JP3718555B2 - Method for diagnosing bovine factor XIII abnormality - Google Patents

Description

【００４３】

【００４４】

【００４５】

【００４６】

【００４７】

【００４８】

【００４９】

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【００５１】

【００５２】

【００５３】

【００５４】

【００５５】

【００５６】

【００５７】

【００５８】

【００５９】

【００６０】

【００６１】

【００６２】

【００６３】

【図面の簡単な説明】
【図１】図１は、塩基の置換による制限酵素ＭｂｏIIの切断部位の発生を示す模式図である。
【図２】図２は、正常ウシ由来のＤＮＡ断片及び第ＸIII 因子異常症ウシ由来のＤＮＡ断片をＭｂｏII消化後に電気泳動に付して得られた結果を示す図である。
【図３】図３は、遺伝子型と血清中の第ＸIII 因子の活性の相関を示すグラフを表した図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for diagnosing factor XIII abnormality, which is a bovine hemorrhagic disease. The present invention also relates to a diagnostic kit for bovine factor XIII abnormality using such a diagnostic method.
[0002]
[Prior art]
In southern Kyushu, there are many bleeding diseases of Wagyu beef. Among these bleeding diseases, there are often multiple genetic diseases such as Chediak-Higashi syndrome, von Willebrand disease, and factor XIII abnormality. There are many livestock damages due to severe symptoms. In the future, if the production of Japanese black beef is continued by the same crossing as before, there is a concern that the productivity will be further lowered due to an increase in hereditary hemorrhagic diseases including factor XIII abnormality.
In particular, cattle that have developed factor XIII abnormalities often develop umbilical cord bleeding in the neonatal period and often die from massive intraperitoneal bleeding. In surviving individuals, repeated hemorrhage, etc. results in poor growth, and in severe cases, blood loss occurs. To date, there have been over 100 cases of this disease.
[0003]
Factor XIII (fibrin stabilizing factor, plasma transglutaminase) is fibrinogen, fibrin, fibronectin, α₂-Plasmin inhibitor, alpha₂-A precursor of the SH enzyme that catalyzes the formation of amide bridges between proteins such as macroglobulin, actin and denatured collagen.
[0004]
Plasma factor XIII is composed of two subunits, A and B.₂B₂Is a heterotetramer. The expression of the activity mainly requires limited hydrolysis with thrombin, which changed the subunit A having a molecular weight of 75,000 to A 'having a molecular weight of 71,000.₂B₂Is converted to At this time, Ca²⁺A 'by the presence of₂And B₂Dissociates into SH, and the SH activity center in the A ′ subunit is exposed, and the enzyme activity is expressed. The activated factor XIII thus stabilizes the fibrin clot by forming an amide bond between fibrin molecules generated by the blood coagulation cascade, and completes the blood coagulation reaction.
[0005]
Until now, factor XIII abnormality has been diagnosed by measuring factor XIII activity in plasma. Factor XIII activity can be measured by (1) checking the acid solubility of fibrin produced using purified fibrinogen, (2) neutralizing factor XIII in a dilution series using fibrinogen in the sample (plasma) as a substrate. (3) A method for determining an acid-insoluble fibrin formation boundary value in the presence of an antibody, (3) A method for measuring acid solubility using purified fibrin monomer as a substrate, (4) A method for measuring incorporation of a fluorescent amine compound into casein, (5 There are those that use a radioisotope instead of the fluorescent amine compound, (6) those that quantify the release of ammonia from the substrate casein, and (7) those that use a completely synthetic substrate. When measuring the enzyme activity of factor XIII in bovine plasma, (2) and (7) are mainly used among the above measurement methods.
[0006]
However, the above factor XIII activity measuring method has drawbacks such as the time required for preparation of the substrate, the quality of the substrate is not constant, and the activity value obtained varies depending on the state of enzyme purification in the sample. have. In addition, it is estimated that factor XIII abnormality has autosomal recessive inheritance, and about the bovine of genetically factor XIII abnormality carrier that has a gene related to abnormality only on one chromosome The conventional method based on enzyme activity measurement cannot know the abnormality. Therefore, in the diagnosis by the above method, even when cattle that do not have any abnormality are mated, abnormalities may appear in calves that are born, and this has a problem in preventing the occurrence of this disease.
[0007]
One method for preventing bovine factor XIII abnormality is to avoid using the carrier of the gene responsible for the disease for mating. For this purpose, it is necessary to diagnose existing bovine factor XIII abnormality at the gene level and to quickly find a carrier of the disease gene. If it is possible to clarify how the disease gene of an abnormal bovine disease is mutated compared to a normal one, and to obtain a means that can quickly detect the mutated gene by various genetic engineering techniques, A genetic diagnostic method can be established.
In humans, mutations in the subunit A gene are known to be the main cause [Blood, 80, 937-941 (1992); i., 84, Pp. 517-525 (1994)] is not known as to which of the two subunits, A and B, is associated with bovine factor XIII abnormalities.
[0008]
[Problems to be solved by the invention]
Accordingly, it is an object of the present invention to provide a bovine factor XIII gene. Another object of the present invention is to provide a genetic diagnosis method capable of rapidly diagnosing bovine factor XIII abnormality using the gene. Still another object of the present invention is to provide a kit for diagnosing bovine factor XIII gene abnormality that can be easily diagnosed and is used in such a diagnostic method.
[0009]
[Means for Solving the Problems]
As a result of intensive studies to achieve the above object, the present inventors have found that a specific base substitution on the gene encoding factor XIII subunit A is responsible for this disease. It was found that a recognition site for a specific restriction enzyme was formed on the gene due to the mutation, and the present invention was completed.
[0010]
  That is, the gist of the present invention is as follows.
[1] A bovine factor XIII abnormality factor XIII subunit A gene represented by SEQ ID NO: 2;
[2] Step (a): A step of obtaining a bovine nucleic acid sample,
Step (b): subjecting the nucleic acid sample obtained in step (a) to a gene amplification reaction,Position 248 of the base sequence of bovine factor XIII subunit A geneObtaining a nucleic acid fragment in which the region containing is amplified, and
Step (c): About the nucleic acid fragment of step (b)A nucleotide sequence having a mutation in the amino acid sequence in which the 83rd phenylalanine in the amino acid sequence of bovine factor XIII subunit A protein is substituted with serine;Examining the presence of the mutation,
A method for diagnosing bovine factor XIII abnormality comprising
[3] Gene amplification reaction is performed by PCR (polymerase chain reaction) method[2]The diagnostic method described,
[4]Base sequenceDetect mutations by detecting restriction fragment length polymorphism (RFLP)[2] or [3]The diagnostic method described,
[5] It is characterized by examining RFLP using restriction enzyme MboII[4]The diagnostic method described,
[6] The nucleic acid sample is a sample containing genomic DNA, cDNA, or mRNA.[2] to [5]Any of the diagnostic methods,And
[7][4]A kit for diagnosing bovine factor XIII abnormality by the described diagnostic method,
(A):Position 248 of the base sequence of bovine factor XIII subunit A genePrimers for amplifying the region containing
(B): By RFLPA nucleotide sequence having a mutation in the amino acid sequence in which the 83rd phenylalanine in the amino acid sequence of bovine factor XIII subunit A protein is substituted with serine;Restriction enzymes for detecting mutations,
It is related with the kit characterized by including.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
1. About normal and abnormal bovine factor XIII subunit A gene
In order to examine the relationship between genetic variation and this disease, first, a gene encoding normal bovine factor XIII subunit A is isolated and its nucleotide sequence is clarified. Although an example in which the gene was isolated has not been reported so far, it can be isolated using homology with the human factor XIII subunit A gene. That is, the base sequence of the human factor XIII subunit A gene [Proceedings
CDNA prepared from Holstein using primers prepared based on the National Academy of Science of the USA (USA), Volume 83, pages 8024-8028 (1986)] A DNA reaction having a homology with the human factor XIII subunit A gene can be amplified by performing a PCR reaction using as a template.
[0012]
Next, the base sequence of each of the obtained DNA fragments was determined to confirm homology with the human factor XIII subunit A gene, and these base sequences were compared and analyzed to obtain a bovine factor XIII subunit A gene. The entire base sequence can be known. The base sequence of the cDNA encoding the normal bovine factor XIII subunit A thus obtained is shown in SEQ ID NO: 1 in the sequence listing.
[0013]
To clarify the mutation in the gene causing bovine factor XIII abnormality by comparing the nucleotide sequences of normal and factor XIII subunit A genes present in cattle that have developed factor XIII abnormality Can do. That is, based on the base sequence encoding the normal bovine factor XIII subunit A described above, an appropriate primer that can be amplified by dividing the entire base sequence into several regions can be synthesized. Next, using the primer, PCR reaction was performed using cDNA prepared from a cattle that developed factor XIII abnormality as a template, and the base sequence of the obtained amplified DNA fragment was determined and compared with each other. It is possible to confirm the mutation that is present on the bovine gene that developed factor XIII abnormality and that causes the disease.
[0014]
In the present invention, as shown in SEQ ID NO: 2 in the sequence listing, the 248th thymine (compared with the base sequence of the normal bovine factor XIII subunit A gene) on the cDNA of the bovine that developed factor XIII abnormality. T) was found to be mutated to cytosine (C), and the 261st cytosine was mutated to thymine. Of these, the latter is not accompanied by amino acid substitution, but in the former, the codon TTC encoding phenylalanine is changed to TCC encoding serine. It is shown that factor XIII subunit A is expressed. This phenylalanine is present in most mammalian transglutaminases whose amino acid sequences have been clarified so far, such as human factor XIII subunit A, and this gene mutation causes factor XIII abnormality. It can be seen that it is. Further, it can be seen that this mutation generates a new restriction enzyme MboII cleavage site on the base sequence. In human factor XIII abnormality, the 681st arginine of subunit A [Blood, 80, 937-941, (1992)], the 661th arginine or the 242nd methionine [Blood]. (Blood), Vol. 84, pp. 517-525, (1994)], which was different from the bovine mutation found in the present invention.
[0015]
2. About the diagnostic method of bovine factor XIII abnormality
As described above, since the mutation on the gene causing the factor XIII abnormality has been elucidated, the mutation can be used to diagnose the disease.
[0016]
As a specific method for diagnosing bovine factor XIII abnormality, an embodiment having the following steps is exemplified. That is,
Step (a): obtaining a bovine nucleic acid sample;
Step (b): subjecting the nucleic acid sample obtained in step (a) to a gene amplification reaction to obtain a nucleic acid fragment in which a region containing a mutation site that may be present in the factor XIII gene is amplified; and
Step (c): A step of examining the presence of mutation in the nucleic acid fragment of step (b).
[0017]
About process (a)
The bovine nucleic acid sample used in the present invention is not particularly limited as long as it contains the bovine factor XIII subunit A gene, and includes genomic DNA, cDNA, mRNA and the like. Preparation of genomic DNA and the like from bovine can be performed by a commonly known method.
[0018]
About step (b)
By this step, a region containing a mutation site that may be present in the bovine factor XIII gene is amplified, and a desired nucleic acid fragment can be obtained. The method of gene amplification reaction used in this step is not particularly limited as long as it can amplify the region, but PCR method, nucleic acid amplification method using RNA polymerase (Japanese Patent Laid-Open Nos. 2-5864 and 7). -209999) and strand displacement amplification methods (JP-B-7-114718, JP-A-7-88242) can be used. Of these, the PCR method is preferably used.
The region including the mutation site to be amplified is not particularly limited as long as it includes the position 248 of the base sequence of the bovine factor XIII subunit A gene. Preferably, a sequence that becomes a recognition site for the restriction enzyme MboII when a mutation occurs, that is, a region containing positions 244 to 248 of SEQ ID NO: 1 for normal cattle and SEQ ID NO: 2 for affected cattle. is there.
[0019]
The primer used in the PCR method is not particularly limited as long as it can amplify a mutation site, that is, a DNA fragment containing position 248 of the base sequence of bovine factor XIII subunit A gene. Also, the PCR conditions are not particularly limited, and may be known conditions that are usually performed.
[0020]
About process (c)
In this step, the presence of mutation is examined for the nucleic acid fragment obtained in step (b). The method for detecting the presence of the mutation is not particularly limited, but a method of detecting and examining a restriction fragment length polymorphism (RFLP) is preferably used. The restriction enzyme used for examining RFLP is not particularly limited as long as it can detect a gene mutation, and examples thereof include restriction enzyme MboII and its isoschizomer, or restriction enzyme BbrII and its isoschizomer. Digestion with the restriction enzyme MboII can be performed using a composition suitable for MboII digestion, eg, 10 mM Tris-HCl, pH 7.5, 10 mM MgCl.₂A reaction solution composed of 1 mM DTT is used, and the amount of MboII added and the reaction time are appropriately adjusted according to the amount of DNA to be digested. RFLP can be detected by a known method such as gel electrophoresis using a normal bovine nucleic acid sample treated in the same manner as the bovine nucleic acid sample to be diagnosed as a control.
[0021]
As a diagnostic method other than the PCR-RFLP method, there is a method in which the step (c) of the embodiment exemplified above is changed to another mutation detection method. For the detection of mutation, for example, a known mutation detection method such as a hybridization method using an appropriate DNA fragment containing a mutation site as a probe or the SSCP method can be used. Japanese Patent Application Laid-Open No. 7-327698 discloses a method for detecting a mutation using MutS protein, and this method is also applicable to the diagnostic method of the present invention. Furthermore, mutation can also be detected by determining the base sequence of the amplified DNA cloned into an appropriate vector, or by determining the base sequence using the amplified fragment itself as a template.
[0022]
According to the method for diagnosing bovine factor XIII abnormality of the present invention, not only cattle that cause factor XIII abnormality but also cattle that are expected to develop disease, and cows that are carriers of factor XIII abnormality are diagnosed. be able to. Therefore, bovine factor XIII abnormality as used in the present invention means that it is genetically abnormal, and it should be interpreted in a broad sense including the presence or absence of a symptom.
[0023]
3. About bovine factor XIII abnormality diagnosis kit
By utilizing the above diagnostic method, the kit for diagnosing abnormalities can be provided.
Specifically, a kit for diagnosing bovine factor XIII abnormality by the diagnostic method described above,
(A): a primer for amplifying a region containing a mutation site that may be present in the factor XIII gene; and
(B): a restriction enzyme for detecting mutations by RFLP,
It is characterized by including.
By using such a kit, bovine factor XIII abnormality can be easily diagnosed. Examples of the primers and restriction enzymes used in the kit of the present invention include those described in the above-mentioned section of the diagnostic method.
[0024]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by these Examples.
[0025]
Example 1
Determination of the base sequence of normal bovine factor XIII subunit A gene
A cDNA serving as a template for obtaining a DNA fragment containing the bovine factor XIII subunit A gene by PCR was prepared from blood collected from Holstein by the method described below.
First, platelet-rich plasma (PRP, Platelet rich plasma) is separated from 200 ml of bovine whole blood (sodium citrate added) by specific gravity centrifugation (about 100 g x 20 min.), And further centrifuged by about 1500 g x 5 min. Fractions were prepared. After washing 3 times with phosphate buffered saline (PBS), RNAzol^TMTotal RNA was extracted using B (Biotex Laboratories). 1 μg of the total RNA thus obtained was used to synthesize cDNA using a cDNA cycle kit (Invitrogen), and finally 40 μl of TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM). A cDNA (Pt cDNA) solution dissolved in EDTA was obtained.
[0026]
Proceedings of the National Academy of Science of the USA, Vol. 83, pp. 8024-8028 (1986), as well as the base sequence of human factor XIII subunit A gene, as well as the base sequence of transglutaminase derived from other animals. Primers 1S, 2S, 3R, 5R, 6S, and 7R were chemically synthesized using homology. The base sequences of primers 1S, 2S, 3R, 5R, 6S, and 7R are shown in SEQ ID NO: 3 to SEQ ID NO: 8, respectively.
PCR reactions carried out in this example and in the following examples were carried out in reaction solutions having the following composition unless otherwise specified: 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂0.25 mM dATP, 0.25 mM
dCTP, 0.25 mM dGTP, 0.25 mM dTTP, 0.001% gelatin, 2.5 units Taq DNA polymerase (Takara Shuzo). The total volume of the reaction solution was 100 μl, and two primers each having a final concentration of 10 μg / ml were added together with an appropriate amount of template DNA.
[0027]
That is, 100 μl of a PCR reaction solution containing 3 μl of the above Pt cDNA solution and primers 2S and 7R (each final concentration of 10 μg / ml) was prepared. From 94 ° C. for 1 minute, 42 ° C. for 1 minute, and 72 ° C. for 2 minutes. The PCR reaction of 35 cycles was performed with the following step as one cycle. A portion of this reaction solution was subjected to agarose gel electrophoresis, and then a DNA fragment corresponding to about 1400 bp recovered from the gel was transformed into plasmid vector pCR.^TMA recombinant plasmid incorporated into II (manufactured by Invitrogen) was prepared. Using the obtained recombinant plasmid as a template, the base sequence of the DNA fragment incorporated in this recombinant plasmid was determined using a fluorescent DNA sequencer (manufactured by ABI). The obtained base sequence is shown in SEQ ID NO: 9 in the sequence listing.
[0028]
In addition, 100 μl of a PCR reaction solution containing 3 μl of Pt cDNA solution and primers 6S and 3R was prepared, and the cycle consisting of 94 ° C. for 1 minute, 37 ° C. for 1 minute, and 72 ° C. for 2 minutes was 35 cycles. PCR reaction was performed. After isolating the amplified DNA fragment of about 700 bp by agarose gel electrophoresis, the same PCR reaction solution containing a part thereof as a template was prepared, and it was 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. The PCR reaction of 35 cycles was performed with 1 step consisting of 2 minutes. The obtained PCR product was transformed into plasmid vector pCR.^TMA recombinant plasmid incorporated into II was prepared, and the base sequence of the DNA fragment incorporated into this recombinant plasmid was determined as described above. The obtained base sequence is shown in SEQ ID NO: 10 in the sequence listing.
[0029]
When the two base sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10 in the sequence listing were compared, both sequences contained a common base sequence of about 200 bp, and both covered a base sequence of about 1900 bp. .
Two types of primers, 8R and 9S, were further synthesized based on the above-described nucleotide sequences. The base sequences of primers 8R and 9S are shown in SEQ ID NO: 11 and SEQ ID NO: 12 in the sequence listing, respectively.
[0030]
In order to clarify the nucleotide sequence of the bovine factor XIII subunit A gene not covered by the above two fragments, 100 μl of a PCR reaction solution containing 3 μl of the above Pt cDNA solution and primers 1S and 3R was prepared at 94 ° C. A 30-cycle PCR reaction was carried out with 1 cycle consisting of 1 minute, 2 minutes at 37 ° C and 2 minutes at 72 ° C. Next, 100 μl of a PCR reaction solution containing 1 μl of the reaction solution after the PCR reaction and primers 1S and 8R is prepared, and one cycle consisting of 94 ° C. for 1 minute, 52 ° C. for 2 minutes, and 72 ° C. for 30 seconds. The PCR reaction of 30 cycles was performed. A part of this reaction solution was subjected to 2% agarose gel electrophoresis, and a DNA fragment of about 300 bp was recovered to obtain the above plasmid vector pCR.^TMAfter subcloning into II, the base sequence was examined by the dideoxy method, and it was confirmed that the base sequence of this DNA fragment (269 bp) had homology in the 5 'region of the human factor XIII subunit A gene. The nucleotide sequence of this DNA fragment is shown in SEQ ID NO: 13 in the sequence listing.
[0031]
Subsequently, 100 μl of a PCR reaction solution containing 3 μl of the above Pt cDNA solution and primers 4S (SEQ ID NO: 14) and 5R was prepared, consisting of 94 ° C. for 1 minute, 37 ° C. for 2 minutes, and 72 ° C. for 2 minutes. A 30-cycle PCR reaction was performed with the process as one cycle. Next, 100 μl of a PCR reaction solution containing 1 μl of the reaction solution after the PCR reaction and primers 9S and 5R is prepared, and one cycle of the process consisting of 94 ° C. for 1 minute, 52 ° C. for 2 minutes, and 72 ° C. for 30 seconds. The PCR reaction of 30 cycles was performed. The DNA fragment recovered from this reaction solution was transformed into the above plasmid vector pCR.^TMAfter subcloning into II, the base sequence was examined by the dideoxy method. The nucleotide sequence of this DNA fragment is shown in SEQ ID NO: 15 of the sequence listing.
The nucleotide sequences of the various DNA fragments thus obtained were compared and combined to determine the full-length nucleotide sequence (2196 bp) encoding bovine factor XIII subunit A. The base sequence of 2196 bp is shown in SEQ ID NO: 1 in the sequence listing. The amino acid sequence of normal bovine factor XIII subunit A is shown in SEQ ID NO: 16 in the sequence listing.
[0032]
Example 2
Confirmation of mutation on bovine factor XIII subunit A gene
The base sequence of the bovine factor XIII subunit A gene shown in SEQ ID NO: 1 in the sequence listing is divided into three regions, each of which can be amplified by PCR, primers AS, AR, BS, BR, CS, CR was synthesized. The base sequences of primers AS, AR, BS, BR, CS, CR are shown in SEQ ID NO: 17 to SEQ ID NO: 22 in the sequence listing, respectively.
[0033]
CDNA was prepared from bovine blood that developed factor XIII abnormality by the same procedure as in Example 1, and using this as a template, primer pairs of primers AS and AR, primers BS and BR, primers CS and CR were used. The PCR reaction used was performed. A step of preparing 100 μl of a PCR reaction solution containing the above template and primer pair with the same composition as used in Example 1 and consisting of 94 ° C. for 1 minute, 60 ° C. for 1 minute, and 72 ° C. for 2 minutes. A cycle of 35 cycles of PCR reaction was performed. The base sequence of the amplified DNA fragment was determined in the same manner as in Example 1, and each was compared with the base sequence of SEQ ID NO: 1 in the sequence listing. Further, the determined nucleotide sequence of the factor XIII subunit bovine factor XIII subunit A cDNA is shown in SEQ ID NO: 2 in the sequence listing.
[0034]
As a result, there was no difference in the base sequence of the region amplified with primer pair BS, BR and CS, CR, but the N-terminal side of factor XIII subunit A amplified with primer pair AS, AR. In the region coding for, a difference was found in the nucleotide sequence between normal and affected cattle. That is, in the affected cattle, it was shown that 248th thymine and 261st cytosine were mutated to cytosine and thymine, respectively, in the base sequence shown in SEQ ID NO: 1 in the sequence listing. Of these, the mutation from 248th thymine to cytosine is accompanied by a mutation in the amino acid sequence in which the 83rd phenylalanine is substituted with serine in the amino acid sequence of the factor XIII subunit A protein. The affected cattle are thought to express a mutant form of factor XIII subunit A having this amino acid substitution, which has been shown to be the cause of this disease.
[0035]
Example 3
Diagnosis of bovine factor XIII abnormality by PCR-RFLP method
Of the mutations in the factor XIII abnormal cattle clarified in Example 2, the base substitution from 248th thymine to cytosine resulted in the recognition and cleavage of a new restriction enzyme MboII on the gene (Fig. 1). The primers AS and 8R described above were used as primers for amplifying a 141 bp region containing this base substitution site.
[0036]
Peripheral blood mononuclear cells were isolated from whole blood of normal and factor XIII abnormal cows (total 23) using specific gravity centrifugation and 0.15 M ammonium chloride, edited by T. Maniatis et al., Molecular cloning published by Cold Spring Harbor Laboratory: Genomic DNA was isolated according to the method described in Molecular Cloning: A Laboratory Manual, 2nd edition, 9.16-9.23. 50 μl of PCR reaction solution (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl) containing 50 ng of this genomic DNA and the above primer pair (each final concentration 20 μg / ml)₂, 0.2 mM each dNTP, 1 unit Taq DNA polymerase), followed by incubation at 94 ° C. for 5 minutes, followed by 1 step at 94 ° C. for 1 minute, 60 ° C. for 1 minute, 72 ° C. for 30 seconds. 30 cycles of reaction were performed.
[0037]
After the reaction, the PCR product was recovered by ethanol precipitation, and using 1/5 of the amount, 10 μl of MboII digestion reaction solution (10 mM Tris-HCl, pH 7.5, 10 mM MgCl) was used.₂1 mM DTT), 4 units of MboII (Takara Shuzo) were added, and incubation was performed at 37 ° C. for 1 hour. After electrophoresis of the reaction solution on a 3% agarose gel, the separated DNA fragment was analyzed by staining the gel with ethidium bromide. As a result, as shown in the upper stage of FIG. 2, a single band of about 140 bp was detected in the DNA fragment amplified from genomic DNA derived from normal cattle (No. 18-23), In cattle with XIII factor abnormality, the band was decomposed into two bands of about 80 bp and about 60 bp (No. 1 to 4), indicating that a recognition cleavage site of MboII was generated in the amplified fragment. It was.
[0038]
No. 5 and no. 6 is the analysis result of cattle clinically showing no symptoms of factor XIII abnormality. In these samples, a total of three bands of about 140 bp and about 80 bp and about 60 bp were confirmed, indicating that these cows are heterozygotes for normal and mutant alleles. Therefore, it was found that the diagnostic method of the present invention enables detection of apparently normal bovine carriers of factor XIII abnormality.
In addition, No. in the lower row of FIG. 7-17 is a figure which shows the result of the electrophoresis obtained by diagnosing with the same method about 11 cattle whose blood relations with onset cattle are clear. From this result, it can be seen that 5 out of 11 are carriers of factor XIII abnormality.
[0039]
Sample No. About 1-23, the correlation of the diagnostic result by PCR-RFLP method and the activity of factor XIII in serum was investigated.
The activity of factor XIII is Iatro-FLF. It was measured by a monodansyl cadaverine (MDC) uptake method using XIII (manufactured by Yatron). The results are shown in FIG.
[0040]
The horizontal axis of FIG. 3 shows each genotype related to this mutation,-/-indicates the homozygous genotype, +/- indicates the normal / mutant heterozygous, + / + indicates the normal homozygous Show. The vertical axis shows the activity of factor XIII in serum.
From this figure, it can be seen that there is a clear correlation between the genotype for this mutation and the factor XIII activity in serum.
[0041]
【The invention's effect】
According to the present invention, bovine factor XIII abnormality and its carrier can be detected easily and rapidly.
[0042]
[Sequence Listing]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[Brief description of the drawings]
FIG. 1 is a schematic diagram showing the generation of a restriction enzyme MboII cleavage site by base substitution.
FIG. 2 is a view showing the results obtained by subjecting a normal bovine-derived DNA fragment and a factor XIII abnormal cow-derived DNA fragment to electrophoresis after digestion with MboII.
FIG. 3 is a graph showing a correlation between genotype and factor XIII activity in serum.

Claims (7)

  The bovine factor XIII abnormality factor XIII subunit A gene shown in SEQ ID NO: 2. Step (a): obtaining a bovine nucleic acid sample;
Step (b): The nucleic acid sample obtained in step (a) is subjected to a gene amplification reaction to obtain a nucleic acid fragment in which the region containing position 248 of the base sequence of bovine factor XIII subunit A gene is amplified. Step and Step (c): Regarding the nucleic acid fragment of Step (b) , the nucleotide sequence having a mutation on the amino acid sequence in which the 83rd phenylalanine in the amino acid sequence of bovine factor XIII subunit A protein is substituted with serine Examining the presence of the mutation,
A method for diagnosing bovine factor XIII abnormality. The diagnostic method according to claim 2 , wherein the gene amplification reaction is performed by a PCR (polymerase chain reaction) method. The diagnostic method according to claim 2 or 3 , wherein the presence of a mutation in the base sequence is examined by detecting a restriction fragment length polymorphism (RFLP). The diagnostic method according to claim 4 , wherein RFLP is examined using a restriction enzyme MboII. The diagnostic method according to claim 2 , wherein the nucleic acid sample is a sample containing genomic DNA, cDNA, or mRNA. A kit for diagnosing bovine factor XIII abnormality by the diagnostic method according to claim 4 ,
(A): a primer for amplifying a region containing position 248 of the base sequence of bovine factor XIII subunit A gene , and (b): the 83rd position in the amino acid sequence of bovine factor XIII subunit A protein by RFLP A restriction enzyme for detecting a nucleotide sequence mutation, with a mutation in the amino acid sequence in which phenylalanine is substituted with serine ,
A kit comprising:

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