WO2005085446A1 - Method of selecting bird resistant to rna virus - Google Patents

Abstract

By studying the relationship between the Mx genotype in a chick individual and the influenza-resistance of the chick individual, it is clarified that resistance against an RNA virus is observed in the case where the amino acid at the 631-position in the amino acid sequence of chick Mx protein has the asparagine homo-structure or the asparagine/serine hetero-structure compared with the case of having the serine homo-structure. Thus, the resistance to an RNA virus such as influenza virus in chick can be judged by detecting a DNA encoding the amino acid at the above-described site.

Description

 Specification

 Selection method for RNA virus resistant birds

 Technical field

 The present invention relates to a method for selecting an avian influenza virus-resistant individual from poultry such as -avian or duck, and a method for producing an RNA virus vaccine using the method.

 Background art

 [0002] In 1997, the avian influenza virus was rampant in Hong Kong! /, Which became a major problem due to human infection and death. Avian influenza virus disease has continued to spread in the United States and Europe, and has become a major problem in the poultry industry. By the way, the infectious disease caused by the virulent strain of influenza virus is called poultry plague, and when it spread in Japan, the power that has been feared to panic became real in early 2004. Until now, the development of vaccines has been considered as the first priority in controlling viral infectious diseases. However, there are no existing vaccines that are effective against emerging epidemics! There are many cases.

 [0003] On the other hand, it is well known empirically that there are individual differences and varieties in the incidence rate of infectious diseases, and the presence of such phenotypic differences and the genetic level at If the differences can be clarified scientifically, flu-resistant varieties can be created. However, to date, there has been little help to elucidate the genotype specific to -bird and the resistance of -bird individuals to influenza virus. Thus, the group of the present inventors has heretofore been known to inhibit the growth of RNA viruses in mice as an interferon (IFN) -inducible protein. The experiment was begun with.

[0004] Prior to the above-mentioned research on chickens, the following findings were obtained. When influenza virus, one of the representative RNA viruses, was used in infection experiments on inbred mice for experiments, most mice were lethal. Only two strains, A2G and SL mice, resisted Showed sex. The cause is the Mxl gene Have been reported (see Non-Patent Documents 1 and 2). That is, experimental inbred mice that are lethal to influenza virus are

It was identified that some exons were deleted! /, And that they were associated with a base force that caused a stop codon in the middle.

 Therefore, the present inventors examined strains derived from wild mice in various parts of the world, and as a result, confirmed that they all had a resistant Mxl gene. Furthermore, in the process of examining the lineage derived from wild mice, the world's first detectable functional Mx2 gene was discovered. The expression of the Mxl gene is localized in the nucleus, indicating an inhibitory effect on influenza virus growth, whereas the expression of the Mx2 gene is localized in the cytoplasm, which has an inhibitory effect on vesicular stomatitis virus Pennanta virus. Indicated. Therefore, it was presumed that the Mxl and 2 genes share the virus responsible for each other.

 [0006]-In birds, there is only one type of Mx gene, and there is no distinction like Mxl and Mx2. From this, it was unclear whether it was clearly responsible for antiviral activity. For example, a German research group performed cloning of Mx cDNA (see Non-Patent Document 3), but reported that it was a susceptibility gene without antiviral activity. However, the report found only one example of one breed (only the German White Leghorn)!

[0007] Accordingly, the present inventors have developed more than 25 varieties including practical chickens (commercial chickens) such as White Leghorn and Rhode Island Red and native Japanese species such as Shamo and Nagoya. Were cloned, and their entire nucleotide sequences were determined. As a result, the-ヮ bird Mx gene was extremely diverse, and a total of 25 base substitutions were observed. Among them, amino acid substitutions were detected in 15 of all 706 amino acids. Subsequently, several -Avian Mx cDNAs were integrated into an expression vector, and the expression of the Mx gene was deficient. After transfection into mouse 3T3 cultured cells, GFP-expressing mutant vesicular stomatitis virus (VSV) and In vitro infection experiments using avian influenza virus showed that the Mx gene derived from Nagoya sp., Which showed no antiviral activity similarly to the above-mentioned German white leghorn species, whereas the Mx gene derived from shamo Showed clearly high antiviral activity. Furthermore, as a result of conducting a number of transfected cell infection experiments and organizing amino acid substitutions, only one of the 15 amino acid substitutions was responsible for the critical difference in the resistance susceptibility trait. It was possible. That is, when the amino acid at position 631 was Asn (asparagine) (AAT in the base sequence), it was considered to be resistant, and in the case of Ser (serine) (AGT in the base sequence), it was considered to be sensitive (Non-patent Document 4). reference).

[0008] However, in the case of live birds, it is a big question whether or not the influenza resistance of the birds differs due to a difference in only one portion corresponding to amino acid position 631 of the Mx gene. Was. Antiviral mechanisms also involve general humoral immunity, not just the Mx gene. Also, like the Mx gene, the genes that are expressed in cells by type I interferon (IFN) to prevent virus growth include PKR (Protein Kinase R), which causes apoptosis in virus-infected cells, and virus-derived single chains. There is 2-5A (2, 5,-Oligoadenilates) synthetase that degrades RNA, and it is expected that the effects of these other genes will be greater than changes in the ability to suppress virus growth due to differences in the exon sequence of the Mx gene. Was. Even if influenza resistance varies depending on the amino acid composition of the Mx protein, the degree of influenza resistance is very weak compared to the effects of immune mechanisms other than Mx, and the influenza resistance of the living body is significantly reduced. In other words, differences in the sequence of the Mx gene may not have any meaning as a phenotype. In addition, even if the sequence of the exon portion of the Mx gene is the same, the expression level of the gene is different, so it is considered that the disease-causing ability may vary greatly between individuals. For example, the ability of interferon to express the Mx gene and suppress virus growth In humans, the effect of interferon on virus growth suppression is due to differences in the nucleotide sequence of the Mx gene expression control portion. It is said that the difference in the sequence of the cDNA portion was too powerful to explain. In other words, whether the difference at amino acid position 631 in the Mx gene may be useful information for selecting individuals with influenza resistance may be determined by comparing individuals with amino acid position 631 in the Mx gene that are genetically different. However, it was not clear unless a test was conducted to directly infect avian influenza virus.

 [0009] Non-patent document 1: Jin, H.K. et al., 4 authors, "Characterization and expression of the Mxl gene in wild mouse species.", Biochem. Genet, 1998, Vol 36, p. 311-322.

 Non-patent document 2: Jin, H.K. et al., 4 authors, `` Identification of the murine Mx2 gene:

Interfern— induced expression of the Mx2 protein from the fetal mouse gene confers resistance to vesicular stomatitis virus-", J. Virol., 1999, Vol. 73 p. 4925-4930.Non-patent literature reference 3: Bernasconi, D. et al.,` `The interferon- induced Mx protein of chickens lacks antiviral activity., J. Interferon Res., 1995, Vol.l5, p.47-53.Non-specialized literature 4: Joe-Hon Ko et al., 11 authors, `` Polymorphysms and the differential anitiviral activity of the chicken Mx gene. Genome Research ^ 2002, Vol.l2, p.595-601. Disclosure of the Invention

 Problems to be solved by the invention

 [0010] The present invention has been made in view of such circumstances, and an object of the present invention is to clarify the relationship between the Mx genotype of an avian individual and influenza virus resistance of the avian individual, In particular, it is an object of the present invention to provide a method for selecting avian influenza virus-resistant individuals from poultry such as birds and birds. Another object of the present invention is to provide a method for producing an RNA virus vaccine using the method.

 Means for solving the problem

[0011] The present inventors have carried out an in vitro experiment in which the Mx gene is extracted for each varieties of birds, the gene is introduced into mouse cells, and various RNA viruses are transmitted thereto. However, it was necessary to clarify whether the disease resistance or susceptibility of individuals differed only by differences in the nucleotide sequence encoding the Mx genotype, particularly the type at amino acid position 631.

 [0012] Thus, the present inventors conducted an experiment to clarify the power at which amino acid position 631 of the Mx protein separates resistance and susceptibility to a disease-causing RNA virus. In other words, Koshamo (KS) and Nagoya (N) have serine at the amino acid at position 631 and are susceptible to RNA virus. Sugar beetle 2 (S2) and hokkaido shomo (HS) often have asparagine at position 631. Thought resistant. Then, a mutant cDNA was constructed so that the amino acid at position 631 was replaced, and it was examined whether the sensitivity and the resistance were switched between influenza virus-sensitive mouse 3T3 cells transfected with these genes.

[0013] As a result, it was confirmed that the cells into which the Mx gene of KS or N, which was considered to be susceptible to the virus, was introduced were susceptible, and that S2, which was considered to be resistant, was relatively resistant. It was confirmed that there was. On the other hand, even in the sensitive Mx protein, only amino acid 631 is serine It was shown that the power was changed to VSV resistance by replacing it with asparagine. Furthermore, even in the resistant Mx protein, it was shown that the amino acid at position 631 replaced asparaginka with serine, but changed to VSV sensitivity.

 [0014] From these facts, at least one of the resistances of the -Pet transgenic cells to VSV, a disease-causing RNA virus, is attributable to the Mx protein, and among them, only the position 631 of the -Pet Mx protein is replaced. It turned out that sensitivity and resistance were switched. Influenza virus and Newcastle disease virus are RNA viruses like VSV, and it is highly likely that Mx protein plays a role in suppressing the growth of these pathogenic RNA viruses. Furthermore, as shown here, it is highly likely that the ability to suppress the growth of the Mx protein at the amino acid position 631 against the RNA virus among the Mx proteins will be determined.

 [0015] Further, the present inventors preliminarily distinguished the group of -Avians of the same cultivar with the 63rd amino acid type of the Mx gene, and developed A / Hong Kong Using the / 483/97 (H5Nl) strain, infection tests were performed on individuals with different Mx genotypes. The Trinfluenza virus, which was confirmed in 2004 in Japan, Korea and Southeast Asia, is also the same type of H5N1 virus.

 As a result, it was found that some individuals who had amino acid position 631 of the Mx protein in Asn-type homozygotes! /, And those who had heterozygous heterozygotes were more resistant to avian influenza than those in Ser-type homozygotes.

 [0016] From the above, by knowing the type of the gene portion corresponding to amino acid position 631 of the chicken Mx gene, it is possible to know whether or not the individual is capable of S-flu resistance. By applying this genetic marker single selection method to breeding flocks, birds with influenza resistance can be bred in a short time.

[0017] The H5N1 avian influenza virus is rampant in the United States, South Korea, Southeast Asia, and Japan, causing many birds to die. However, not all birds are killed at one time on one farm. The spread of the influenza virus has killed a significant number of individuals, while some have not. These phenomena are thought to be due to differences in genetic resistance among individuals within the same line, that is, differences in the type of the gene within the same-ヮ bird group. [0018] In fact, the present inventors experimentally discriminated the population of the Livestock Improvement Center Okazaki Ranch based on the genetic information presented in the present invention. The strains used were 6 White Leg Horns, 2 Rhode Island Reds, 1 White Plymouth Rock, 1 Macular Plymouth Rock, 1 Light Sussex, 1 Mikawa, 1 Nagoya, 1 Koshou Chicken, 1 Aro There are a total of 16 lines, one line per kana.

 [0019] White Leghorn MA strains have 9 resistant homozygotes, 1 heterozygous one, 2 S52 strains resistant to 2 homozygotes, 2 heterozygous, 6 susceptible homozygous, and 11 strains resistant to homozygous. 3 birds, 2 heterozygous birds, resistant homozygous wings in the MK strain, 5 heterozygous birds, 1 susceptible homozygous, 6 strains in the LA strain with resistant homozygous, 2 heterozygous, 1 susceptible homozygous, In the MC line, there were three resistant homozygous wings, three heterozygous wings and three susceptible homozygous wings.

 [0020] Next, in the Rhode Island Red YS line, all 10 birds were susceptible homologues, and in the YA line, all 10 birds were susceptible homologues. White Plymouth Rock has 6 resistant homozygous, 2 heterozygous, 2 susceptible homozygous, 2 laterally Plymouth Rock breed, all 10 susceptible homozygous, Light Sussex all 5 susceptible homozygous, Mikawa 4 resistant homozygotes, heterozygous wings, 3 heterozygous birds in Nagoya breeds, 2 susceptible homozygotes, 5 susceptible homozygotes in the crow bone chicken, 3 resistant homozygotes in the Aroucana breed, There were two heteros.

 [0021] Some deviations were observed depending on the strains. Overall, the resistance Mx gene and the susceptibility gene were almost 50% each. These observations are considered to support the fact that "even under the condition that a considerable number of plants die at the production site, not all of them die at the same time, reflecting the genotype difference."

 As described above, the present inventors succeeded in elucidating, for the first time, the relationship between the Mx genotype of an avian individual and the influenza virus resistance of the avian individual, and completed the present invention. Was.

As described above, the fact that the susceptibility to an RNA virus, particularly an avian influenza virus, dramatically changes depending on the type of the amino acid at position 631 of the avian Mx protein was first proved by the present inventors. The findings found in (1) have made it possible to actually implement a method for determining influenzae resistance. That is, if there is no knowledge found by the intensive studies by the present inventors, RNA viruses and It is extremely difficult to determine the resistance to avian influenza virus in Japan, and it is also difficult to efficiently produce avian influenza virus vaccines and influenza vaccines for humans and other livestock. It can be said that.

 The present invention relates to a method for selecting an avian influenza virus-resistant individual for birds, particularly for poultry such as birds and birds, more specifically,

[1] DNA resistance to avian avian Mx gene, comprising the step of determining the base type of DNA encoding the amino acid at position 633 in the amino acid sequence of SEQ ID NO: 2, A method of determining whether or not

 (2) the method according to (1), wherein the test bird is determined to be resistant to RNA virus when the base species is a base encoding asparagine;

 (3) The method according to (1), wherein the test bird is determined to be susceptible to an RNA virus when the base species is a base encoding serine, and when the base is detected as a homozygote,

[4] DNA of the avian Mx gene, which comprises a step of determining the nucleotide type at position 2032 in the nucleotide sequence set forth in SEQ ID NO: 1; How to determine,

 [5] The method according to [4], wherein the test bird is determined to be resistant to an RNA virus when the base species is adenine.

 (6) When the base species is guanine and the base is detected as homozygous,

The method according to (4), which is determined to be susceptible to RNA virus,

 (7) the method according to any one of (1) to (6), wherein the bird is-ヮ,

 (8) the method of any one of (1) to (7), wherein the RNA virus is an avian influenza virus;

 [9] an avian RNA virus resistance determination reagent comprising the following oligonucleotide (a) or (b):

 (a) an oligonucleotide probe having a chain length of at least 15 nucleotides, which is a site on the avian Mx gene and hybridizes to DNA containing the position at position 2032 in the nucleotide sequence of SEQ ID NO: 1;

(b) a site on the avian Mx gene, position 2032 in the nucleotide sequence of SEQ ID NO: 1 Oligonucleotide primers for amplifying DNA containing a site

 [10] The reagent according to [9], wherein the RNA virus is an avian influenza virus.

 In a preferred embodiment of the present invention, the following method is provided.

[A] The above-mentioned determination method comprising the following steps (a) to (c).

 (a) Preparation of DNA from test birds

 (b) DNA on the avian Mx gene, which encodes the amino acid at position 631 in the amino acid sequence of SEQ ID NO: 2 (eg, DNA at position 2032 in the nucleotide sequence of SEQ ID NO: 1) Amplifying DNA containing

 (c) determining the base sequence of the amplified DNA

 [B] The above-mentioned determination method, comprising the following steps (a) to (e).

 (a) Preparation of DNA from test birds

 (b) DNA on the avian Mx gene, which encodes the amino acid at position 631 in the amino acid sequence of SEQ ID NO: 2 (eg, DNA at position 2032 in the nucleotide sequence of SEQ ID NO: 1) Amplifying DNA containing

 (c) Step of dissociating the amplified DNA into single strands

 (d) Separating the dissociated single-stranded DNA on a non-denaturing gel

 (e) comparing the mobility of the separated single-stranded DNA on the gel with a control

[C] The above-mentioned determination method comprising the following steps (a) to (f).

 (a) Preparation of DNA from test birds

 (b) DNA on the avian Mx gene, which encodes the amino acid at position 631 in the amino acid sequence of SEQ ID NO: 2 (eg, DNA at position 2032 in the nucleotide sequence of SEQ ID NO: 1) Amplifying DNA containing

 (c) providing a substrate on which the nucleotide probe is immobilized;

 (d) Step of bringing the DNA of step (b) into contact with the substrate of step (c)

 (e) a step of detecting the intensity of hybridization between the DNA and the nucleotide probe immobilized on the substrate.

(f) comparing the intensity detected in step (e) with a control [0025] By using the above method of the present invention, an RNA virus vaccine can be produced. For the production of RNA virus vaccines, fertilized eggs of -bird are generally used. It is considered that the fertilized eggs can produce a vaccine more efficiently and efficiently with RNA virus-sensitive birds than with RNA virus-resistant birds. Therefore, if it is possible to select RNA virus-sensitive birds efficiently, it would be very useful in the production of actin. Until now, there has been no known method for determining whether a given bird is resistant to RNA virus.

 [0026] The present inventors have provided a method for judging whether or not a bird has RNA virus resistance as described above, and for the first time, have developed a very efficient and effective method for producing an RNA virus vaccine. Was done. That is, the present invention further provides

 [11] a method for producing an RNA virus vaccine, comprising the following steps (a) to (e):

 (a) A step of selecting a bird determined to be not resistant to RNA virus or susceptible to RNA virus by the method described in [7].

 (b) Step of obtaining embryonated chicken eggs of the bird selected in step (a)

 (c) a step of injecting an RNA virus into the general urine fluid (chorion fluid) of the embryonated chicken eggs

 (d) Step of incubating the chicken eggs obtained in step (c)

 (e) Step of collecting general urine fluid from the eggs obtained in step (d)

 [12] The method according to [11], wherein the RNA virus is an avian influenza virus.

[0027] Further, the present invention provides

 [13] an RNA virus vaccine produced by the method of [11] or [12], [14] a DNA encoding an Mx protein in which the amino acid at position 631 in the amino acid sequence of SEQ ID NO: 2 is serine. Containing an RNA virus (eg, avian influenza virus) for vaccine production-Petri,

 [15] use of the-ヮ bird according to [14] for producing an RNA virus (eg, avian influenza virus) vaccine,

A method for preventing avian influenza, which comprises a step of administering an effective amount of an RNA virus vaccine obtained by the production method according to [16] to an individual (human, domestic animal, poultry, or the like). To provide.

 Brief Description of Drawings

 FIG. 1 is a graph showing VSV infectivity of mouse 3T3 cells into which a chicken Mx gene and a mutant Mx gene have been introduced. The vertical axis shows the VSV infection ratio (%) of the cells, and the horizontal axis shows the type of the transfected cells. 3T3: mouse 3T3 cells without gene transfer. pCI: mouse 3T3 cells transfected only with beta. KS: Mouse 3T3 cells that express mRNA of the Koxamo Mx gene. N: Mouse 3T3 cells expressing mRNA of Nagoya Mx gene. S2: Mouse 3T3 cells expressing the mRNA of the summer 2 Mx gene. KS / N631: Mouse 3T3 cells expressing mRNA of Mx gene of Koshamo in which amino acid number 63 1 is replaced with asparagine. N / N631: Mouse 3T3 cells expressing the Nagoya Mx gene mRNA with amino acid position 631 replaced by asparagine. S2 / S631: Mouse 3T3 cells expressing the mRNA of the Mx gene of Satsumadori 2 in which amino acid position 631 has been replaced with serine. The number in the box indicates the number of tests.

 [FIG. 2] FIG. 2 is a view showing an amino acid sequence of Mx of -Petri (PubMed: Z23168). The 631 position of amino acid is the underlined S (Ser: serine) moiety. N (Asn: asparagine) in resistance-Petria.

 [Fig. 3] Fig. 3 is a view showing a nucleotide sequence (PubMed: Z23168) of a cDNA of the -Avian Mx gene. The portion corresponding to amino acid position 631 (agt) is underlined. This part is aat in the resistance-petri.

 BEST MODE FOR CARRYING OUT THE INVENTION

[0029] The present inventors have found that individuals having Asn homozygous or heterozygous amino acids at position 631 of the avian Mx gene have higher resistance to RNA viruses, particularly avian influenza virus, than individuals having Ser homozygous. It was found to show sex. Therefore, by determining the base type encoding the amino acid at position 631 in the gene, it can be determined whether or not the test bird is resistant to the RNA virus. According to the method of the present invention, it is possible to easily determine whether or not a test bird is resistant to an RNA virus, and thereby it is possible to select (select) birds that are resistant to an RNA virus. It is possible, and the present invention is considered to be effectively used for breeding and the like in the poultry industry. In addition, since fertilized eggs produced from virus-sensitive birds are suitable for efficient production of vaccines, the present invention is considered to be effectively used for vaccine production for humans, livestock, and poultry.

 [0030] In the present invention, "birds" are usually birds, preferably poultry. The poultry is, for example, -Avian, turkey, pigeon, duck, goose and the like, and most preferably -Avian and duck in the present invention.

 In the present invention, “RNA virus resistance” means, for example, “resistance to RNA virus-derived disease”. The “RNA virus” in the present invention includes, for example, Myxovirus. In the present invention, the RNA virus preferably includes influenza virus, Newcastle disease virus and the like. The RNA virus in the present invention is not particularly limited, but is usually an influenza virus, preferably an avian influenza virus.

 The influenza virus belongs to the family Orthomyxoviridae, and the virus of Newcastle disease belongs to the family Paramyxoviridae. Both are myxoviruses belonging to the same genus. The relationship between the polymorphism of the tri-Mx gene (amino acid mutation on the Mx protein) and the influenza virus resistance Z susceptibility of the present invention is considered to be directly applicable to the avian resistance Z susceptibility to Newcastle disease virus. Can be Therefore, in the present invention, it is possible to suitably determine whether or not a test bird has resistance to Newcastle disease virus.

 [0032] Influenza is an acute infectious respiratory disease, caused by the influenza virus. Influenza virus is a virus belonging to the Orthomyxoviridae influenza genus and invades the upper respiratory mucosa and affects the respiratory tract.

[0033] As an example of the avian Mx gene in the present invention, the nucleotide sequence of the cDNA of -Avian Mx gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the nucleotide sequence is shown in SEQ ID NO: 2.

The data on the entire nucleotide sequence of the avian Mx gene of the present invention, or any partial sequence, and the amino acid of the protein encoded by the gene can be obtained by a person skilled in the art using a public gene bank or a literature database. And obtain it as appropriate Is possible. For example, the-ヮ bird Mx gene can be easily obtained from PubMed by accession number: Z23168. -For poultry other than birds, for example, the Mx protein and exon sequences in ducklings can be found in GenBank accession number Z21549 and in literature (Bazzigher, L., bchwarz.A. And Staeheli'P., No enhanced influenza virus resistance of murine and avian cells expressing cloned duck Mx protein ", Virology 195 (1), 100-112 (1993)).

[0034] DNA usually has a double-stranded DNA structure complementary to each other. Therefore, in this specification, even if a DNA sequence on one strand is shown for convenience, it is construed that a sequence complementary to the sequence (base) is also disclosed. If a person skilled in the art knows one DNA sequence (base), the sequence (base) complementary to the sequence (base) is obvious.

 [0035] Since chromosomes generally exist in pairs, each chromosome usually has two DNA sites corresponding to a test DNA site. The determination of the “base type of the DNA on the avian Mx gene, which encodes the amino acid at position 631 in the amino acid sequence set forth in SEQ ID NO: 2” of the present invention refers to at least one of To determine.

[0036] It should be noted that those skilled in the art, based on the information on the base sequence represented by SEQ ID NO: 1 disclosed in the present specification, may be a DNA on the avian Mx gene as appropriate and described in SEQ ID NO: 2. It is easy to know the actual position on the genome corresponding to position 631 in the amino acid sequence. For example, by referring to a published genome database, etc., the position of the DNA on the avian Mx gene of the present invention at position 631 in the amino acid sequence described in SEQ ID NO: 2 in the genome can be known. Can be. That is, even if there is a slight difference in base sequence (substitution, deletion, insertion, etc.) between the base sequence listed in the sequence table and the actual base sequence on the genome, The DNA on the avian Mx gene of the present invention, which encodes the amino acid at position 631 in the amino acid sequence of SEQ ID NO: 2, by conducting homology search or the like with the genome sequence based on the base sequence listed in It is possible to know the exact position of the DNA on the actual genome. Further, even when the position on the genome cannot be specified, the present invention can be carried out from the information in the sequence listing described in the present specification. The present invention is characterized by determining the type of the amino acid at position 631 of the Mx protein (for example, the amino acid sequence set forth in SEQ ID NO: 2) in a test bird, It is a method of determining whether or not the above is true.

 In the method of the present invention, when the amino acid at position 631 is asparagine, the test bird is determined to be resistant to RNA virus. That is, the determination method of the present invention can be performed by determining the base type of the DNA site on the Mx gene encoding the amino acid at position 631.

 [0038] In a preferred embodiment of the present invention, a DNA encoding the amino acid at position 631 in the amino acid sequence set forth in SEQ ID NO: 2 which is DNA on the avian Mx gene (in the present specification, the DNA The corresponding site on the Mx gene may be referred to as “test DNA site.” “DNA containing test DNA site” usually refers to Mx gene DNA containing the test DNA site or a fragment thereof. And b) determining whether the test bird has RNA virus resistance or not.

 More specifically, the present invention relates to the DNA of the avian Mx gene, wherein the base type of the DNA encoding the amino acid at position 631 in the amino acid sequence of SEQ ID NO: 2 is asparagine (Asn). This method is used to judge that the test bird is resistant to RNA virus if it is an encoded base.

 Since the gene codon encoding asparagine is “aat” or “aac”, for example, “aat” or “aac” was detected as DNA (codon) encoding the amino acid at position 631 In some cases, the test bird is determined to be resistant to RNA virus. More specifically, in the nucleotide sequence of SEQ ID NO: 1, (i) the nucleotide species at position 2032 is “a”, or (ii) the nucleotide species at position 2032 is “a” and If the base species is "c", the test bird is determined to be resistant to RNA virus.

 [0041] The term "sensitivity" in the present invention means that a test bird is susceptible to a disease caused by an RNA virus!

In this method, the base species may be detected in a heterozygous (hetero type) in the test bird. That is, in the present invention, the base type in the above DNA site is determined for one chromosome of the test bird, and the base may be a base encoding asparagine. On the spot If so, the test bird is determined to be virus resistant. Further, in the present invention, a test can also be performed on two chromosomes to be paired with a test bird by determining the base type at the DNA site.

 [0042] In another embodiment of the method of the present invention, the base species of the DNA on the avian Mx gene and encoding the amino acid at position 631 in the amino acid sequence of SEQ ID NO: 2 is serine (Ser). ), And when the base is detected as homozygous, the test bird is determined to be susceptible to RNA virus.

 [0043] The gene codon encoding serine is known to have "agt", "agc", etc. Preferably, if "agt" is detected, the test bird is determined to be susceptible to RNA virus Is done. More specifically, when the nucleotide at position 2032 in the nucleotide sequence of SEQ ID NO: 1 is “g”, the test bird is determined to be susceptible to RNA virus.

 [0044] In the above method, preferably, a test tree in which the above-mentioned base species is detected in a homo (homo type) is determined to be virus-sensitive. For example, for both pairs of two chromosomes to be paired with the test bird, if the base species of the DNA encoding the amino acid at position 631 in the amino acid sequence of SEQ ID NO: 2 is serine, the test bird is an RNA virus Determined to be susceptible.

 [0045] As described above, in the present invention, by determining the base type at position 2032 in the DNA of the avian Mx gene in the base sequence set forth in SEQ ID NO: 1, the test bird is tested for RNA virus resistance. It is possible to determine whether or not sex is present. Therefore, the present invention relates to a method for determining whether or not a test bird is an RNA virus resistant DNA, comprising a step of determining the nucleotide type at position 2032 in the nucleotide sequence of SEQ ID NO: 1 which is DNA on the avian Mx gene. It provides a method for determining whether or not to do so.

 [0046] In the above method, for example, when the base species is adenine (A) as described above, the test bird is determined to be resistant to RNA virus. Alternatively, if the base species is guanine (G) and the base species is detected as homozygous, the test bird is determined to be susceptible to RNA virus.

[0047] In the above method of the present invention, the nucleotide type or genotype at position 2032 in the nucleotide sequence of SEQ ID NO: 1 in the DNA of the avian Mx gene is determined. . Generally, “genotype” refers to the presence of an allele or allele at the locus of interest. That is, “genotype” refers to a combination of genes (base species) at a certain locus. In the present specification, when expressing a genotype, it is described as x / x (x is a base type). In the present invention, the nucleotide type is determined for the DNA on the avian Mx gene present as a pair on two chromosomes and at position 2032 in the nucleotide sequence of SEQ ID NO: 1 (that is, For example, when the genotype at position 2032 in the nucleotide sequence of SEQ ID NO: 1 is AZA or AZG, the test bird is resistant to RNA virus, and GZG If, the test specimen is determined to be susceptible to RNA virus.

[0048] In the method of the present invention, "determination of base type" can be performed by those skilled in the art by various methods. As an example, it can be carried out by directly determining the nucleotide sequence of the DNA containing the test DNA site of the present invention.

 In this method, first, a DNA sample is prepared from a test bird. In the present invention, the DNA sample is based on, for example, chromosomal DNA, cDNA, RNA, or the like extracted from blood, skin, oral mucosa, excised tissues or cells, body fluids collected for the purpose of examination, etc. Can be prepared.

 In this method, the DNA containing the test DNA is then isolated. The isolation of the DNA can also be performed by PCR using chromosomal DNA, cDNA or RNA as a type II, using a primer that hybridizes to the DNA containing the test DNA.

 In this method, the nucleotide sequence of the isolated DNA is determined. The determination of the base sequence of the isolated DNA can be easily performed by those skilled in the art using a DNA sequencer or the like. In the present invention, as described above, if the position 2032 is A, the test bird is determined to be virus-resistant, and if G, the test bird is determined to be virus-sensitive. Is done.

[0049] Also, various methods for determining a base type are known. The method for determining a base type according to the present invention is not particularly limited. For example, TaqMan PCR, Acyclo Prime method, MALDI-TOF / MS method, and the like have been put to practical use as an analysis method applying the PCR method. Independent of PCR! Invader method and RCA method are known as methods for determining base type! Further, the base type can be determined using a DNA array. The following briefly describes these methods. Any of the methods described here can be applied to the determination of the base type in the present invention.

 [0050] [TaqMan PCR method]

 The principle of the TaqMan PCR method is as follows. The TaqMan PCR method is an analysis method using a primer set capable of amplifying a region containing an allele and a TaqMan probe. TaqMan probes are designed to hybridize to the region containing the allele amplified by this primer set.

 If the target base sequence is hybridized under the conditions close to the Tm of the TaqMan probe, the hybridization efficiency of the TaqMan probe is significantly reduced due to the difference of one base. If PCR is performed in the presence of the TaqMan probe, the extension reaction from the primer will eventually reach the hybridized TaqMan probe. At this time, the TaqMan probe is degraded from its 5 'end by the 5'-3' exonuclease activity of DNA polymerase. By labeling the TaqMan probe with a reporter dye and quencher, the degradation of the TaqMan probe can be tracked as a change in the fluorescent signal. In other words, when the TaqMan probe is degraded, the reporter dye is released and the distance to the quencher is increased, thereby generating a fluorescent signal. If the hybridization of the TaqMan probe decreases due to a single base difference, the degradation of the TaqMan probe does not proceed and no fluorescent signal is generated.

[0051] If a TaqMan probe corresponding to the test DNA site of the present invention is designed and a different signal is generated by the decomposition of each probe, the genotype can be determined at the same time. For example, as a reporter dye, 6-carboxy-fluorescein (FAM) is used as a TaqMan probe for allele A of a certain allele, and VIC is used as a probe for allele B. In a state where the probe is not decomposed, the fluorescence signal generation of the reporter dye is suppressed by the quencher. When each probe hybridizes to the corresponding allele, a fluorescence signal corresponding to the hybridization is observed. That is, it becomes clear that the signal of either FAM or VIC is stronger than the other! On the other hand, when the allele is heterologous, both signals will be detected at almost the same level. Separation on gel by using TaqMan PCR method PCR and genotyping can be performed simultaneously on genomes without the time-consuming steps described above. Therefore, the TaqMan PCR method is useful as a method for genotyping a large number of subjects.

[0052] [Acyclo Prime method]

 The Acyclo Prime method has also been put to practical use as a genotyping method using the PCR method. The Acyclo Prime method uses one set of primers for genomic amplification and one primer for SNPs detection. First, a region containing SNPs in the genome is amplified by PCR. This step is the same as for normal genomic PCR. Next, the obtained PCR product is annealed with a primer for detecting SNPs, and an extension reaction is performed. Primers for SNPs detection are targeted for detection.

V is designed to anneal to the region adjacent to the SNPs.

 At this time, a nucleotide derivative (terminator) labeled with a fluorescent polarizing dye and blocking 3′-OH is used as a nucleotide substrate for the extension reaction. As a result, only one base complementary to the base at the position corresponding to the SNPs is incorporated, and the extension reaction is stopped. Incorporation of the nucleotide derivative into the primer can be detected by an increase in Fluorescence polarization (FP) due to an increase in molecular weight. If two types of labels with different wavelengths are used for the fluorescent polarization dye, specific SNPs can be used for the two types of bases.

V, it can be specified whether it is a deviation. Because the level of fluorescence polarization can be quantified, a single analysis can determine whether an allele is homo or hetero.

 [0053] [MALDI—TOF / MS method]

The genotype can also be analyzed by analyzing the PCR product with MALDHTOF / MS. MALDI-TOF / MS can be used in various fields as an analytical method that can clearly identify small differences in protein amino acid sequences and DNA base sequences because it can determine the molecular weight extremely accurately. Being done. In order to determine genotype by MALD TOF / MS, the region containing the allele to be analyzed is first amplified by PCR. The amplified product is then isolated and its molecular weight is determined by MALDI-TOF / MS. Since the nucleotide sequence of the allele is preliminarily determined, the nucleotide sequence of the amplification product is uniquely determined based on the molecular weight. Specifically, first, DNA is prepared from a test bird, and then the DNA containing the test DNA site is amplified. Next, the amplified DNA is subjected to a mass spectrometer to measure the molecular weight. Next V. Compare the measured molecular weight with the control.

 Genotyping using MALDI-TOF / MS requires a step of separating PCR products. However, accurate genotyping can be expected without using labeled primers or labeled probes.

 [SNPs-specific labeling method using lis type restriction enzyme]

 There have also been reports of methods that can perform genotyping at higher speeds using the PCR method. For example, genotyping of SNPs has been performed using lis-type restriction enzymes. In this method, a primer having a recognition sequence for a lis type restriction enzyme is used for PCR. A general restriction enzyme (type II) used for gene recombination recognizes a specific nucleotide sequence and cuts a specific site in the nucleotide sequence. In contrast, lis-type restriction enzymes recognize a specific nucleotide sequence and cut sites away from the recognized nucleotide sequence. The number of bases between the recognition sequence and the cleavage site is determined by the enzyme. Therefore, if the primer containing the recognition sequence for lis-type restriction enzyme is annealed at a position separated by this number of bases, the amplification product can be cleaved at the site of SNPs by the lis-type restriction enzyme. . A cohesive end containing the bases of SNPs is formed at the end of the amplification product cleaved with the lis type restriction enzyme. Here, an adapter consisting of a base sequence corresponding to the cohesive end of the amplification product is ligated. The adapters can be labeled with different fluorescent dyes, each having a different base sequence containing bases corresponding to SNPs. Finally, the amplification product is labeled with a fluorescent dye corresponding to the base at the SNPs site.

 If PCR is performed by combining a primer containing the lis type restriction enzyme recognition sequence with a capture primer, the amplification product can be fluorescently labeled and immobilized using the capture primer. For example, if a biotin-labeled primer is used as a capture primer, the amplification product can be captured on avidin-conjugated beads. By tracking the fluorescent dye of the amplification product thus captured, the genotype can be determined.

 [Multiplexing SNPs typing using magnetic fluorescent beads]

The following method for polymorphic site (SNPs) typing can also be applied to the determination of the base type at the test DNA site of the present invention. Techniques are also known in which a plurality of alleles can be analyzed in parallel in a single reaction system. Analyzing multiple alleles in parallel is called multiplexing. In general, a typing method using a fluorescent signal requires fluorescent components having different fluorescent wavelengths for multiplexing. There are not so many fluorescent components that can be used for actual analysis. On the other hand, when a plurality of types of fluorescent components are mixed with resin or the like, various types of fluorescent signals that can be distinguished from each other can be obtained even with limited types of fluorescent components. Furthermore, if a component that is magnetically adsorbed is added to the resin, the resin emits fluorescence and can be separated into beads by magnetism. Multiplexed SNPs typing using such magnetic fluorescent beads has been devised (Bioscience and Bioindustry, Vol.60 No.12, 821-824).

 In multiplexed SNPs typing using magnetic fluorescent beads, a probe having a base complementary to the polymorphic site of each allele at the end is immobilized on the magnetic fluorescent beads. The two are combined such that each allele corresponds to a magnetic fluorescent bead having a unique fluorescent signal. On the other hand, when the probe fixed to the magnetic fluorescent beads hybridizes to the complementary sequence, a fluorescently labeled oligo DNA having a complementary base sequence in a region adjacent to the allele is prepared.

 The region containing the allele is amplified by asymmetric PCR, the above-described probe for immobilizing magnetic fluorescent beads and the fluorescent-labeled oligo DNA are hybridized, and both are ligated. When the end of the magnetic fluorescent bead immobilization probe has a base sequence complementary to SNPs, ligation is performed efficiently. Conversely, if the terminal bases are different due to polymorphism, the ligation efficiency of both will decrease. As a result, the fluorescent-labeled oligo DNA binds to each magnetic fluorescent bead only when the sample has a genotype complementary to the magnetic fluorescent bead.

The genotype is determined by collecting the magnetic fluorescent beads by magnetism and detecting the presence of fluorescently labeled oligo DNA on each magnetic fluorescent bead. Since the fluorescent signal can be analyzed for each magnetic fluorescent bead using a flow cytometer, it is easy to separate the signals even when various types of magnetic fluorescent beads are mixed. In other words, “multiplexing” is achieved in which multiple types of SNPs are analyzed in parallel in a single reaction vessel. [0057] [Invader method]

 The following method for polymorphic site (SNPs) typing can also be applied to the determination of the base type at the test DNA site of the present invention.

 Methods for genotyping that do not depend on the PCR method have also been put to practical use. For example, in the Invader method, genotyping is achieved using only three types of oligonucleotides, namely, an Arenole probe, an Invader probe, and a FRET probe, and a special nuclease called cleavase. Of these probes, only the FRET probe needs to be labeled.

 The allele probe is designed so as to be in a region adjacent to the allele to be detected. On the 5 ′ side of the allele probe, a flap that has a base sequence that is unrelated to hybridization is linked. The allele probe has a structure that hybridizes to the 3 ′ side of the polymorphic site and is connected to a flap above the polymorphic site.

 On the other hand, the invader probe is composed of a nucleotide sequence that hybridizes to the 5 ′ side of the polymorphic site. The nucleotide sequence of the invader probe is designed by hybridization so that the 3 'end corresponds to the polymorphic site. The base at the position corresponding to the polymorphic site in the Invader probe may be arbitrary. That is, the base sequences of both the invader probe and the allele probe are designed so that they hybridize adjacent to each other across the polymorphic site.

 [0058] When the polymorphic site is a base complementary to the base sequence of the allele probe, when both the invader probe and the alenorre probe hybridize to the alenorre probe, the invader probe is replaced with a base corresponding to the polymorphic site of the alenorreprobe. A structure is formed in which the probe has invaded. Cleavase cleaves the penetrated side strand of the thus formed oligonucleotides forming the interstitial structure. Since the cut occurs on the intrusion structure, the result is that the flap of the allele probe is cut off. On the other hand, if the base at the polymorphic site is not complementary to the base of the allele probe, the invader probe will not compete with the allele probe at the polymorphic site to form an invasive structure. Therefore, the flap is not cut by cleavase.

[0059] The FRET probe is a probe for detecting the flap thus separated. The FRET probe has a self-complementary sequence at the 5 'end, and forms a hairpin loop in which a single-stranded portion is arranged at the 3' end. The single-stranded portion located at the 3 'end of the FRET probe also has a nucleotide sequence complementary to the flap, and the flap can hybridize there. Flap force When hybridized to the SFRET probe, both base sequences are designed so that a structure in which the 3 ′ end of the flap invades the 5 ′ end of the self-complementary sequence of the FRET probe is formed. Cleavase recognizes and cuts invasive structures.

If a portion of the FRET probe that is cleaved by the cleavase is sandwiched and labeled with a reporter dye and quencher similar to that of TaqMan PCR, the cleavage of the FRET probe can be detected as a change in the fluorescent signal.

 Theoretically, the flap should still hybridize to the FRET probe even if it is not cleaved. However, in practice, there is a large difference in the binding efficiency to FRET between the cleaved flap and the flap existing in the state of the allele probe. Therefore, it is possible to specifically detect cleaved flaps using FRET probes

In order to determine the genotype based on the Invader method, two types of allele probes containing base sequences complementary to allele A and allele B may be prepared. At this time, the base sequences of both flaps are different base sequences. If two types of FRET probes for flap detection are prepared and each reporter dye can be identified, the genotype can be analyzed in the same way as in the TaqMan PCR method.

 The advantage of the Invader method is that the only oligonucleotide that needs to be labeled is the FRET probe. The same oligonucleotide can be used as the FRET probe regardless of the nucleotide sequence to be detected. Therefore, mass production is possible. On the other hand, it is not necessary to label the allele probe and the invader probe! Therefore, reagents for genotyping can be produced at a low cost.

[0061] [RCA method]

An RCA method can be mentioned as a method for genotyping that does not depend on the PCR method. DNA polymerase with strand displacement action Long circular single-stranded DNA Rolling Circle DNA amplification method based on complementary strand synthesis reaction

 Amplification (RCA) method (Lizardri PM et al, Nature Genetics 19, 225, 1998). In the RCA method, an amplification reaction is configured using a primer that anneals to circular DNA to start complementary strand synthesis and a second primer that anneals to a long complementary strand generated by this primer. .

 The RCA method utilizes a DNA polymerase having a strand displacement action. Therefore, the portion that has become double-stranded by complementary strand synthesis is replaced by a complementary strand synthesis reaction initiated by another primer that has been annealed to the 5 ′ side. For example, a complementary strand synthesis reaction in which a circular DNA is a type II does not end in one round. The complementary strand synthesis continues while replacing the previously synthesized complementary strand, producing a long single-stranded DNA. On the other hand, the second primer anneals to the long single-stranded DNA generated as a circular DNA as the 铸 type, and the complementary strand synthesis starts. Since the single-stranded DNA generated by the RCA method has a circular DNA as a type III, its base sequence is a repetition of the same base sequence. Thus, the continuous generation of a long single strand results in a continuous annealing of the second primer. As a result, a single-stranded portion where the primer can be annealed without undergoing the denaturation step is continuously produced. Thus, DNA amplification is achieved.

 [0062] If the circular single-stranded DNA required for the RCA method is generated according to the bases of the SNPs, the SNPs can be typed using the RCA method. For this purpose, a linear, single-stranded padlock probe is used. The nodlock probe has complementary nucleotide sequences on both sides of the SNPs to be detected at the 5 'end and the 3' end. These base sequences are linked by a part consisting of a special base sequence called a backbone. If the SNPs portion has a nucleotide sequence complementary to the terminal of the padlock probe, the terminal of the padlock probe hybridized to the allele can be ligated by DNA ligase. As a result, the linear padlock probe is circularized, triggering the RCA reaction. The reaction efficiency of DNA ligase is significantly reduced when the terminal portions to be ligated are not completely complementary. Therefore, it is possible to determine the genotype of SNPs by confirming the presence or absence of ligation by the RCA method.

[0063] In the RCA method, a signal cannot be generated with the force capable of amplifying DNA. Ma If only the presence or absence of amplification is used as an index, the genotype cannot be determined unless a reaction is performed for each allele. Methods that improve these points for genotyping are known.

. For example, molecular beacons can be used to perform genotyping in one tube based on the RCA method. The molecular beacon is a signal generation probe using a fluorescent dye and quencher, as in the TaqMan method. The molecular beacon is composed of complementary base sequences at the 5 'and 3' ends and forms a hairpin structure by itself. If both ends are labeled with a fluorescent dye and quencher, a fluorescent signal cannot be detected while the hairpin structure is formed. If a part of the molecular beacon is used as a base sequence complementary to the RCA amplification product, the molecular beacon will hybridize to the RCA amplification product. The hybridization breaks down the hairpin structure and generates a fluorescent signal.

 [0064] The advantage of the molecular beacon is that by using the base sequence of the backbone portion of the padlock probe, the base sequence of the molecular beacon can be shared regardless of the detection target. By changing the base sequence of the backbone for each allele and combining two types of molecular beacons with different fluorescence wavelengths, genotyping can be performed with one tube. Since the cost of synthesizing fluorescently labeled probes is high, it is an economical advantage to be able to use a common probe regardless of the measurement target.

 [0065] [Single molecule fluorescence spectroscopy]

 liUfemtoliter) and a system that enables fluorescence analysis of microscopic regions have been put to practical use! Using this system, the extension of fluorescently labeled primers

(translations diffiision time). Prepare primers each having a base sequence complementary to the allele to be typed. Each of the primers has a fluorescent label that can be identified. PCR is performed using these primers, and the amplified product is measured for fluorescence by Fluorescence Correlation Spectroscopy. If the sample has a base sequence complementary to the primer, the primer is extended by PCR. The extended primer causes a fluctuation in fluorescence because the molecule becomes large. This fluctuation in fluorescence is detected as an increase in translational diffusion time. The base sequence complementary to the primer is included in the sample Otherwise, there will be no change in fluorescence since no PCR amplification product is generated. Specifically, PCR is performed on the two alleles A and B in the same reaction mixture using primers having different fluorescent labels. In the fluorescence measurement of the amplification product, if a change in the fluorescent signal of either A or B is observed, it can be confirmed that either homozygous or heterozygous if both fluorescent signals change (PharmaGenomics, July / August 46-48, 2003). It is evaluated as an accurate and quick analysis method.

 [0066] These methods are methods developed for high-speed genotyping of samples with large amounts of deviation. Except for MALDI-TOF / MS, it is necessary to prepare some kind of labeled probe for each method. On the other hand, gene type determination that does not rely on labeled probes has been performed for a long time.

 [0067] As one of such methods, for example, a method using restriction fragment length polymorphism (ZRFLP), a PCR-RFLP method, and the like can be mentioned. These methods can also be applied to the determination of the base type at the test DNA site of the present invention.

 RFLP utilizes the fact that mutation of a recognition site of a restriction enzyme or insertion or deletion of a base in a DNA fragment resulting from treatment with a restriction enzyme can be detected as a change in the size of the fragment generated after treatment with the restriction enzyme. If there is a restriction enzyme that recognizes the nucleotide sequence containing the polymorphism to be detected, the base at the polymorphic site can be known by the RFLP principle.

[0068] Specifically, when a mutation is present at the recognition site of the restriction enzyme, the size of the fragment generated after the treatment with the restriction enzyme changes as compared with the control. By amplifying the portion containing this mutation by the PCR method and treating it with each restriction enzyme, these mutations can be detected as a difference in band movement after electrophoresis. Alternatively, the presence or absence of a mutation can be detected by treating chromosomal DNA with these restriction enzymes, electrophoresing, and performing Southern blotting using probe DNA. The restriction enzyme to be used can be appropriately selected according to each type. In this method, in addition to genomic DNA, RNA that has also been adjusted for test bird power can be converted into cDNA with reverse transcriptase, and this can be directly cut with restriction enzymes, followed by Southern blotting. In addition, this cDNA is After amplifying the DNA containing the test DNA site of the present invention and digesting it with a restriction enzyme, the difference in mobility can be examined.

 [0069] As a method that does not require a labeled probe, a method of detecting a difference in base using a change in the secondary structure of DNA as an index is also known. PCR-SSCP utilizes the fact that the secondary structure of single-stranded DNA reflects the difference in its base sequence (Cloning and polymerase chain reaction— single—strand conformation polymorphism analysis of anonymous Am repeats on chromosome 丄 丄Genomics. 1992 Jan 1; 12 (1): 139-146., Detection of p53 gene mutations in human brain tumors by single-strand conformation polymorphism analysis of polymerase chain reaction products.Oncogene.1991 Aug 1; 6 (8): 1313- 1318., Multiple fluorescence-based PCR-SS, P analysis with postlabeling., PCR Methods Appl. 1995 Apr 1; 4 (5): 275-282.). This method has advantages that the operation is relatively simple and the amount of the test sample can be reduced, and is particularly suitable for screening a large number of DNA samples. The principle of the PCR-SSCP method is as follows. When a double-stranded DNA fragment is dissociated into single strands, each strand forms a unique higher-order structure depending on its base sequence. When the dissociated DNA strands are electrophoresed in a polyatarylamide gel containing no denaturing agent, single-stranded DNAs of the same complementary length move to different positions according to the difference in their higher-order structures. The higher-order structure of the single-stranded DNA also changes by the substitution of a single base, and shows a different mobility in polyacrylamide gel electrophoresis. Therefore, by detecting the change in mobility, the presence of a mutation due to a deletion or the like in the DNA fragment can be detected.

[0070] Specifically, first, a DNA sample is prepared from a test bird. Next, the DNA containing the test DNA site of the present invention is amplified by PCR or the like. As a range to be amplified, usually, a length of about 200 to 400 bp is preferable. PCR can be performed by those skilled in the art by appropriately selecting reaction conditions and the like. In PCR, the amplified DNA product can be labeled by using a primer labeled with an isotope such as ³² P, a fluorescent dye, or biotin. Alternatively, the amplified DNA product can be labeled by performing PCR by adding a substrate base labeled with an isotope such as ³² P, a fluorescent dye, or biotin to the PCR reaction solution. Furthermore, after PCR reaction, isotope such as ³² P, Labeling can also be performed by adding a substrate base labeled with a photo dye or biotin to the amplified DNA fragment. The labeled DNA fragment thus obtained is denatured by applying heat or the like, and electrophoresis is performed on a polyacrylamide gel containing no denaturing agent such as urea. At this time, the conditions for separating DNA fragments can be improved by adding an appropriate amount (about 5 to 10%) of glycerol to the polyacrylamide gel. In addition, electrophoresis conditions vary depending on the nature of each DNA fragment.Generally, the reaction is performed at room temperature (20 to 25 ° C), and when the desired separation cannot be obtained, optimal mobility is provided at a temperature of 4 to 30 ° C. Consider the temperature. After electrophoresis, the mobility of the DNA fragment is detected by autoradiography using X-ray film or a scanner that detects fluorescence, and analyzed. If a band with a difference in mobility is detected, this band can also be directly excised from the gel force, re-amplified by PCR, and sequenced directly to confirm the presence of the mutation. Even when labeled DNA is not used, bands can be detected by staining the gel after electrophoresis with ethidium bromide, silver staining, or the like.

 [0071] Other methods that do not require a labeled probe include, for example, denaturant gradient gel electrophoresis (DGGE method) and the like.

 The DGGE method is a method in which a mixture of DNA fragments is electrophoresed in a polyacrylamide gel having a concentration gradient of a denaturing agent, and the DNA fragments are separated based on differences in their instabilities. When an unstable DNA fragment with a mismatch migrates to a certain denaturant concentration in the gel, the DNA sequence around the mismatch is partially dissociated into single strands due to the instability. The mobility of the partially dissociated DNA fragment becomes very slow, and can be separated from the mobility of the complete double-stranded DNA without dissociated parts, because both differ.

Specifically, first, a region including the test DNA site of the present invention is amplified by a PCR method or the like. The amplified product is hybridized with a probe DNA whose base sequence is known to form a double strand. This is electrophoresed in a polyacrylamide gel, which gradually increases as the concentration of a denaturant such as urea moves, and is compared with a control. In a DNA fragment in which a mismatch has occurred due to hybridization with the probe DNA, the DNA fragment becomes single-stranded at a lower concentration of the denaturant, and the migration speed becomes extremely slow. Detecting the difference in mobility that occurs Thus, the presence or absence of a mismatch can be detected.

 [0073] Furthermore, the genotype can be determined using a DNA array (cell engineering separate volume "DNA Microarray and Latest PCR Method", Shujunsha, 2000.4 / 20, pp97-103, " Analysis ”, Shinichi Kajie). The DNA array hybridizes sample DNA (or RNA) to a large number of probes arranged on the same plane, and scans the plane to detect hybridization for each probe. For example, a DNA array is useful for analyzing a large number of SNPs at the same time, because reactions to many probes can be observed simultaneously.

 Generally, a DNA array is composed of thousands of nucleotides printed on a substrate at high density. Normally, these DNAs are printed on the surface of a non-porous substrate. The surface of the substrate may also use a porous membrane, typically glass, for example a -trocellulose membrane.

 [0075] In the present invention, an array based on oligonucleotides developed by Aifymetrix can be exemplified as a method for immobilizing (arraying) nucleotides. In an array of oligonucleotides, the oligonucleotides are usually synthesized in situ. For example, in situ synthesis of oligonucleotides by photolithographic technology (Aifymetrix) and ink jet (Rosetta Inpharmatics) technology for immobilizing chemical substances are already known, and both technologies are applicable to the substrate of the present invention. It can be used for fabrication.

[0076] The oligonucleotide is composed of a base sequence complementary to the region containing the SNPs to be detected. The length of the nucleotide probe to be bound to the substrate is usually 10-100 base, preferably 10-50 base, and more preferably 15-25 base when the oligonucleotide is immobilized. Furthermore, in the DNA array method, a mismatch (MM) probe is generally used in order to avoid an error due to cross-hybridization (non-specific hybridization). The mismatch probe forms a pair with an oligonucleotide having a nucleotide sequence completely complementary to the target nucleotide sequence. Oligonucleotides whose base sequence is completely complementary to the mismatched probe are called perfect match (PM) probes. Signals observed with mismatched probes during data analysis Eliminating the nulls can reduce the effects of cross-hybridization.

 [0077] A sample for genotyping by the DNA array method can be prepared by a method well known to those skilled in the art based on a biological sample collected from a test bird. The biological sample is not particularly limited. For example, a DNA sample can be prepared from chromosomal DNA extracted from blood, skin, tissue or cells, feces or feathers of a test bird. A specific region of chromosomal DNA is amplified using primers for amplifying a region containing the test DNA site to be determined. At this time, a plurality of regions can be simultaneously amplified by the multiplex PCR method. Multiplex PCR is a PCR method in which a plurality of primer sets are used in the same reaction solution. When analyzing multiple test DNA sites, multiplex PCR is useful.

 [0078] In the DNA array method, in general, a DNA sample is amplified by a PCR method, and an amplification product is labeled. A labeled primer is used to label the amplification product. For example, first, genomic DNA is amplified by a PCR method using a primer set specific to the region containing the test DNA site of the present invention. Next, the biotin-labeled DNA is synthesized by the labeling PCR method using the biotin-labeled primer. The biotin-labeled DNA thus synthesized is hybridized to an oligonucleotide probe on the chip. The reaction solution and reaction conditions of the hybridization can be appropriately adjusted according to conditions such as the length of the nucleotide probe fixed to the substrate and the reaction temperature. One skilled in the art can design the appropriate conditions for the hybridization. Avidin labeled with a fluorescent dye is added to detect the hybridized DNA. The array is analyzed with a scanner, and the presence or absence of hybridization is confirmed using fluorescence as an index.

[0079] In addition to the above method, an allele-specific oligonucleotide (ASO) hybridization method can be used to detect a base at a specific site. An allele-specific oligonucleotide (ASO) is composed of a base sequence that hybridizes to a region where a test DNA site to be detected exists. When ASO is hybridized to sample DNA, if a mismatch occurs in a test DNA site due to a polymorphism (base mutation), the efficiency of hybrid formation decreases. Mismatch can be detected by Southern blotting or special fluorescent reagents. It can be detected by a method utilizing the property of quenching by intercalating in the gap of the hybrid. Further, the mismatch can also be detected by the ribonuclease A mismatch cleavage method. Specifically, the DNA containing the test DNA site of the present invention is amplified by PCR or the like, and this is hybridized with a labeled RNA prepared by capillary such as cDNA of the Mx gene in which the DNA is incorporated into a plasmid vector or the like. Do. Since the hybrid has a single-stranded structure at the portion where the mutation is present, the presence of the mutation can be detected by cleaving this portion with ribonuclease A and detecting this by autoradiography or the like. .

 [0080] Another embodiment of the above-described test method of the present invention is a method of performing a test using an expression product of an avian Mx gene as an index. Here, “expression” includes transcription and translation. Thus, "expression products" include mRNAs and proteins.

 [0081] The present invention provides a method for determining an avian resistant to an RNA virus (for example, an avian influenza virus), comprising detecting an expression product of an avian Mx gene. In a preferred embodiment of the method, first, a protein sample is prepared from a test bird, and the amount of Mx protein contained in the protein sample at position 631 as asparagine or Mx protein at position 631 as serine Measure the amount of protein.

 [0082] Examples of such a method include SDS polyacrylamide electrophoresis, Western blotting, dot blotting, immunoprecipitation, and enzyme-linked immunoassay using an antibody that recognizes the above-mentioned protein. When the Mx protein having asparagine at position 631 is detected by the above method, the test bird is resistant to an RNA virus (e.g., avian influenza virus). Is determined. On the other hand, when the Mx protein in which position 631 is asparagine is not detected (or when the Mx protein in which position 631 is serine is detected), the test bird is susceptible to an RNA virus (for example, avian influenza virus). Is determined.

[0083] As described above, the forces exemplified in various detection methods are not particularly limited to these. Further, those skilled in the art can implement the method of the present invention by appropriately modifying the above method. [0084] The present invention also provides probes or primers for the method of the present invention. The present invention has clarified the relationship between the nucleotide type of the DNA encoding the amino acid at position 631 in the amino acid sequence of SEQ ID NO: 2 and the RNA virus resistance in the DNA on the avian Mx gene. Therefore, a probe or primer for clarifying the base at the site is useful in the method of the present invention. Various methods such as those described above can be applied to a method for clarifying a base at a certain site. In these techniques, a primer for amplifying a region containing the site or a probe hybridizing to the site is generally used.

 [0085] The present invention also provides a determination reagent for use in the method of the present invention for determining avians having RNA virus resistance.

 The reagent of the present invention hybridizes with a DNA region containing a test DNA site of the present invention, and amplifies an oligonucleotide having a chain length of at least 15 nucleotides or a DNA containing a test DNA site of the present invention. It is a reagent containing a primer.

 [0086] A preferred embodiment of the reagent of the present invention is an avian RNA virus resistance determination reagent containing the following oligonucleotide (a) or (b).

 (a) an oligonucleotide probe having a chain length of at least 15 nucleotides, which is a site on the avian Mx gene and hybridizes to DNA containing the position at position 2032 in the nucleotide sequence of SEQ ID NO: 1;

 (b) an oligonucleotide primer for amplifying DNA containing a site at position 2032 in the nucleotide sequence of SEQ ID NO: 1, which is a site on the avian Mx gene

[0087] The oligonucleotide of the present invention specifically hybridizes to DNA containing the test DNA site of the present invention. As used herein, the term “specifically hybridizes” means under ordinary hybridization conditions, preferably under stringent hybridization conditions (ί column X, Samhunorec et al., Molecular Cloning, Cold Spring Harbor Laboratory). Press, New York, USA, 2nd Edition, 1989) means that no significant cross-hybridization occurs with DNA encoding other proteins. If specific hybridization is possible, the oligonucleotide will be completed with respect to the nucleotide sequence described in (a) or (b) above in the gene to be detected or in the DNA region near the gene. Need not be completely complementary.

 [0088] Stringent hybridization conditions are, specifically, usually conditions of about "lxSSC, 0.1% SDS, 37 ° C", and more stringent conditions are "0.5xSSC, 0.1% SDS". , 42 ° C ”, and as a more severe condition, a condition of“ 0.2xSSC, 0.1% SDS, 65 ° C ”can be exemplified. However, the combination of the above SSC, SDS and temperature conditions is an example, and those skilled in the art will recognize the above or other factors (eg, probe concentration, probe length) that determine the stringency of the hybridization. , Hybridization reaction time, etc.) as appropriate, it is possible to realize the same stringency as described above.

 When the oligonucleotide is used as a primer, its length is usually 15 bp to 100 bp, preferably 17 bp to 30 bp. The primer is not particularly limited as long as it can amplify at least a part of the DNA containing the position at position 2032 in the nucleotide sequence of SEQ ID NO: 1 of the present invention.

 [0090] In the present invention, a probe that hybridizes to a region containing the test DNA site refers to a probe that can hybridize with a polynucleotide having the nucleotide sequence of the region containing the above site. More specifically, a probe containing a test DNA site (for example, position 2032 in the nucleotide sequence of SEQ ID NO: 1) in the nucleotide sequence of the probe is preferable as the probe of the present invention. Alternatively, the probe may be designed so that the end of the probe corresponds to the base adjacent to the polymorphic site, depending on the method of analyzing the base at the site. Therefore, a probe which does not include a test DNA site in the base sequence of the probe itself but includes a base sequence complementary to a region adjacent to the test DNA site can also be indicated as a desired probe in the present invention. .

[0091] In other words, a probe that can hybridize to a region on the genomic DNA adjacent to the test DNA site of the present invention is preferable as the probe of the present invention. In the probe of the present invention, the modification of the base sequence, the addition of the base sequence, or the modification is allowed as in the case of the primer. For example, a probe used in the Invader method has a nucleotide sequence that is unrelated to the genome constituting the flap. Such a probe is also included in the probe of the present invention as long as it hybridizes to the region containing the test DNA site. Construct the probe of the present invention The base sequence can be designed according to the analysis method based on the base sequence of the DNA region around the test DNA site of the present invention in the genome.

[0092] On the other hand, in the present invention, primers for amplifying a region containing the test DNA site include a DNA containing the test DNA site as a 铸 type and complementary strand synthesis toward the test DNA site. Also included are primers that can initiate PCR. The primer can also be expressed as a primer for providing a replication origin on the 3 ′ side of the test DNA site in the DNA containing the test DNA site. The distance between the region where the primer hybridizes and the test DNA site is arbitrary. A suitable number of bases can be selected for the interval between the two in accordance with the method of analyzing bases at the test DNA site. For example, if a primer is used for analysis using a DNA chip, the primer should be designed so that an amplified product with a length of 20 to 500, usually 50 to 200 bases is obtained as the region containing the test DNA site. Can be. Those skilled in the art can design primers according to the analysis method based on the information on the surrounding nucleotide sequence including the polymorphic site. The base sequence constituting the primer of the present invention can be appropriately modified as well as the base sequence completely complementary to the base sequence of the genome.

 [0093] The primer of the present invention can have an arbitrary base sequence added thereto in addition to the base sequence complementary to the base sequence of the genome. For example, a primer to which a recognition sequence for a lis-type restriction enzyme is added is used as a primer for a polymorphism analysis method using a lis-type restriction enzyme. Such a primer whose base sequence has been modified is included in the primer of the present invention. Further, the primer of the present invention can be modified. For example, a primer labeled with a fluorescent substance or a binding affinity substance such as biotin or digoxin is used in various genotyping methods. Primers having these modifications are also included in the present invention.

[0094] The primer or probe of the present invention can be synthesized by any method based on the base sequence constituting it. The length of the nucleotide sequence complementary to the genomic DNA of the primer or probe of the present invention is usually 15-100, generally 15-50, usually 15-30. Techniques for synthesizing oligonucleotides having the given base sequence based on the given base sequence are known. Furthermore, in the synthesis of oligonucleotides, a fluorescent dye Arbitrary modifications can also be introduced into oligonucleotides using nucleotide derivatives modified with thiophene or biotin. Alternatively, a method of binding a fluorescent dye or the like to the synthesized oligonucleotide is also known.

 [0095] The reagent of the present invention can be combined with various enzymes, enzyme substrates, buffers, and the like, depending on the genotyping method. Examples of the enzyme include enzymes required for various analysis methods exemplified as the above-mentioned methods for genotyping, such as DNA polymerase, DNA ligase, and lis restriction enzyme. As the buffer, a buffer suitable for maintaining the activity of the enzyme used in these analyzes is appropriately selected. Further, as the enzyme substrate, for example, a substrate for complementary strand synthesis or the like is used.

 [0096] Further, a control in which the base in the test DNA site is clear can be attached to the reagent of the present invention. As a control, a genome whose genotype is known in advance, or a fragment of the genome can be used. The genome may be extracted from cells, or cells or cell fractions may be used. If cells are used as a control, the results of the control can prove that the genomic DNA extraction operation was performed correctly. Alternatively, DNA having a base sequence including a test DNA site can also be used as a control. Specifically, a YAC vector or a BAC vector containing a DNA derived from a genome in which the base in the test DNA site of the present invention has been identified is useful as a control. Alternatively, a vector into which only a few hundred b containing the test DNA site has been cut out and inserted can be used as a control.

 [0097] The present invention also provides a method for producing an RNA virus vaccine using the above-described determination method of the present invention. According to the determination method of the present invention, -Pet with high RNA virus susceptibility suitable for vaccine production can be selected, and fertilized eggs (preferably embryonated chicken eggs) of the -Pet can be obtained. That is, the present invention relates to a method for producing an RNA virus vaccine, comprising the following steps (a) to (e).

 (a) a step of selecting a bird determined to be not RNA virus-resistant or RNA virus-sensitive by the determination method of the present invention;

 (b) Step of obtaining embryonated chicken eggs of the bird selected in step (a)

 (c) a step of injecting an RNA virus into the general urine fluid of the embryonated chicken eggs

(d) Step of incubating the chicken eggs obtained in step (c) (e) Step of collecting general urine fluid from the eggs obtained in step (d)

 In the method for producing the virus vaccine of the present invention, the RNA virus is preferably an avian influenza virus.

 Normally, virus vaccines can be produced using eggs from chickens, more specifically embryonated chicken eggs. The method for producing and preparing a virus vaccine using embryonated chicken eggs is a technique known to those skilled in the art. In a preferred embodiment of the vaccine production method of the present invention, the above steps (a) to (e) are performed in order. As an example, the ability to produce and prepare a vaccine by the following method is specifically not limited to the following method for producing a peptide of the present invention.

 (1) According to the determination method of the present invention, -viruses susceptible to RNA virus are selected, and fertile eggs (fertilized eggs) of the -viruses are warmed for about 10 days using an incubator. Thereafter, a hole is made in the eggshell, and an RNA virus (preferably avian influenza virus) to be vaccinated is injected into the general urine fluid in the egg, and the hole is closed. Then, use the incubator again and warm for about 3 days.

 (2) Collect the urine fluid in the eggs, remove impurities such as blood, and centrifuge. The virus solution that has settled into a paste is appropriately treated with formalin or the like to kill the virus, and used as a vaccine stock solution.

 According to the method of the present invention, for example, fertilized eggs (embryonic chicken eggs) suitable for vaccine production can be obtained by selecting and crossing RNA virus-sensitive male and female birds.

 Further, the RNA virus vaccine produced by the above method is also included in the present invention. Furthermore, the present invention provides a DNA encoding an Mx protein in which the amino acid at position 631 in the amino acid sequence of SEQ ID NO: 2 is serine. The present invention provides a bird for the production of an RNA virus (eg, avian influenza virus) vaccine.

 The bird can be used for production of RNA virus (eg, avian influenza virus) as described above.

Accordingly, the present invention relates to the use of the above-mentioned vaccine-producing bird for the production of an RNA virus (eg, avian influenzae virus) vaccine. Further, the vaccine of the present invention is effective for preventing diseases caused by RNA viruses. Accordingly, the present invention provides an RNA virus (eg, avian influenza virus) comprising a step of administering an effective amount of an RNA virus vaccine obtained by the vaccine production method of the present invention to an individual (human, domestic animal, poultry, or the like). A method for preventing a disease caused by Administration to an individual can be generally performed by a method known to those skilled in the art, such as, for example, intraarterial injection, intravenous injection, and subcutaneous injection. The dose can be appropriately selected by a person skilled in the art (eg, a physician or veterinarian) in consideration of the weight and age of the individual, the administration method, and the like.

 All prior art documents cited in this specification are incorporated herein by reference.

 Example

 [0099] Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

 (Example 1)

 The present inventors have constructed two types of mutagenized-ヮ avian Mx gene cDNA mutants that clarify whether amino acid position 631 of the Mx protein separates resistance and susceptibility to a disease-causing RNA virus. Infection experiments were performed using vesicular stomatitis virus (R-VSV), which was transfected into influenza virus-sensitive mouse 3T3 cells and recombinant with GFP. In other words, Koshamo (KS) and Nagoya (N) thought that they had serine at the amino acid at position 631, and that they were susceptible to RNA virus. Satsumadori-2 (S2) and beetle squid (HS) were considered resistant because they had asparagine at position 631. Then, a mutant cDNA was constructed so that the amino acid at position 631 was replaced, and it was examined whether susceptibility and resistance could be switched between cells transfected with these genes.

[0100]-The bird Mx gene encodes 705 amino acids! The 2011 to 2016 positions of the cDNA were changed so that the amino acid sequence from the 625th position including the 631 position to the final 707th position was replaced. That is, the p-CI-neo vector containing the KS, N, S2, and HS cDNAs was digested with the restriction enzyme Aatl to obtain fragments of 6300 bp and 1716 bp. The 1716 bp fragment S-encoded amino acids are the same between (KS, N) and (S2, HS) except for position 631, 631 is different from Serine and Asparagine. 631 position is the same serine between KS and N, and 631 position is the same asparagine between S2 and HS. These fragments were ligated to different 6300 bp fragments using a T4 ligase system (Promega) to obtain three mutant cDNAs. That is, the first 6300 bp are derived from KS and the second 1716 bp are derived from S2 (KSZS2), N / HS and S2 / KS. Since the same base sequence is used in all varieties except for the base portion encoding amino acid position 631 in the latter 1716 bp, a cDNA mutant producing Mx protein in which only amino acid position 631 has been replaced is obtained.

 [0101] Plasmid pCI-neo having these mutant cDNAs or not! /, Was introduced into mouse 3T3 cell line. 3T3 cells are derived from the mouse BALB / c strain, which has a defect in the function of the Mx gene, and cannot produce Mx protein by itself. The transfected cells were cultured in a DMEM (Dulbecco, modified Eagle's medium) culture medium containing 10% fetal calf serum (Gibco-BRL). The introduction into 3T3 was performed using FuGene loposome (Roche), and the introduced cells were selected using a culture solution containing 500 μg / ml G418.

 The virus infection intensity on the cells was measured by counting the number of cells expressing GFP after infecting RSV to which the GFP gene was bound (see Non-Patent Document 4).

 [0102] The results are shown in FIG. Assuming that the infection rate of virus-sensitive 3T3 cells is 100%, on average, cells transfected with the intact Mx gene of Koshamo (KS) or Nagoya (N) considered to be susceptible are about 75% or 110%, respectively. % Were infected and confirmed to be susceptible. In addition, the infection rate of Satsuma-dori 2 (S2), which was considered to be resistant, averaged about 10%, confirming that S2 is relatively resistant.

 [0103] On the other hand, a mutant of susceptible KS but amino acid 631 is a resistant S2 mutant (KSZ S2), and a similarly sensitive N but amino acid 631 is a mutant of resistant squirrel (HS) Infected 3T3 cells (N / HS) both showed infection rates of 10% or less. This indicates that even in the power-sensitive Mx protein, only amino acid position 631 is changed to VSV resistance by replacing serine with asparagine.

[0104] Further, only the amino acid position 631 of S2, which is resistant, was replaced with KS, which is sensitive. For 3T3 transfected with Utant cDNA (S2 / KS), the average infection rate was about 95%. In other words, it was shown that even in the resistant Mx protein, the amino acid at position 631 was replaced by asparagine-potassium serine, but the VSV sensitivity was changed.

 [0105] From these facts, at least one of the resistances of the -Pet transgenic cells to VSV, a disease-causing RNA virus, is attributable to the Mx protein, and among them, only the position 631 of the -Pet Mx protein is replaced. It turned out that sensitivity and resistance were switched.

 [Example 2]

 A group of birds of the same breed with the 631 amino acid type of the Mx gene was divided in advance, and A / Hong, a virulent strain of avian influenza virus that was endemic in Hong Kong.

 Using the Kong / 483/97 (H5Nl) strain, infection tests were performed on individuals with different Mx gene types.

First, an in vivo infection experiment was performed to determine the 50% lethality (LD50 value) of 4-week-old Rhode Island Red in which the amino acid at position 631 of the Mx gene is homozygous for Ser. The virus-infected chicken embryo urine was diluted to 10 ^{1 to} 10 ⁶ and inoculated intranasally 100 1 per bird. Four birds were used for each dilution. After the inoculation, the cells were observed for 2 weeks, and the LD50 value of the bird was determined. As a result, the LD50 was 3.5!

Next, from the population of Rhode Island Red species of 5 weeks old, those with the nucleotide sequence of amino acid No. 631 (AAT in the nucleotide sequence) and those with the nucleotide sequence in Ser 631 (AGT in the nucleotide sequence) things, each one of these heteroatoms picked three birds, infection chick embryo urine 100 1 10 ^{3 5} / ml is a LD50 value determined previously by nasal inoculation, performed in vivo infection experiments, 2 Observed for a week.

 The results were as shown in Table 1 (observation of 5-week-old Rhode Island Red infected with A / Hong Kong / 483/97 (H5Nl)).

[Table 1] Group (631 amino acids)

 Vigilance (Asn / Asn) 3 / No abnormalities

 Hetero (Asn / Ser) 3/3 No abnormalities

Raw (Ser / Ser) 1/3 dead, 1/3 fine abnormalities, 1/3 no abnormalities [0108] That is, there was no abnormality in both Asn homo (Asn / Asn) and hetero type (Asn / Ser). However, one homozygous Ser homozygous (Ser / Ser) had died, one had minor clinical symptoms (feather handstand), and one had no abnormality. From this, it was found that individuals having the Asn-type homozygous or the heterozygous individuals having the 631st position of the Mx gene were more resistant than individuals having the Ser-type homozygous. Industrial applicability

[Influenza resistance-breeding of birds and ducks]

 Currently, the -Pet strains that are already reared often have variations in the Mx genotype within the same strain. This indicates that it is possible to easily select and breed influenza-resistant birds using the genetic information disclosed in the present invention. That is, at the stage of the original breeding chicken or the breeding hen, a resistant type (Asn type) homo is selected, and this may be used for production of commercial chickens (utility chickens). Even when resistant homozygotes are not found in the original breeding chickens, it is possible to obtain resistant homozygotes by combining heterozygous types.

 [Production of avian fertilized eggs for virus vaccine production]

 Fertilized eggs of-ヮ birds are used for virus vaccine production. High antiviral activity

V. It is considered that vaccine production is more efficient and more efficient when fertilized eggs are used than when fertilized eggs are used. There was no way to know if you were high or low. However, according to the present invention, it is possible to select "poultry such as birds that can produce fertilized eggs capable of producing vaccines with higher efficiency", and to produce vaccines efficiently for humans and animals. Can contribute.

 [Maintenance of consumption of chicken and eggs]

、た多くの -ヮトリが殺処分されている。これらは、最終的にはヒトへの感染を防止するためにと られた措置である。しかし、消費者はヒトへのウィルス感染を危惧し、鶏肉および鶏卵 の購入を控えることになる。このようなことによる、養鶏産業の経済的被害は総計では 莫大なものと考えられる。 -ヮトリへの遺伝的抵抗性の導入は、インフルエンザの発 生を防止することになり、その養鶏産業への恩恵は計り知れない。 〔ヒトに対して〕 To prevent the spread of the influenza virus,

Many-Petri have been killed. These are the measures that were ultimately taken to prevent transmission to humans. Consumers, however, are concerned about human transmission of the virus and will refrain from purchasing chicken and eggs. The economic damage to the poultry industry due to these factors is considered to be enormous in total. -The introduction of genetic resistance into birds will prevent the outbreak of influenza, and its benefits to the poultry industry are enormous. [For humans]

 -If the spread of influenza virus in birds is long, the probability of acquiring mutations that can easily infect humans has increased, and humans have met! / ヽ Possibility of emergence of a new influenza virus Also increase. A group of birds that are susceptible to influenza will spread the influenza virus in the body for a period of time until infection with influenza and immediate death, while infecting the next individual. At this time, the influenza virus sometimes grows as a new type of influenza virus with genetic mutation. A new influenza virus, such as the influenza virus (H1N1) in the Spanish flu, which spread between 1917 and 1918 and killed more than 40 million people worldwide and 300,000 in Japan alone, could also emerge. is there. If the avian influenza virus becomes highly infectious to humans, it will cause unprecedented damage. According to the present invention, the selection of -triafil, which has a high ability to suppress the growth of influenza virus, suppresses the growth of influenza virus in the group and reduces the possibility of the occurrence of a new influenza virus. This could potentially avoid one of the threats to humanity of the emergence of a new influenza virus.

Claims

The scope of the claims  [I] An avian Mx gene, comprising the step of determining the nucleotide type of DNA encoding the amino acid at position 631 in the amino acid sequence of SEQ ID NO: 2,方法 How to determine whether or not.  [2] The method according to claim 1, wherein the test bird is determined to be resistant to RNA virus when the base species is a base encoding asparagine. [3] The method according to claim 1, wherein the test species is determined to be susceptible to an RNA virus when the base species is a base encoding serine and the base is detected as homozygous. [4] DNA of the avian Mx gene, which comprises a step of determining the nucleotide type at position 2032 in the nucleotide sequence of SEQ ID NO: 1, and determining whether or not the test bird has RNA virus resistance or not. How to determine.  [5] The method according to claim 4, wherein the test bird is determined to be resistant to RNA virus when the base species is adenine. [6] The method according to claim 4, wherein the test bird is determined to be susceptible to an RNA virus when the base species is guanine and the base is detected as homozygous. [7] The method according to any one of claims 116, wherein the bird is-ヮ. [8] The method according to claim 17, wherein the RNA virus is an avian influenza virus.  [9] An avian RNA virus resistance determination reagent comprising the following oligonucleotide (a) or (b):  (a) an oligonucleotide probe having a chain length of at least 15 nucleotides, which is a site on the avian Mx gene and hybridizes to DNA containing the position at position 2032 in the nucleotide sequence of SEQ ID NO: 1;  (b) an oligonucleotide primer for amplifying DNA containing a site at position 2032 in the nucleotide sequence of SEQ ID NO: 1, which is a site on the avian Mx gene  [10] The reagent according to claim 9, wherein the RNA virus is an avian influenza virus.  [II] A method for producing an RNA virus vaccine, comprising the following steps (a) to (e). (a) not being resistant to RNA virus or RNA virus by the method of claim 7; The process of selecting a bird that is determined to be sensitive  (b) Step of obtaining embryonated chicken eggs of the bird selected in step (a)  (c) a step of injecting an RNA virus into the general urine fluid of the embryonated chicken eggs  (d) Step of incubating the chicken eggs obtained in step (c)  (e) Step of collecting general urine fluid from the eggs obtained in step (d)  The production method according to claim 11, wherein the RNA virus is an avian influenza virus,