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T cell receptor and b cell receptor repertoire analysis system, and use of same in treatment and diagnosis

A kind of cell receptor, B cell technology, applied in the field of therapy and diagnosis, genetic information system, can solve the problem of inability to obtain the frequency of use of V chain and so on

Pending Publication Date: 2016-11-09
REPERTOIRE GENESIS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is almost impossible to obtain any information other than the frequency of V-chain usage

Method used

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  • T cell receptor and b cell receptor repertoire analysis system, and use of same in treatment and diagnosis
  • T cell receptor and b cell receptor repertoire analysis system, and use of same in treatment and diagnosis
  • T cell receptor and b cell receptor repertoire analysis system, and use of same in treatment and diagnosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0703] The preferred embodiments of the present invention are described below. The embodiments are provided to better understand the present invention. It should be understood that the scope of the present invention should not be limited to the following description. Further, it is obvious that those skilled in the art can easily make modifications within the scope of the present invention by referring to the description herein. With regard to such embodiments, those skilled in the art can appropriately combine any of the embodiments.

[0704] (Unbiased sample amplification)

[0705] The present invention can use next-generation sequencing technology to prepare samples for quantitative analysis of the T cell receptor (TCR) or B cell receptor (BCR) variable region library. Such sequencing technology can obtain one million or more decoded genetic codes from samples at a reasonable cost. By using these techniques in a specific and unbiased manner, even genotypes that exist at a lo...

preparation example 1

[1068] (Preparation Example 1: Analysis of BCR Library in Peripheral Blood of Healthy Individuals) In this example, BCR library analysis was performed on the peripheral blood of healthy individuals.

[1069] (Materials and Method)

[1070] Sample: Mononuclear cells from peripheral blood of healthy individuals

[1071] method:

[1072] (1, RNA extraction)

[1073] Collect 5 mL of whole blood from healthy individuals in a blood collection tube containing heparin. Peripheral blood mononuclear cells (PBMC) were separated by sucrose density gradient centrifugation. Using RNeasy Lipid TissueMini Kit (QIAGEN, Germany), from the separated 5×10 6 Extract / purify total RNA from one PBMC. Using an absorption spectrometer, the obtained RNA was quantified by measuring the absorbance of A260. The concentration in 30 μL of eluate is 232 ng / μL.

[1074] (2. Synthesis of complementary DNA and double-stranded complementary DNA)

[1075] Using the extracted RNA sample, perform adaptor ligation PCR. Firs...

preparation example 2

[1169] (Preparation example 2: TCR library analysis in the peripheral blood of healthy individuals)

[1170] In this example, TCR library analysis was performed on the peripheral blood of healthy individuals.

[1171] (Materials and Method)

[1172] (sample)

[1173] Peripheral blood mononuclear cells of 10 healthy individuals

[1174] (method)

[1175] (1, RNA extraction)

[1176] Collect 5 mL of whole blood from 10 healthy individuals in a blood collection tube containing heparin. Peripheral blood mononuclear cells (PBMC) were separated by sucrose density gradient centrifugation. Using RNeasy Lipid Tissue Mini Kit (QIAGEN, Germany), total RNA was extracted / purified from isolated PBMC. Using Agilent 2100 bioanalyzer (Agilent), the obtained RNA was quantified. The amount of RNA obtained is shown in Table 1-4 below.

[1177] [Table 1-4]

[1178]

[1179] (2. Synthesis of complementary DNA and double-stranded complementary DNA)

[1180] Using the extracted RNA sample, perform adaptor ligat...

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Abstract

The repertoire of the variable region of T cell receptors (TCR) or B cell receptors (BCR) is quantitatively analyzed using non-biased gene sequence analysis. The present invention provides the following: a method for quantitatively analyzing the repertoire of the variable region of the T cell receptors (TCR) or B cell receptors (BCR) of a subject by using a database, wherein the method includes (1) a step for providing a nucleic acid sample containing the nucleic acid sequence of T cell receptors (TCR) or B cell receptors (BCR) amplified in a non-biased manner from the subject; (2) a step for determining the nucleic acid sequence contained in the nucleic acid sample; and (3) a step for calculating the frequency of appearance of each gene or combination thereof on the basis of the determined nucleic acid sequence and deriving the TCR or BCR repertoire of the subject.

Description

Technical field [0001] The present invention relates to a technology for amplifying genes generated by gene rearrangement from biological samples without applying deviation, a system for analyzing the obtained genetic information, and treatment and diagnosis thereof. Background technique [0002] The biological defense mechanism of the immune system relies heavily on the specific immunity provided by T cells and B cells. T cells and B cells do not respond to their own cells or molecules and can specifically recognize and attack foreign pathogens, such as viruses or bacteria. For this reason, T cells and B cells have a mechanism that can recognize and distinguish self-antigens and various antigens derived from other organisms through receptor molecules expressed on the cell surface. T cell receptor (TCR) and B cell receptor (BCR) function as antigen receptors in T cells and B cells, respectively. Intracellular signals are transmitted through stimulation by such antigen receptors...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12Q1/68G06F19/22G16B30/10
CPCC12Q2600/156C12Q2600/16G16B30/00C12Q1/6881C12Q1/6886C12Q2600/158C12Q2600/172G16B30/10
Inventor 铃木隆二新井理
Owner REPERTOIRE GENESIS
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