Method for analyzing intermediate product of secondary rearrangement of Igk gene and kit

A gene and product technology, applied in the field of capturing and amplifying secondary rearrangement intermediate products of Igκ gene, can solve the problems of low sensitivity and specificity, no modification of downstream primers, and uncertain whether the VκJκ gene fragment is secondary rearranged by Igκ gene Eliminate problems such as gene fragments to achieve the effect of improving sensitivity and specificity

Inactive Publication Date: 2016-12-07
NANKAI UNIV
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Problems solved by technology

[0003] The secondary rearrangement of the Igκ gene is the main pathway of receptor editing, and the molecular mechanism for initiating this pathway has been unclear. Researchers have attempted to analyze the molecular mechanism of this pathway by amplifying the intermediate product of the secondary rearrangement of the Igκ gene by PCR. However, because the secondary rearrangement of the Igκ gene occurs instantaneously, the target fragment can only be amplified when there is sufficient template amount. Therefore, the researchers used the southern blot method combined with lm-PCR to amplify the

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  • Method for analyzing intermediate product of secondary rearrangement of Igk gene and kit
  • Method for analyzing intermediate product of secondary rearrangement of Igk gene and kit
  • Method for analyzing intermediate product of secondary rearrangement of Igk gene and kit

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Embodiment Construction

[0045] 1. Take MRL.FAS lpr 、B6.FAS lpr Bone marrow single cell suspension of C3H mice was used as experimental material, and bone marrow B lymphocytes of different developmental stages were obtained by flow cytometry technology.

[0046] Prepare mouse bone marrow single cell suspension, and design antibody staining strategy according to the cell types and their specific surface antigens in the mouse bone marrow single cell suspension, as well as the specific surface antigens of B cells at various developmental stages, so as to sort wild type mice (C3H) and lupus erythematosus mice (B6.FAS lpr , MRL.FAS lpr ) pro-B (B220 + CD43 + IGM - IGD - ), small pre-B (B220 + CD43 - IGM - IGD - ) and immature-B (B220 + CD43 - IGM + IGD - )cell.

[0047] 2. Extraction of genomic DNA from pro-B, small pre-B and immature-B cells Extract genomic DNA from pro-B, small pre-B and immature-B cells obtained by sorting. Due to the number of cells obtained by sorting Less and subseque...

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Abstract

The invention relates to a method for analyzing an intermediate product of secondary rearrangement of an Igk gene and a kit. The kit consists of an artificially designed oligodeoxynucleotide linker, 6 pairs of upstream primers and a downstream primer; a first rearrangement fragment VkJk-intron-RS which is replaced in the secondary rearrangement of the Igk gene is captured and amplified by using a ligation mediated-PCR (lm-pcr) technology. The kit disclosed by the invention can be applied to in vitro analysis of the generation efficiency of the secondary rearrangement of the Igk gene and research on a molecular mechanism which generates the secondary rearrangement of the Igk gene.

Description

technical field [0001] The invention relates to the field of immunoglobulin gene rearrangement, in particular to the method lm-pcr for capturing and amplifying the secondary rearrangement intermediate product of Igκ gene. Background technique [0002] The process of bone marrow B lymphocyte differentiation and development is accompanied by rearrangements of immunoglobulin heavy chain (IgH) and light chain (Igκ) germline genes and the expression of functional B cell surface receptors (BCR), V(D)J gene The rearrangements generate a rich and diverse immunoglobulin gene repertoire. However, the V(D)J gene rearrangement process also produces autoreactive BCRs. In the process of negative selection, self-reactive B lymphocytes will be apoptotic, or through heavy chain gene replacement (IgH replacement) and Igκ gene secondary rearrangements (secondary Igκ rearrangements) to change the specificity of self-reactive BCR to achieve immune resistance. Tolerance (immune tolerance), so a...

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q2521/131C12Q2531/101
Inventor 刘菲菲李树芳向荣
Owner NANKAI UNIV
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