens having the resistance.

FIG. 1 shows the positions of LEI0258 in the MHC-B region located on chromosome 16 and 35 single base mutations (SNPs) found in the region by the present inventors. FIG. 2 is an electrophoretic image diagram of LEI0258 in each haplotype (WLA, WLB, WLC, RIRa, RIRb, RIRc) in the WL11 strain amplified by the PCR reaction. Each lane shows the following sample. L: 100 bp to 1,000 bp ladder (marker), 1: WLA homo (443 bp homo), 2: WLB homo (260 bp homo), 3: RIRa homo (310 bp homo), 4: RIRb homo (310 bp homo), 5: RIRc homo (357 bp homo), 6: WLB, RIRa hetero (260 bp / 310 bp), 7: WLA, WLB hetero (260 bp / 443 bp), 8: WLA, RIRa hetero (310 bp / 443 bp), 9: WLA, RIRc hetero ( 357 bp / 443 bp), 10: WLB, RIRa hetero (260 bp / 310 bp), 11: WLA, RIRc hetero (357 bp / 443 bp), 12 WLA homo (443 bp homo). FIG. 3-1 shows synonymous single base substitutions based on the direction of genes present in the MHC-B regions of the four haplotypes RJF, B6, B24, RIRa, the length of CDS, the number of amino acids and the WLA haplotype, and Indicates the number of non-synonymous single base substitutions. (Continuation of Fig. 3-1) FIG. 4-1 shows the base sequences of the TAPBP gene present in five different MHC-B haplotypes. In the nucleotide sequence, nucleotides surrounded by squares indicate synonymous substitutions, and nucleotides indicated by underlining indicate non-synonymous substitutions. (Continued from Fig. 4-1) (Continued from Fig. 4-2) (Continued from Fig. 4-3) (Continued from Fig. 4-4) (Continued from Fig. 4-5)

  The present invention provides a method for assessing resistance to viral infection in chickens.

  In the present invention, “virus” includes avian leukemia virus (Avian Leukosis Virus: hereinafter referred to as “ALV”) and Rous sarcoma virus (hereinafter referred to as “RSV”). Both ALV and RSV are cancer viruses, and are classified into 10 types, A, B, C, D, E, F, G, H, I and J, based on their capsid structure (Payne L. N. et. al. (1992) Journal of General Virology, 73: 2995-2977). Preferably, the “virus” in the present invention is RSV, more preferably RSV-J.

  In the present invention, “resistance to viral infection” means that the virus is infected but the increase in virus is suppressed by the action of cellular immunity and / or humoral immunity. In the present invention, the “resistance” can be evaluated by tumor regression caused by viral infection, preferably RSV infection, more preferably RSV-J infection. “Tumor regression” means that a tumor caused by viral infection in a chicken shrinks or disappears in about 20 to 45 days. Therefore, in this specification, “resistance to viral infection” may be expressed by terms such as “tumor regression” and “tumor regression ability”, but these terms are all used interchangeably. .

  In the present invention, “chicken” is not particularly limited. Preferably, it is a chicken derived from the WL11 strain or the WL11 strain. “Chick derived from the WL11 strain” is not limited to chickens obtained by crossing with the WL11 strain (F1 generation), but F2 and F3 generations (particularly not limited thereto) Chickens obtained by crossing with these chickens are also included.

  In the present invention, evaluation of resistance to viral infection can be performed based on chicken gene information. That is, it can be performed by detecting and evaluating a polymorphism of the microsatellite marker LEI0258 in at least the MHC-B region located on chromosome 16 in a sample containing chicken DNA.

  The “sample containing chicken DNA” used in the present invention is prepared from any cells (except germ cells), tissues, organs, etc. isolated from chickens. Preferably, it can be prepared from peripheral blood (white blood cells or mononuclear cells).

  In the present specification, the “gene polymorphism” includes a so-called single nucleotide polymorphism (single nucleotide polymorphism: hereinafter referred to as “SNP”), and polymorphism over a plurality of consecutive nucleotides. Includes both. That is, in a population, there are mutations such as substitution, deletion, insertion, translocation, inversion, etc. of one or more nucleotides at a specific site in one or more individual genomes based on the genome sequence of one individual. If the mutation is statistically certain that the mutation is not a mutation that has occurred in the individual or individuals, or it is not an intra-individual mutation and is present in the population at a frequency of 1% or more If it is proved by the above, the mutation is referred to as “gene polymorphism”.

  LEI0258 is known as a microsatellite located in the MHC-B region located on chromosome 16, and is conventionally used to determine the haplotype in the chicken MHC region (Fulton et al. Immunogenetics 58: 407-421 (2006)). The base sequence of LEI0258 is described in, for example, McConnell, S .; K. et al. , Anim. Genet. 30 (3), 183-189 (1999).

  As described in detail in the Examples below, by utilizing the LEI0258 polymorphism, 50% of the haplotype of the MHC-B region (hereinafter referred to as “MHC-B haplotype”) is analyzed. In the WL11 strain in which an individual exhibits tumor regression ability, three types of MHC-B haplotypes, that is, haplotypes significantly associated with tumor regression ability (hereinafter referred to as “WLA”) and tumor regression ability are not significantly related 2 Two haplotypes (hereinafter referred to as “WLB” and “WLC”) can be determined. In addition, using the LEI0258 polymorphism, three types of MHC-B haplotypes, that is, haplotypes with high tumor progression (hereinafter referred to as “RIRa” and “RIIRb”) in the RIRYS line of Rhode Island Red where tumors progress. ) And haplotypes (denoted “RIRc”) that are not significantly related to tumor progression.

  There is a correlation between the MHC-B haplotype and tumor regression ability, and chickens with WLA homozygous have tumor regression ability with a probability of 90-100%. In addition, a chicken having WLA in heterogeneity has a tumor regression ability with a probability of 80 to 90% unless one allele is RIRa.

  Therefore, by detecting the LEI0258 polymorphism and determining the MHC-B haplotype in the chicken, specifically by detecting the presence or absence of WLA, the chicken's resistance to viral infection can be evaluated. .

  A polymorphism of LEI0258 can be detected by measuring the base length. For example, LEI0258 polymorphisms having a base length of 261 bp, 295 bp, 307 bp, 309 bp, 310 bp, 321 bp, 333 bp, 345 bp, 357 bp, 367 bp, 369 bp, 381 bp, 393 bp, 408 bp, 420 bp or 443 bp are known ( Fulton et al. (2006) supra).

  The relationship between the base length of LEI0258 and the MHC-B haplotype is shown below.

  Since only the base length of LEI0258 in WLA is 443 bp, it can be distinguished from other MHC-B haplotypes based on the base length.

  The measurement of the base length of LEI0258 can be performed by a general method known to those skilled in the art, and can be performed, for example, using a PCR method.

  In addition to the detection of the above-mentioned LEI0258 polymorphism, the method of the present invention further comprises 1 selected from the group consisting of the Blec2 gene, the TAPBP gene, the BLB2 gene, the DMB1 gene and the TAP2 gene, which are present in the MHC-B region as in the case of LEI0258. Alternatively, the method includes a step of detecting and evaluating gene polymorphisms of a plurality of genes. In addition to the LEI0258 polymorphism, WLA can be selected with high accuracy by detecting the gene polymorphism of the above gene.

  Conventionally, detection and evaluation of a gene polymorphism of the BF2 gene have been performed in order to determine the MHC-B haplotype of a chicken. Based on the gene polymorphism of the BF2 gene, a very large number of haplotypes (B1-24) ) Have been detected and identified (Livant E. J. and S. J. Edward (2005) Animal Genet. 36: 432-434.).

  Among these, the haplotype called B6 does not have the same base sequence as the MHC-B region in WLA, but the base length of the LEI0258 is 443 bp, which is the same as the size of LEI0258 in WLA. For this reason, WLA cannot be distinguished from B6 only by the base length of LEI0258.

  As described in detail in the examples below, the polymorphisms of the Blec2 gene, the TAPBP gene, the BLB2 gene, the DMB1 gene and the TAP2 gene differ at least between WLA and B6, and the Blec2 gene in WLA and B6, By detecting and evaluating gene polymorphisms of one or more genes selected from the group consisting of TAPBP gene, BLB2 gene, DMB1 gene and TAP2 gene, both can be distinguished. The Blec2 gene, the TAPBP gene, the BLB2 gene, the DMB1 gene and the TAP2 gene are known and are registered in GenBank as BAG69318.1, BAG69321, BAG69320, BAG69325 and BAG69329, respectively, and these gene information can be used. In addition, the base sequence of each gene is described in, for example, Hosomichi, K. et al. et al. , J .; Immunol. 181 (5), 3393-3399 (2008).

  Preferably, a gene polymorphism of the TAPBP gene is detected and evaluated.

  The gene polymorphism of the TAPBP gene is present at the 944th nucleotide in the base sequence of the TAPBP gene represented by SEQ ID NOs: 2 to 6. The gene polymorphism of the TAPBP gene is different between WLA and B6. That is, in the TAPBP gene of WLA, the 944th nucleotide is adenine (SEQ ID NO: 2), whereas in B6, the 944th nucleotide is guanine (SEQ ID NO: 4).

  Therefore, WLA and B6 can be distinguished by detecting and evaluating the gene polymorphism of the TAPBP gene.

  The method for detecting and evaluating the gene polymorphism of the TAPBP gene can be performed by a general method known to those skilled in the art. For example, the most direct method of target region base sequencing (sequencing method) or the purpose of detecting only base substitution at a specific position is effective for synthetic oligonucleotides of about 20 bases and target base sequences. Allele-specific oligonucleotide hybridization method (MASA method) using the presence or absence of hybridization, treatment of hybrid of standard RNA probe and standard DNA fragment with ribonuclease A, and presence of base substitution by cleavage of probe RNA at mismatch position When the DNA sequence is amplified by PCR, a GC-rich base sequence (GC clamp) is attached to one end of the DNA sequence to determine the approximate position. Electrophoresis (denaturing gradient gel) lectrophoresis: DGGE) may be used a method of detecting a single nucleotide substitution performed (PCR-DGGE method).

  In addition, a method of detecting single base substitution as a difference in mobility by dissociating a double-stranded DNA fragment into single strands and performing polyacrylamide gel electrophoresis on a unique higher order structure depending on the base sequence (PCR) -SSCP method), hybridization method using a nucleotide sequence specific probe (SSOP) (PCR-SSOP method), modified methods (PCR-RD method and PCR-MPH method), PCR method for certain regions, etc. PCR-RFLP method in which a site where a point mutation has occurred is treated with a restriction enzyme that recognizes it as a cleavage site and then detected as an allele having a size different from that of the DNA that did not cause a point mutation. -308999 gazette), annealing the detection primer to the nucleic acid sequence adjacent to the nucleotide site at the 3 ′ end of the target DNA, and DNA polymerase Used, complementary to nucleotides of detecting, a method for extending a primer according with nucleotide triphosphates single label (mini sequencing method) can also be used.

  It can also be detected by various known SNP typing methods such as TaqManPCR method and invader method.

  Any method capable of detecting a single-base mutation can be used in the present invention without being limited to the above method.

  By evaluating the resistance of a chicken to viral infection using the method of the present invention, it is possible to select a chicken having resistance to viral infection. That is, using the method of the present invention, a chicken having a homozygous or heterozygous WLA (however, one allele is not RIRa) is selected as having resistance to viral infection.

  The present invention also provides a primer for use in the above-described method for detecting a polymorphism of LEI0258 and the method for detecting a gene polymorphism of the TAPBP gene.

  The primer pair for detecting the polymorphism of LEI0258 needs to be a primer designed to sandwich at least LEI0258 on the chicken genome sequence. Here, “sandwich” means that the base sequence of the target gene is included in the base sequence of a DNA fragment amplified by a primer pair consisting of a forward (reverse) primer and a reverse (reverse) primer. The primer is usually 10 mer to 100 mer, preferably 15 mer to 40 mer, more preferably 18 mer to 30 mer. The DNA region amplified by the primer is not particularly limited as long as the base length of WLA and other haplotype LEI0258 can be distinguished, but preferably the length of the amplified DNA fragment is 100 bp to 1,500 bp, Preferably it is 200 bp-700 bp.

  Preferably, a primer for detecting the base length of LEI0258 can be preferably designed based on a common base sequence in the gene polymorphism, and those skilled in the art will be able to design such a base length based on the base sequence of LEI0258. Primers can be easily designed. Examples of the primer for detecting the base length of LEI0258 include, but are not limited to, primers including the base sequences represented by SEQ ID NOs: 7 and 8.

  In addition, the primer pair for detecting the gene polymorphism of the TAPBP gene hybridizes to the DNA encoding the TAPBP gene or its complementary strand, and represents the base sequence of the target polymorphism site (WLA and B6 TAPBP gene, respectively) It is necessary that the primer is designed so as to sandwich the nucleotide 944 in SEQ ID NO: 2 or SEQ ID NO: 4. “Stuck” is as defined above. The primer is usually 10 mer to 100 mer, preferably 15 mer to 40 mer, more preferably 18 mer to 30 mer. The length of the DNA region amplified by the primer is 50 bp to 5,000 bp, preferably 100 bp to 1,500 bp, more preferably 200 bp to 700 bp.

  Primers for detecting the gene polymorphism of the TAPBP gene can be preferably designed based on the common base sequence in the gene polymorphism, and those skilled in the art will be able to design such a primer based on the base sequence of the TAPBP gene. Primers can be easily designed. Examples of the primer for detecting a gene polymorphism of the TAPBP gene include, but are not limited to, primers including the nucleotide sequences represented by SEQ ID NOs: 11 and 12.

  Additional primers or primer pairs can be designed within the region sandwiched by these primers. Such a second primer can be used in sequencing and the like. Such a second primer is represented by, for example, SEQ ID NO: 13, but is not limited thereto.

  The primer can be hybridized with the template DNA under conditions usually used in this field. As long as the primer satisfies the two conditions of being capable of forming a complementary base pair bond and providing an —OH group as a starting point for complementary strand synthesis at its 3 ′ end, the main chain constituting the primer is a phospho group. It is not limited to those by diester bonds (for example, DNA). For example, it may be composed of a phosphothioate body having sulfur (S) as a backbone instead of phosphorus (P), or a peptide nucleic acid based on a peptide bond. Moreover, the base should just be a thing which enables complementary base pair coupling | bonding. In nature, it is composed of five types of adenine, guanine, cytosine, thymine, and uracil, and may be an analog such as bromodeoxyuridine.

  The present invention also provides an oligonucleotide for use in the method for detecting a gene polymorphism of the TAPBP gene. Such an oligonucleotide preferably hybridizes specifically to a region containing the polymorphic site of the TAPBP gene. Here, “specific” means that it hybridizes to a region containing the polymorphic site of the TAPBP gene and does not hybridize to other regions. Such hybridization conditions can be appropriately selected by those skilled in the art. Examples of hybridization conditions include low stringency conditions. The low stringent conditions are, for example, conditions of 42 ° C., 5 × SSC, 0.1% SDS, and preferably 50 ° C., 2 × SSC, 0.1% SDS in washing after hybridization. is there. More preferable hybridization conditions include highly stringent conditions. High stringent conditions are, for example, 65 ° C., 0.1 × SSC, and 0.1% SDS. However, factors affecting the stringency of hybridization include multiple factors such as temperature and salt concentration, and those skilled in the art can realize the same stringency by selecting these factors as appropriate. is there.

  As long as the oligonucleotide in the present invention hybridizes to the DNA region containing the polymorphic site, the chain length is not particularly limited, but is preferably 10 mer to 200 mer, more preferably 15 mer to 100 mer, still more preferably. Is a 15-mer to 30-mer oligonucleotide.

  The oligonucleotide of the present invention can be used as a probe for detecting the polymorphism and as an adsorbing ligand for purifying DNA containing the polymorphism.

  The oligonucleotide of the present invention may be immobilized on a solid phase. Examples of such a solid phase material include, but are not limited to, cellulose derivatives such as cellulose and nitrocellulose, sepharose, agarose, metal, glass, ceramic, resin, and the like. The shape and material of the solid phase are not particularly limited.

  The primers and / or oligonucleotides described above can be used as test reagents for evaluating resistance to viral infection of chickens. Furthermore, these reagents can be packaged and supplied as a kit.

  The primers and probes can be produced by methods known in the art. For example, the DNA primers and DNA probes can be produced using the phosphotriethyl method, the phosphodiester method or their automated methods. It can be conveniently synthesized according to the base sequence disclosed in the present invention using an automatic DNA synthesizer or the like.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further, this invention is not limited to these Examples.

Example 1 Determination of MHC-B Haplotypes Microsatellite marker LEI0258 polymorphism in the MHC-B region, BF2 gene polymorphism, and 35 types of SNPs found by the present inventors in the MHC-B region ( From FIG. 1), MHC-B haplotypes were determined in the WL11 strain considered to have tumor regression ability and the RIRYS strain of Rhode Island Red in which the tumor progressed. In addition, an infection test of RSV-J was conducted to clarify the relationship between each MHC-B haplotype and the regression ability against tumors derived from RSV-J infection (K. Suzuki et al. Poultry Science 89: 651-657 ( 2010)).

Venoject II EDTA vacuum blood collection tubes (TERMO, Tokyo, Japan) or equivalent from individuals of WL11 strain (n = 300) and RIRYS strains (n = 200) (both cultivated at the National Livestock Improvement Center) Was collected with 2% W / V sodium citrate, and DNA was extracted and purified by any one of the following three methods.
1. 50 μl of blood was diluted to 2 ml with 1 × PBS (−) (GIBCO, USA), and DNA was extracted and purified with QuickGene DNA whole Blood kit L (FUJIFILM, Tokyo, Japan).
2. 5 μl of blood was diluted to 200 μl with 1 × PBS (−), and DNA was extracted and purified with QIAmp DNA Mini Kit (QIAGEN, Tokyo, Japan).
3. Within 0.5 mg of the feather root was cut finely, and DNA was extracted and purified with a DNA extractor FM kit (Wako, Tokyo, Japan).

  In addition, even if DNA is prepared using any method, the following experiment is not affected at all.

  Using the DNA obtained from each individual, the gene polymorphisms of LEI0258 and BF2 were determined by the PCR method using the following primers, respectively.

Primer forward primer for amplification of LEI0258: 5′-CACGCAGCAGACTACTGGTAAGG-3 ′ (SEQ ID NO: 7)
Reverse primer: 5′-AGCTGTGCTCCAGCTCTCAGTGGC-3 ′ (SEQ ID NO: 8)
Primer forward primer for BF2 amplification: 5′-GCAGAGCTCCATACCCTGCGGTA-3 ′ (SEQ ID NO: 9)
Reverse primer: 5′-GCCGGTCTGGTTGTTAGGCCG-3 ′ (SEQ ID NO: 10)

The PCR reaction was performed with the following composition. In a volume of 20 μl, 10-20 ng DNA / 5 pmol each primer / 0.2 mM dNTP / 10 mM Tris-HCl [pH 8.3] / 50 mM KCl / 1.5 mM MgCl ₂ /0.2 U AmpliTaq-Gold polymerase (Applied Biosystems, Foster City, CA, USA).

  The reaction conditions were 94 ° C for 10 minutes, and then 94 ° 30 seconds, 60 ° 30 seconds, 72 ° 30 seconds in 40 cycles.

  After completion of the reaction, each sample was electrophoresed using 2% w / v agarose, and the size of the PCR product was calculated from the DNA size marker. The type of BF2 was determined based on a known method (Livant E. J. and S. J. Edward (2005) listed above).

  Further, the 35 SNP types described above were subjected to matrix-assisted laser desorption / ionization time-of-flight mass spectrometry based on a known method (Okamura N. et al. Anim. Sci. J. 79: 303-313 (2008)). Determined by.

  RSV-J infection tests consisted of inoculating RSV-J (HPRS-103; Payne et al, J. Gen. Virol. 72: 801-807 (1992)) subcutaneously into wing buds of 3-week-old chickens. The size of each tumor was measured on days 7, 10, 14, 28 and 42.

  As a result, three types of haplotypes were identified in the WL11 line (referred to as WLA, WLB, and WLC), and three types (referred to as RIRa, RIRb, and RIRc) in the RIRYS line, and each haplotype was derived from RSV-J infection. The relationship of the regression capacity for tumors was also revealed.

  Table 1 below shows the type of LEI0258 and BF2 and the ability of individuals with each haplotype to regress on tumors due to RSV-J infection.

  From this result, it became clear that among the MHC-B haplotypes, WLA characterized as having a base length of LEI0258 of 443 bp is significantly associated with the ability to regress tumors due to RSV-J infection.

Example 2: Influence on tumor progression ability by combination of MHC-B haplotype In order to examine the influence on tumor progression ability by combination of MHC-B haplotype, an experimental family was constructed by crossing WL11 strain and RYRY strain. In response to this, an RSV-J infection test was conducted.

  A total of 23 F1 generation males and females were produced from the cross of 3 WL11 males and 7 females of the RYRY line, and 91 F2 males and females were produced by crossing F1 males and females. Furthermore, males and females of WLA homozygote and RIRa homozygote were selected and mated from the F2 generation to produce a total of 28 F3 generation males and females.

  The RSV-J infection test was performed in the same manner as in Example 1.

  DNA was extracted from a plurality of individuals of the F2 generation in the same manner as described in Example 1 above. Using the obtained DNA, the base length of LEI0258 of each individual was detected by the PCR method in the same manner as in Example 1, and the combination of MHC-B haplotypes in each individual was determined based on the results in Table 1 above.

  The results are shown in FIG. By detecting the base length of LEI0258 by the PCR method, the combination of MHC-B haplotypes in each individual (especially the presence or absence of WLA) can be easily determined.

  The relationship between the combination of MHC-B haplotypes found in the F2 and F3 generations of experimental families and tumor regression caused by RSV-J infection is shown in Table 2 below.

  From this result, it became clear that almost 100% of chickens having WLA homozygote exhibit a regression ability against tumors caused by RSV-J infection. In addition, even in chickens having WLA heterozygously, if another allele is not RIRa, it has been revealed that about 85% of individuals show regression ability against tumors caused by RSV-J infection. Furthermore, it was found that almost 90% of chickens having RIRa homozygously form large tumors that are fatal.

  From this result, it became clear that the regression ability for tumors caused by RSV-J infection in chickens can be evaluated using WLA and RIRa as indices.

Example 3: Identification of WLA and B6 haplotypes When the MHC-B haplotypes were classified based on the base length of LEI0258, both B6, which are one of the haplotypes of WLA and BF2, detected 443 bp of LEI0258. Cannot be distinguished. In addition, no difference was found between the 35 types of SNPs (FIG. 1) found by the inventors in the MHC-B region.

  Until now, it has not been clarified whether B6 affects the regression ability against tumors caused by virus infection, but the line with B1 corresponding to B6 has a low rate of progression of RSV-B-derived tumors. Have been reported (Schierman, LW et al. (1977) Immunogenetics 5, 325-332). Therefore, by identifying WLA and B6, it is possible to detect WLA with high accuracy, and as a result, it is possible to evaluate with high accuracy the regression ability against tumors caused by RSV-J infection in chickens, and a gene that can distinguish both Searched for markers.

  A BAC library was constructed from the genomic DNA of a chicken individual having a homozygous WLA, and the base sequence of the 167,464 bp region including the MHC-B region was determined for WLA and RIRa. Then, the base sequence of WLA was compared with the base sequences of RIRa, RJF (Red Jungle Fowl) (World Livestock Dictionary: Supervised by Yoichi Shoda, published by Toyoshobayashi, p. 344-345 (2006)), B6 and B24.

  The results are shown in FIGS.

  For comparison between WLA and B6, 14 genes (BG1, Blec2, Blec1, BLB1, TAPBP, BLB2, BRD2, DMA1, DMB1, DMA2, BF1, TAP1, TAP2, Comparison of the amino acid coding sequence (CDS) of BF2) was performed.

  As a result, one place of synonymous substitution and one place of non-synonymous substitution (abbreviated as 1-1 hereinafter), TAPBP gene (1-1), BLB2 gene (22-19), DMB1 gene (2-1) In addition, 49 SNPs in the TAP2 gene (1-0) were found to be different between WLA and B6 (FIGS. 3-1 and 2).

  This revealed that the MHC-B regions of WLA and B6 are not identical.

  Among the 14 gene CDSs, there were 49 SNPs that differed between WLA and B6. However, as a genetic marker that makes it possible to distinguish between them, both can be amplified in the same way, and detection is easy. To genes with a small number of SNPs are preferred. Considering this point, the Blec2 gene and TAPBP (TAP Binding protein) gene having one non-synonymous substitution SNP per gene between WLA and B6 can be used as genetic markers.

  Since the Blec2 gene has 39 SNPs between WLA, B24, and RIRa, each of them can be amplified in the same manner, and therefore there are fewer SNPs (3 sites each). TAPBP gene having one) is judged to be effective as a target gene marker.

  The comparison results of the base sequences in the amino acid coding region of the TAPBP gene in WLA, RIRa, RJF, B6 and B24 are shown in FIGS.

  In the TAPBP gene, the position of the SNP that exists at one position between WLA and B6 is the 944th nucleotide, adenine (codon is CAC, amino acid is H: histidine) in WLA, and guanine (codon is CGC, amino acid in B6) Is R: arginine).

  Therefore, WLA and B6 can be distinguished by detecting and evaluating the SNP present in the TAPBP gene.

  The SNP was detected by sequencing a region containing the 944th nucleotide of the TAPBP gene in each individual.

Genomic DNA of chicken individual and the following TAPBP gene amplification primer: forward primer: 5′-ATGAGGGCACCTACATCTGC-3 ′ (SEQ ID NO: 11)
Reverse primer: 5′-TTGGGACATAGTGACATGCTC-3 ′ (SEQ ID NO: 12)
PCR was performed in the same manner as in Example 1, and the obtained PCR product was treated with Pre-Sequencing Kit (USB, USA), and 2 μl of BigDye Terminator kit v. The sequence was determined using 3.1 (Applied Biosystems, Foster City, CA, USA) and sequencing primer: 5'-GGACCGGGTGGTATCTCAT-3 '(SEQ ID NO: 13). Whether the nucleotide at the position of the SNP is adenine was evaluated to identify whether it was WLA.

  From the above results, WLA can be detected with high accuracy by combining the detection and evaluation of the SNP present in the TAPBP gene with the detection and evaluation of the base length of LEI0258.

  According to the present invention, resistance to viral infection in chickens can be evaluated, and it becomes possible to select chickens having the resistance. Therefore, the present invention is used for breeding new chicken lines having viral disease resistance. It is highly expected.

Claims (10)

  A method for evaluating resistance to viral infection in a chicken, the method comprising detecting and evaluating a polysatellite LEI0258 polymorphism in the MHC-B region located on chromosome 16.   The method of claim 1, wherein the virus is Rous sarcoma virus (RSV).   The method of claim 2, wherein the virus is avian RSV-J.   The method according to any one of claims 1 to 3, wherein when the base length of the microsatellite LEI0258 is 443 bp, the microsatellite LEI0258 is judged to have resistance to viral infection.   Furthermore, the gene polymorphism of one or a plurality of genes selected from the group consisting of Blec2 gene, TAPBP (TAP Binding protein) gene, BLB2 gene, DMB1 gene and TAP2 gene in the MHC-B region located on chromosome 16 The method according to claim 1, comprising detecting and evaluating a mold.   The method of Claim 5 which detects and evaluates the gene polymorphism of a TAPBP (TAP Binding protein) gene.   The method according to claim 6, wherein when the 944th nucleotide is adenine in the amino acid coding sequence of a TAPBP (TAP Binding protein) gene, it is judged to be resistant to viral infection.   The method of selecting the chicken which has resistance with respect to viral infection using the method of any one of Claims 1-7.   The method according to any one of claims 1 to 8, wherein the kit comprises a primer for amplifying microsatellite LEI0258 by a PCR reaction, for evaluating resistance to viral infection in chickens.   Furthermore, the reagent kit of Claim 9 containing the primer or oligonucleotide for detecting the gene polymorphism of a TAPBP gene.

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