JP3852059B2 - HLA type discrimination database creation method and HLA type discrimination method. - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The human leukocyte antigen (HLA) gene, which is a human tissue-synthesizing gene complex, is strongly involved in the immune response and is deeply involved in the appropriate synthesis of transplanted organs during organ transplantation. ing. The present invention relates to an HLA Class I and Class II type determination method and creation of a database to be used.
[0002]
[Prior art]
Conventionally, HLA typing has been performed using serological or cytological methods. That is, DR, DQ antigens of HLA Class I antigens A, B and C and Class II antigens are a lymphocyte disorder test method which is one of serological methods using specific antibodies and complements for each antigen. Has been judged by. Among the Class II antigens, the DP antigen has been typed by the Primed Limhocyte Typing (PLT) method, which is one of the cell culture methods.
[0003]
However, these methods have problems in the maintenance and supply of specific antibodies in the lymphocyte injury test, and it takes time and labor to make a determination in PLT, and the accuracy of the determination is not always satisfactory.
[0004]
Furthermore, identification of the HLA gene and further cloning of the gene have been carried out, and HLA typing at the gene level has been carried out. However, most of them discriminate HLA types from differences in the restriction enzyme cleavage patterns of genes. In recent years, standard data that has been registered by performing homology search on databases registered in gene databanks (DDBJ, EMBL, Genbank) by direct sequencing of DNA sequences involved in HLA due to advances in molecular biology It is displayed from the thing with high similarity.
[0005]
[Problems to be solved by the invention]
However, this data bank contains only homozygous data and newly registered heterozygous data for the registered HLA Class I and CLASS II. Even if it is desired to use for HLA type determination, completely different data may be retrieved.
[0006]
In view of the above-mentioned problems, the first object of the present invention is to create a database of heterocomplexes composed of known homozygous combinations when using a direct sequence in the HLA type determination method. Therefore, the second problem is to perform homozygous and heterozygous type determination and perform accurate HLA type determination.
[0007]
[Means for Solving the Problems]
The method for creating an HLA type discrimination database according to the invention of claim 1 is characterized in that homozygosity data of antigen gene data is arranged in units of antigens, and amino acids so that there are no undecided portions in the base sequences on the start and end sides. In the unit, the same place is deleted for all alleles up to the maximum region where the base sequence has not been determined, and two data are multiplied to form a heterozygote. According to IUB, the data is replaced with a one-character code to create a hetero-complex data of one-character expression.
[0009]
Furthermore, the HLA type discriminating method according to the invention of claim 2 discriminates the optimum HLA type by extracting from the data created by the above method from the data highly correlated with the target DNA sequence data. It is characterized by.
[0010]
According to the first aspect of the present invention, detailed type recognition data in a type in which the type of HLA includes a hetero complex, which can be searched using a computer, can be provided, and can be classified into many types. it can.
[0012]
In the HLA type discriminating method of claim 2, by performing homology search using the database created by the invention of claim 1, it is possible to accurately determine whether or not the gene type is compatible with organ transplantation. Can be extracted.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention will be described below with reference to the drawings.
[0014]
First, gene data banks (DDBJ, EMBL, Genbank, etc.) to HLA Class I (A, B, C) and Class II (DPA, DPB, DRA, DRB, DQA, DQB, DMA, DMB) and other homozygotes related to necessary regions Obtain base sequence data.
[0015]
Fig. 1 shows a part of the beginning of an example of the obtained homozygous nucleotide sequence data. Here, AGG and the like represent the nucleotide sequence, and each of the nucleotide sequence data, such as AGG GCC, in amino acid units. The left column shows the gene type (HLA type).
[0016]
In the figure, the “-” mark means the same as the uppermost sequence, and the “*” mark means that the base sequence has not been determined.
[0017]
Next, all alleles are removed in amino acid units so that there are no portions where the base sequence is not determined at the beginning and end of the base sequence (up to the portion marked with * in FIG. 1).
[0018]
In the case of FIG. 1, DPB1 * 0801 is not determined up to 21 bases. Therefore, up to 7 amino acids are not determined. In this case, 1 to 7 amino acid units are deleted for all data, and the terminal side is similarly processed in the same manner.
[0019]
This result is shown in FIG. 2, which shows a part of the DPB1 on the start end side, and it can be seen that the sequence portion that cannot be determined from the start end side is removed.
[0020]
In order to synthesize heterojunction data from the state of FIG. 2, the logical sum of the two arrays is calculated in a form including ambiguity in all combinations. This ambiguity performs code substitution based on NC-IUB (Nomenclature Committee of the International Union of Biochemistry) (below). For example, DPB1 * 01011 data is (DPB1 * 01011) * (DPB1 * 01012), (DPB1 * 01011) * (DPB1 * 02011) .... Is regarded as ambiguity (corresponding to multiple amino acids: where two or more base sequences are mixed), and single character substitution is performed based on the following NC-IUB.
[0021]
When there is a difference in the base sequence code expression 2 or three base sequence data at the same position by NC-IUB, one character expression is performed based on the following definition.
[0022]
R = G and A
Y = T and C
W = A and T
S = G and C
M = A and C
K = G and T
B = G and T and C
D = G ans A and T
V = G and A and C
H = A and T and C
N = G and A and T and C
FIG. 3 shows the logical base sequence of two sequences including ambiguity. For example, in (DPB1 * 01011) * (DPB1 * 02011), the logical sum of the first amino acid part is G and C, so S from the above table. T and T become T, and the third becomes G and T and K, which agrees with the result of FIG.
[0023]
The above operation is repeated for all data combinations.
[0024]
The heterozygous data determined in this way is stored in a data format that allows homology search by serially connecting the base sequence data as shown in FIG. 4 together with the homozygous data.
[0025]
By performing a homology search on the target direct sequence data with respect to the data created by the above steps, an allele number having a high matching rate is displayed.
FIG. 5 to FIG. 10 are examples in which a homology search is performed on the data using FASTA.
[0026]
FIG. 5 shows that there is a correlation as the OPT value is higher. Therefore, if the OPT value is equal to or greater than a predetermined value, it is possible to determine that the suitability of the HLA type (the value 11012 arranged on the left side) corresponding to the OPT value is high.
[0027]
Here, the GTGT .... symbol arranged in the line described as dpb1 indicates the target DNA direct sequence data, and the data arranged in the line described below as 11012 etc. Shows the nucleotide sequence data in the database.
[0028]
Since the base sequence data of 11012 is exactly the same as the target data, the correlation is highest. Next, (11011 * 11012) is different because the base sequence data in the database of the 73rd column is M and the target data is A, but M is different according to the above NC-IUB law. Therefore, it can be said that there is some correlation with the target data. Therefore, the correlation value is higher than that of data completely different.
[0029]
For other data, the level of correlation is determined in the same way.
[0030]
On the other hand, when performing the search as described above, the storage medium in which the program shown in claim 2 is recorded is loaded into a computer device that can obtain homozygous data of antigen gene data from the gene data bank. If the homozygous data is taken into the computer and then the program in the storage medium is driven, an HLA type discrimination database can be created.
[0031]
【The invention's effect】
According to the present invention, the homozygous database in the database is obtained, the homozygous data is combined, and the heterozygous data is created. Type recognition data can be provided, and by homology searching the base sequence data obtained from the database and the specimen, HLA type including not only homozygous data but also heterocomplexes can be distinguished. This makes it possible to perform detailed type recognition in a type that includes a heterocomplex consisting of a combination of all homotypes, which can be classified only with homozygous and some known heteroconjugates. .
[Brief description of the drawings]
FIG. 1 is a diagram showing a part of a base sequence obtained from a gene data bank on the start end side.
FIG. 2 is a diagram showing base sequence data obtained by removing a sequence portion that cannot be determined from the base sequence of FIG.
FIG. 3 is a diagram showing data obtained by determining a logical OR base sequence of two sequences by NC-IUB.
4 is a diagram illustrating a state in which the data configured in FIG. 3 is serially arranged. FIG.
FIG. 5 is a diagram showing the results of a homology search performed on the base sequence data configured in the present invention.
FIG. 6 is a continuation view from FIG. 5;
FIG. 7 is a continuation view from FIG. 6;
FIG. 8 is a continuation view from FIG. 7;
FIG. 9 is a continuation view from FIG. 8;
FIG. 10 is a continuation view from FIG. 9;

Claims (2)

The storage means for storing homozygous data of antigen gene data arranged in units of antigen, the database for storing HLA type discrimination data, and the two base sequence data are multiplied by NC- In the method of creating a database for HLA type discrimination using a computer, using arithmetic means for replacing with a one-character code according to IUB,
The computer
In the base sequence on the start and end sides in the data sequence stored in the storage means, all the alleles are the same up to the maximum region where the base sequence is not determined in amino acid units so that there is no portion that has not been determined A first step of deleting a place;
Executing a second step of creating data of a hetero-complex of one character representation of the two data strings out of the data strings created in the first step and storing the data in the database. HLA type discrimination database creation method. The storage means for storing homozygous data of antigen gene data arranged in units of antigen, the database for storing HLA type discrimination data, and the two base sequence data are multiplied by NC- In the method of creating a database for HLA type discrimination using a computer, using arithmetic means for replacing with a one-character code according to IUB,
The computer
In the base sequence on the start and end sides in the data sequence stored in the storage means, all the alleles are the same up to the maximum region where the base sequence is not determined in amino acid units so that there is no portion that has not been determined A first step of deleting a place;
A second step of creating data of a hetero-complex of one character representation of the two data strings out of the data strings created in the first step and storing the data in the database;
An HLA type discriminating method comprising: performing a third step of extracting from data stored in a database in the second step from data highly correlated with target DNA sequence data.

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