Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2000-04-21
2001-09-04
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S091200, C536S024310, C536S024330
Reexamination Certificate
active
06284457
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for judging a possibility of the onset of bovine leukemia caused by bovine leukemia virus BLV and a resistance to the onset of the leukemia.
BACKGROUND ART
The major histocompatibility antigens (MHC antigens) are molecules involved in self-nonself differentiation in the defense mechanism of the living body against infection. They are classified into Class I molecule composed of &agr; chain and &bgr;2M, and class II molecule composed of &agr; chain and &bgr; chain. A groove for trapping an antigen peptide is present on the &agr;1 and &agr;2 domains, and also on the &agr;1 and &bgr;1 domains. They are featured to have the T cell receptor recognize only a fragmented peptide trapped in the groove, thereby achieve cell death (cellular immunity) by CD8+ cells which have recognized the class I antigens, as well as induce mainly antibody production (humoral immunity) by CD4+ cells which have recognized the class II antigens.
The MHC genes constitute a gene group most full of polymorphism, and the locations of pockets, shapes, sizes and properties of the peptide trapping grooves are different among haplotypes. It is considered that association conditions of the trapped fragment peptides may vary depending on these differences, which decide immune response and disease sensitivity of each individual. The correlation between the MHC haplotypes and a resistance to a disease (disease insusceptibility) or a possibility of the onset of a disease (disease susceptibility) has been reported, for example, as to human immune deficiency virus (HIV), human T cell leukemia virus (HTLV) and malaria.
As for the bovine MHC (BoLA) class II genes, existence of DQA, DQB, DRA, DRB, DNA, DOB, DYA, and DYB genes has been estimated. DRB3, inter alia, which is one of the three genes (DRB1 to B3) identified on the DRB genetic locus, has been known to encode a functional protein, and existence of 73 alleles has been revealed so far. However, there is almost no report about correlation between bovine infectious diseases and the bovine MHC (BoLA) haplotypes.
In particular, as to the bovine leukemia virus (BLV), which has the gene PX that regulates virus proliferation in the same manner as the human immunodeficiency virus (HIV) and is a retrovirus most related to HTLV-I, a research group in the United States has reported its relationship with the bovine MHC (BoLA) haplotypes mainly focusing on disease resistance; however, its relationship with possibility of onset of leukemia has not been reported. The ratio of cattle infected by this virus (infection rate in Japan) is 10-20%, and 1-2% of the infected cattle develop extremely malignant endemic bovine leukemia and die after a long latent period of 10-15 years. Therefore, economic loss of stockbreeders caused by the virus is very serious. If a possibility of the onset of leukemia in cattle after BLV infection can be evaluated by the analysis of bovine MHC (BoLA) haplotypes, it becomes possible to preliminarily select disease resistant cattle for breeding, and it is expected that extremely safe cattle breeding can be continued.
Accordingly, an object of the present invention is to elucidate the relationship between the bovine leukemia virus (BLV) and the bovine MHC (BoLA) haplotypes, and to provide a method for convenient judgement of a possibility of the onset of leukemia of a cattle caused by the bovine leukemia virus (BLV) and a resistance to the onset of the leukemia by means of genetic engineering techniques. Another object of the present invention is to provide a primer set useful for the aforementioned method for judgement.
DISCLOSURE OF THE INVENTION
The inventors of the present invention previously analyzed the structure of DRB gene locus among the bovine MHC (BoLA) class II genes, and reported the structures of DRB3 gene (BoLA-DRB3) and the gene product thereof (Biochem. Biophys. Res. Commun., 209, pp.981-988, 1995). The inventors further studied the function of the gene and found that a portion is present, whose amino acid sequence is distinctly different between a cattle developing the leukemia and a cattle not developing the disease, in the gene product from the second exon (&bgr;1 domain) of BoLA-DRB3 showing particularly noticeable polymorphism. They also found that the amino acid substitutions directly correlated with disease susceptibility to BLV and disease resistance The present invention was achieved on the basis of these findings.
The present invention thus provides a method for judging a possibility of the onset of bovine leukemia caused by bovine leukemia virus BLV, wherein a bovine individual, in which an amino acid sequence defined by the amino acid number from 75 to 78 of the &bgr;0 1 domain of the bovine MHC Class II DR&bgr; chain is Val-Asp-Thr-Tyr, (SEQ ID NO:9) is judged to have a possibility of the onset of the leukemia. As preferred embodiments of the method of the present invention, there are provided the aforementioned method which is applied to a cattle infected by the bovine leukemia virus BLV; and the aforementioned method wherein a bovine individual, in which an amino acid sequence defined by the amino acid numbers 75-78 of the &bgr;1 domain of the bovine MHC Class II DR&bgr; chain is Val-Asp-Thr-Tyr (SEQ ID NO:9) in both of the alleles, is judged to have a risk of the onset.
According to another embodiment of the method of the present invention, there is provided a method for judging a possibility of the onset of bovine leukemia caused by the bovine leukemia virus BLV, which comprises the steps of:
(1) amplifying genomic DNA isolated from a bovine individual by the polymerase chain reaction (PCR) to prepare a PCR product containing a DNA coding for a part or full length of the &bgr;1 domain of the bovine MHC Class II DR&bgr; chain, and
(2) judging that the bovine individual, in which an amino acid sequence corresponding to the amino acid number from 75 to 78 of the &bgr;1 domain of the bovine MHC Class II DR&bgr; chain is Val-Asp-Thr-Tyr (SEQ ID NO:9) in the amino acid sequence encoded by the DNA contained in the PCR product, has a possibility of the onset of the leukemia. A preferred embodiment of the aforementioned method comprises a step of digesting the PCR product by using PstI.
According to another aspect of the present invention, there is provided a method for judging a resistance to the onset of bovine leukemia caused by the bovine leukemia virus BLV, wherein a bovine individual, in which an amino acid defined by the amino acid number 78 of the &bgr;1 domain of the bovine MHC Class II DR&bgr; chain is Val, is judged to have resistant to the onset of the leukemia. As preferred embodiments of the method of the present invention, there are provided the aforementioned method which is applied to a cattle infected by the bovine leukemia virus BLV; the aforementioned method wherein the bovine individual, in which the amino acid specified by the amino acid number 78 of the &bgr;1 domain of the bovine MHC Class II DR&bgr; chain is Val in at least one of the alleles, is judged to have a resistance to the onset; and the aforementioned method wherein the bovine individual, in which the amino acid specified by the amino acid number 78 of the &bgr;1 domain of the bovine MHC Class II DR&bgr; chain is Val in both of the alleles, is judged to have a high resistance to the onset.
According to another embodiment of the method of the present invention, there is provided a method for judging a resistance to the onset of bovine leukemia caused by the bovine leukemia virus BLV, which comprises the steps of:
(1) amplifying genome DNA isolated from a bovine individual by the polymerase chain reaction (PCR) to prepare a PCR product containing a DNA coding for a part or full length of the &bgr;1 domain of the bovine MHC Class II DR&bgr; chain, and
(2) judging that the bovine individual, in which an amino acid corresponding to amino acid number 78 of the &bgr;1 domain of the bovine MHC Class II DR&bgr; chain is Val in the amino acid sequence encoded by the DNA contained in the PCR p
Chakrabarti Arun K.
Fredman Jeffrey
Greenblum & Bernstein P.L.C.
The Institute of Physical and Chemical Research
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