Primer group for amplifying B lymphocyte immune repertoire and application of primer group

A technology of B lymphocytes and immune repertoire, applied in recombinant DNA technology, microbial assay/test, biochemical equipment and methods, etc., can solve the problem of unclear pathogenesis of IgAN, achieve high coverage and reduce secondary Structure generation, specificity and sensitivity

Pending Publication Date: 2021-02-02
SOUTH UNIVERSITY OF SCIENCE AND TECHNOLOGY OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the above-mentioned genetic factors, abnormal IgA immune complexes, or viral in...

Method used

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  • Primer group for amplifying B lymphocyte immune repertoire and application of primer group
  • Primer group for amplifying B lymphocyte immune repertoire and application of primer group
  • Primer group for amplifying B lymphocyte immune repertoire and application of primer group

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] This embodiment provides a method for expanding the information of the immune repertoire of B lymphocytes in IgAN patients.

[0057] 1. Patient recruitment and screening

[0058] In this example, the whole blood samples and kidney biopsy samples of 13 IgAN patients were collected, aged between 22 and 62 years old (the median was 26), and 7 normal people were recruited at the same time. According to the method recommended by WHO, blood and urinalysis and renal biopsy were performed on the main components. This study was conducted in accordance with the guidelines of the Endocrinology Department of Shenzhen Nanshan Hospital and was approved by the local medical ethics committee. All patients provided written informed consent.

[0059] 2. PBMC separation

[0060] Dilute 10 mL of blood with an equal amount of PBS and carefully load it into 5 mL of lymph preparation solution. The ratio of sample volume to lymph preparation medium is 2:1. All reagents used are purchased fr...

Embodiment 2

[0082] In this example, data analysis is performed on the BCR library prepared in Example 1.

[0083] A total of 18,976,912 pairs of end reads were generated by the Illumina sequencer, and overlapping paired-end reads were merged using FLASH software to obtain 16,376,727 original reads;

[0084] Merged sequence reads were aligned to V, D, and J gene germline references using IgBLAST, and constant regions were mapped to IgA, IgD, IgE, IgG, and IgM, respectively;

[0085] The reference sequences of the V, D and J gene germlines were obtained from IMGT, and reads with low homology (<70%) to the germline reference line were excluded. After filtering, 11,114,426 reads were obtained for further analysis;

[0086] According to the definition of IMGT, the start and end position, reading frame and productivity of the CDR3 region were determined. The overexpression level of IgAN samples was analyzed by software MixCR. Diversity of the antibody repertoire was evaluated with normalized ...

Embodiment 3

[0088] The length distribution of CDR3 in IgAN and normal candidate (NC) populations was analyzed in this example. The IgAN and NC libraries were constructed according to the method described in Example 1, and the data analysis method was analyzed according to the method described in Example 2.

[0089] (1) Analysis of the length distribution of CDR3 in IgAN and NC populations

[0090] The results showed that the amino acid length of CDR3 in IgAN group was significantly shorter than that in NC group. Among them, in addition, the specific sequences of the top 10 amino acid sequences enriched in IgAN CDR3 (SEQ ID NO.37-46) and the top 10 amino acid sequences (SEQ ID NO.47-56) of the NC group are shown in Table 2 below:

[0091] Table 2

[0092] ranking IgAN panel CDR3 NC group CDR3 1 TIMES ARWDCSRTSCHQFDQ 2 ARPIGGS ARDRGRWYQLLYDPLDV 3 GGTSNGWHEIDS ARYVVLSPALGGSRLDY 4 ASGQQLGH PKIGFGYSYGGGLDV 5 ATGQQLGY AKDRSPRYDVMTGGLDS ...

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Abstract

The invention provides a primer group for amplifying a B lymphocyte immune repertoire and an application of the primer group. The primer group comprises a group of forward primers and a group of reverse primers. The forward primers comprise a linker sequence 1, a bar code sequence 1 and a specific sequence aiming at an immunoglobulin variable region complementation determining region 3, which areconnected in sequence from a 5' end to a 3' end, wherein the specific sequence aiming at the immunoglobulin variable region complementary determining region 3 is shown as any one of SEQ ID NO. 1 to SEQ ID NO. 12; and the reverse primers sequentially comprise a linker sequence 2, a bar code sequence 2 and a specific sequence aiming at an immunoglobulin gene constant region from the 5' end to the 3'end. The primer group is used for amplifying B lymphocytes of an IgAN patient and a normal control group, library information is collected, and the length and diversity changes of CDR3 of the IgAN patient are analyzed through sequencing.

Description

technical field [0001] The invention relates to the field of molecular biology detection, in particular to a primer set for amplifying a B lymphocyte immune repertoire and an application thereof. Background technique [0002] B cell antigen receptor (B cell receptor, BCR) is a membrane surface immunoglobulin (SmIg) that recognizes antigens on B cells, and has antigen-binding specificity. BCR is composed of two heavy chains and two light chains connected, in which the heavy chain is divided into variable region (V region), constant region (C region), transmembrane region and cytoplasmic region; the light chain has only V region and Area C. The V region is composed of two domains, VH and VL, each of which is composed of three complementarity determining regions (CDR1, CDR2 and CDR3). 12 , forming a huge BCR library, endowing individuals with a huge potential to recognize various antigens and produce specific antibodies. These three CDRs are all involved in the recognition of...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12Q1/6869C12N15/11
CPCC12Q1/6806C12Q1/6869
Inventor 李周芳魏闻捷曾健明
Owner SOUTH UNIVERSITY OF SCIENCE AND TECHNOLOGY OF CHINA
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