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

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...

AI Technical Summary

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 secondary rearrangement of the Igκ gene. Intermediate product, but the downstream primer is not modified, the sensitivity and specificity are low, and 32 P-labeled probes have radiation and are harmful to the body. The target fragments cannot be recovered, and subsequent sequencing and information analysis cannot be performed.

In addition, some researchers designed primers for specific Vκ and Jκ to amplify the VκJκ gene fragment by ordinary PCR, but the VκJκ gene fragment amplified in this way cannot determine whether it is a gene fragment excluded by the secondary rearrangement of the Igκ gene

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