Sequencing-based typing method of human leucocyte antigen (HLA)-Cw gene

A leukocyte antigen and gene sequencing technology, which is applied in the field of human leukocyte antigen HLA-Cw gene sequencing and typing for clinical transplantation tissue matching, can solve the problems of affecting high-resolution typing results, lack of data and information, etc., to reduce ambiguity, The effect of easy identification and interpretation of results

Inactive Publication Date: 2012-02-29
SHENZHEN BLOOD CENT
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Problems solved by technology

[0012] As of April 2009, there are 440 HLA-Cw alleles published in the IMGT / HLA database (http: / / www.ebi.ac.uk / imgt / hla / ), among which there are full-length sequences of HLA- There are only 49 Cw alleles, and most of the alleles lack SNPs data and information located in other exons and non-coding regions, which directly affects the results of high-resolution typing

Method used

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  • Sequencing-based typing method of human leucocyte antigen (HLA)-Cw gene
  • Sequencing-based typing method of human leucocyte antigen (HLA)-Cw gene
  • Sequencing-based typing method of human leucocyte antigen (HLA)-Cw gene

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Embodiment 1

[0069] This example provides a solution to the problem of missed detection of the Cw*0706 allele by the AlleleSEQR HLA-C plus imported kit through the human leukocyte antigen HLA-Cw gene sequencing and typing method of the present invention.

[0070] AlleleSEQR HLA-C plus kit, the PCR product has two bands, one band extends from exon 1 to intron 3, and the other band extends from intron 3 to intron 7. The samples of 620 healthy random individuals of the Han nationality were detected by the AlleleSEQR HLA-C plus kit, and we found that 5 samples had no genotypes that completely matched them, and there were single base differences with the closest multiple allele types. Matching, and base heterozygosity in exon 4 indicates heterozygosity, but there is no heterozygous base in the polymorphic-rich exon 2 and exon 3 regions.

[0071] First, take the upstream primer C-SBT-F1 and the downstream primer C-SBT-R1 of the first pair of PCR primers in the present invention to amplify the 1s...

Embodiment 2

[0102] This example provides an example of performing HLA-Cw high-resolution sequencing typing on 156 bone marrow voluntary donor samples.

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Abstract

The invention provides a sequencing-based typing method of human leucocyte antigen (HLA)-Cw genes, comprising the following steps: a. carrying out PCR amplification on a typing target region by two pairs of PCR amplification primers, wherein the first pair of amplification primers contains a sequence from a first exon to a fourth exon, and the second pair of amplification primers contains a sequence from a fifth exon to an eighth exon; and b. carrying out sequencing reaction on the obtained amplified product by twelve forward sequencing primers and reverse sequencing primer, wherein the first exon, the second exon, the third exon, the fourth exon and the fifth exon of the HLA-Cw genes are subject to forward and reverse sequencing respectively, the sixth exon is subject to forward sequencing and the seventh exon is subject to reverse sequencing. The invention is established based on full-length sequence of the HLA-Cw genes and single nucleotide polymorphisms (SNPs) data, solving the problems of missed detection of an allele Cw*0706 occurred in population detection via an AlleleSEQR HLA-Cw plus kit and ambiguous results occurred in sequencing-based typing. The method is applicable to high-resolution horizontal gene typing of the HLA-Cw genes in Chinese population, and is beneficial to various application and basic researches of the HLA-Cw genes.

Description

technical field [0001] The invention relates to biomedicine, in particular to a method for sequencing and typing human leukocyte antigen HLA-Cw gene for clinical transplantation tissue typing. Background technique [0002] The protein encoded by the human leukocyte antigen HLA-Cw gene is a classic HLA class I molecule, which not only presents endogenous antigenic peptides to CD8+ T cells for recognition, mediates the immune response of killer T cells, but also acts as a killer cell immunoglobulin-like The ligand of the receptor (Killer Immunoglobulin-like Receptor, KIR), regulates the killing function of natural killer cells (Natural Killer cells), and plays an important role in transplantation immunity, tumor immunity and anti-infection immunity. The HLA-Cw molecule is composed of a polymorphic α polypeptide chain (45 kDa) and a non-polymorphic ?2 microglobulin (12 kDa) encoded by a gene located on chromosome 15 through non-covalent bonds heterodimer. [0003] With the de...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 邓志辉徐筠娉王大明
Owner SHENZHEN BLOOD CENT
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