Primer and kit for detecting B cell receptor repertoire of midge sensitized immune mouse

Abstract

The invention provides the primer and the detection kit for detecting the culicoides chromogen immune mouse B cell receptor repertoire, the amplified B cells and plasma cell clone can be identified and quantified by screening the BCR repertoire by utilizing the kit, and the kit has the advantages of high throughput, rapid and accurate detection and the like. Experiments show that a basis is provided for the research on the immune mechanism of the culicoides leucoides allergen.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology detection, and particularly relates to a primer and kit for detecting B cell receptor library of immunized mouse by Culex pipiens quinquefasciatus allergen. BACKGROUND

[0002] Culex pipiens quinquefasciatus Culicoides taenianus is a widely distributed insect, especially in tropical and subtropical regions. Its bites not only bring great discomfort to humans and animals, but also can trigger severe allergic reactions and diseases. The saliva of Culex pipiens quinquefasciatus contains a variety of allergens, which can trigger the body's immune response, leading to local or systemic allergic symptoms. Common symptoms include skin redness, itching, hives, and even anaphylactic shock in severe cases. In addition, Culex pipiens quinquefasciatus is also a vector of various pathogens, such as blue tongue virus and African horse sickness virus. These viruses are transmitted to livestock through the bites of Culex pipiens quinquefasciatus, causing huge economic losses to the livestock industry.

[0003] The allergic reaction caused by the bite of Culex pipiens quinquefasciatus is mainly due to the allergens in its saliva that can activate the body's immune system, especially the B cell-mediated humoral immune response. B cells recognize and eliminate foreign antigens such as pathogens and toxins by producing specific antibodies. The function of B cells depends on the B cell receptor (BCR) on their surface, which is a key molecule for B cells to recognize antigens. These allergens can bind to the B cell receptor (BCR) on the surface of B cells, triggering the proliferation and differentiation of B cells, and then producing specific antibodies such as IgE antibodies. The production of IgE antibodies is an important factor in the action of allergic reactions, which can bind to the FcεRI receptor on the surface of mast cells and basophils, causing these cells to release histamine and other inflammatory mediators, triggering allergic symptoms. BCR is a tetramer structure composed of two heavy chains (H chain) and two light chains (L chain, κ or λ chain). Each chain contains a variable region (V region) and a constant region (C region). The variable region is formed by rearrangement of V (Variable), D (Diversity), and J (Joining) gene segments, which determines the antigen specificity of BCR. The variable region of BCR contains three complementarity determining regions (CDR1, CDR2, and CDR3), among which the CDR3 region is the most diverse region and directly involved in antigen binding. The diversity of CDR3 region comes from the randomness of V(D)J gene rearrangement process, as well as the insertion or deletion of nucleotide sequences during the rearrangement process. This diversity allows BCR to recognize almost unlimited types of antigens. After B cells recognize antigens through BCR, they will undergo clonal expansion and somatic hypermutation, further optimizing their affinity for antigens. This process occurs in the germinal center, ultimately producing high-affinity memory B cells and plasma cells. Plasma cells can secrete large amounts of specific antibodies, participating in the body's immune defense.

[0004] B cell receptor repertoire (BCR repertoire) refers to the collection of BCRs expressed by all B cells in an individual. By analyzing the diversity of the BCR repertoire, especially the sequence characteristics of the CDR3 region, the immune status of the body under physiological and pathological conditions can be understood. Analysis of the CDR3 receptor repertoire not only reveals the clonal expansion of B cells, but also provides important information for the diagnosis and treatment of diseases. For example, in autoimmune diseases, certain specific BCR clones may over-expand, leading to the production of autoantibodies and triggering diseases. After biting of the sergentomyia baini, the level of IgE in the body usually increases significantly. This increase in IgE is not only an immune response to the allergen, but also an important marker of allergic reactions. By detecting the level of IgE, the sensitivity of the body to the allergen of sergentomyia baini can be evaluated, thereby providing a basis for the diagnosis and treatment of allergic reactions.

[0005] With the rapid development of high-throughput sequencing technology, the analysis of B cell receptor repertoire has become more efficient and accurate. High-throughput sequencing technology, also known as Next-Generation Sequencing (NGS), can simultaneously sequence hundreds of thousands to millions of DNA molecules, greatly improving the efficiency and accuracy of sequencing. In immunology research, high-throughput sequencing technology is widely used in the analysis of BCR, TCR and other receptor repertoires. Through high-throughput sequencing technology, the diversity of B cell receptor repertoire, especially the sequence characteristics of the CDR3 region, can be comprehensively and dynamically monitored. This technology not only reveals the clonal expansion of B cells, but also provides important information for the diagnosis and treatment of diseases. For example, in autoimmune diseases, certain specific BCR clones may over-expand, leading to the production of autoantibodies and triggering diseases. Through high-throughput sequencing technology, these abnormal BCR clones can be quickly and accurately identified, providing a basis for early diagnosis and individualized treatment of diseases.

[0006] In the sergentomyia baini allergen immunized mouse model, high-throughput sequencing technology can be used to analyze the changes in the mouse B cell receptor repertoire. By comparing the BCR repertoire of immunized mice and healthy mice, the influence of sergentomyia baini allergen on B cell clonal expansion can be revealed, thereby providing new ideas and methods for the mechanism research of allergic reactions. The allergic reaction induced by sergentomyia baini biting is a complex immune process involving B cells, BCR and the diversity of their receptor repertoire. Through high-throughput sequencing technology, the characteristics of the B cell receptor repertoire can be analyzed in depth, revealing the influence of sergentomyia baini allergen on the immune system of the body.

[0007] Currently, most of the B cell receptor (BCR) detection primers are designed based on the conserved regions, which are suitable for the amplification of most human samples, but may have obvious limitations in specific research objects such as chironomus allergen sensitized individuals. First, most of the existing primers are based on the common sequences in public databases, which are difficult to cover the low-abundance, rare or highly variable V(D)J gene rearrangements in the receptor library, resulting in some BCR clonal types not being effectively amplified, affecting the accuracy of diversity analysis. Second, the uneven amplification efficiency between primers may cause bias, resulting in over-representation of high-frequency clones and neglect of low-frequency clones. In addition, some primer designs ignore the genetic polymorphism between species or populations, which may cause off-target or amplification failure in specific populations. For the special immune response induced by the chironomus allergen, these defects will directly limit the comprehensive analysis of the real BCR library composition and dynamic changes. Therefore, optimizing or customizing the primer system to improve the detection sensitivity and specificity is the key prerequisite for carrying out high-quality BCR sequencing research. SUMMARY

[0008] In view of the above technical problems, the purpose of the present application is to provide a primer for detecting B cell receptor library of mice immunized by chironomus allergen.

[0009] Another purpose of the present application is to provide a detection kit containing the above-mentioned primer.

[0010] In order to achieve the purpose of the present application, the specific technical solutions provided by the present application are as follows: A primer for detecting B cell receptor library of mice immunized by chironomus allergen, namely IgH-specific PCR amplification primer, the primer is composed of any one of the following (1)~(16): (1) the DNA molecule shown in SEQ ID NO. 1 and the DNA molecule shown in SEQ ID NO. 17; (2) the DNA molecule shown in SEQ ID NO. 2 and the DNA molecule shown in SEQ ID NO. 17; (3) the DNA molecule shown in SEQ ID NO. 3 and the DNA molecule shown in SEQ ID NO. 17; (4) the DNA molecule shown in SEQ ID NO. 4 and the DNA molecule shown in SEQ ID NO. 17; (5) the DNA molecule shown in SEQ ID NO. 5 and the DNA molecule shown in SEQ ID NO. 17; (6) the DNA molecule shown in SEQ ID NO. 6 and the DNA molecule shown in SEQ ID NO. 17; (7) the DNA molecule shown in SEQ ID NO. 7 and the DNA molecule shown in SEQ ID NO. 17; (8) the DNA molecule shown in SEQ ID NO. 8 and the DNA molecule shown in SEQ ID NO. 17; (9) the DNA molecule shown in SEQ ID NO. 9 and the DNA molecule shown in SEQ ID NO. 18; (10) the DNA molecule shown in SEQ ID NO. 10 and the DNA molecule shown in SEQ ID NO. 18; (11) the DNA molecule shown in SEQ ID NO. 11 and the DNA molecule shown in SEQ ID NO. 18; (12) the DNA molecule shown in SEQ ID NO. 12 and the DNA molecule shown in SEQ ID NO. 18; (13) the DNA molecule shown in SEQ ID NO. 13 and the DNA molecule shown in SEQ ID NO. 18; (14) the DNA molecule shown in SEQ ID NO. 14 and the DNA molecule shown in SEQ ID NO. 18; (15) the DNA molecule shown in SEQ ID NO. 15 and the DNA molecule shown in SEQ ID NO. 18; (16) the DNA molecule shown in SEQ ID NO. 16 and the DNA molecule shown in SEQ ID NO. 18.

[0011] A kit for detecting the B cell receptor repertoire of a Culex pipiens quinquefasciatus allergen immunized mouse, comprising a combination of any one of the primers.

[0012] As a preferred embodiment of the present application, the kit further comprises at least one of a reverse transcription primer Oligo(dT) 18 , a reverse transcriptase, a dNTP mixture, DEPC treated water, a reaction buffer.

[0013] As a preferred embodiment of the present application, the kit further comprises a Culex pipiens quinquefasciatus allergen immunized mouse B cell receptor repertoire standard.

[0014] Use of the primers or the kit in detecting the B cell receptor repertoire of a Culex pipiens quinquefasciatus allergen immunized mouse.

[0015] As a preferred embodiment of the present application, detecting the B cell receptor repertoire of a Culex pipiens quinquefasciatus allergen immunized mouse comprises the following steps: extracting total RNA from a sample; using the extracted RNA as a template, performing reverse transcription to obtain a first strand of cDNA; PCR amplification of IgH was performed using the cDNA as template, and the PCR amplification product was analyzed.

[0016] Further preferably, the reaction conditions for PCR amplification of IgH are as follows: 95℃, 1min; 95℃, 30s, 55℃, 30s, 72℃, 30s, 30 cycles; 72℃, 10min.

[0017] Further preferably, each 50μL reaction system for PCR amplification of IgH comprises: cDNA template 1000ng, DreamTaq Green PCR Master Mix (2x) 25μL, IGHV upstream primer 1.6μL, IGHV downstream primer 1.6μL, DEPC-treated water to make up.

[0018] The present application is based on high-throughput sequencing technology, and a primer and a detection kit for detecting B cell receptor library of a mouse immunized by a striped bark midge allergen are developed through a large number of experiments. The kit can accurately identify specific clonal expansion in the B cell receptor library of a mouse immunized by a striped bark midge allergen. The kit has the advantages of high-throughput, rapid detection, and accuracy. Experiments show that the kit can be used to identify and quantify expanded B cells and plasma cells by screening B cell receptor (BCR) library, thereby providing an important basis for the study of the immunization mechanism of a striped bark midge allergen. DETAILED DESCRIPTION

[0019] In order to enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. If not specifically indicated, the technical means used in the embodiments are conventional means familiar to those skilled in the art, and the raw materials used are commercially available goods.

[0020] Embodiment 1 Design of primer for detecting B cell receptor library of a mouse immunized by a striped bark midge allergen The primer for detecting B cell receptor library of a mouse immunized by a striped bark midge allergen, i.e., IgH-specific PCR amplification primer, is designed, and is shown in Table 1.

[0021] Table 1 IgH-specific PCR amplification primer Embodiment 2 The kit for detecting B cell receptor library of a mouse immunized by a striped bark midge allergen comprises any one or a combination of the primers in Embodiment 1 and a standard product of B cell receptor library of a mouse immunized by a striped bark midge allergen.

[0022] The kit can further comprise at least one of reverse transcription primer Oligo(dT) 18 , reverse transcriptase, dNTP mixture, DEPC treated water, reaction buffer.

[0023] The kit comprises the process of reverse transcription to synthesize the first strand of cDNA and multiplex PCR reaction; (1) Reverse transcription to synthesize the first strand of cDNA System I is prepared as follows: total RNA 500 μg, 5x PrimeScript Buffer 2 ul, PrimeScript RT Enzyme Mix I 0.5 ul, Oligo dT Primer 0.5 ul, Random 6 mers 0.5 ul, RNase Free dH2O to a total system of 10 ul; 37℃ incubate for 15 min, 85℃, 5s stop reaction, and cool on ice; (2) Multiplex PCR reaction: The primer combination is shown in Table 2.

[0024] Table 2 Multiplex PCR primer combination System II is prepared according to Table 3: Table 3 Composition of system II The PCR reaction conditions of IgH amplification primer are: 95℃ 1 min, 95℃ 30 s, 55℃ 30 s, 72℃ 30 s, 30 cycles, 72℃ 10 min; 4℃ storage.

[0025] The kit is used to detect the B cell receptor library of the immunized mouse by the bit library of the midge allergen, which comprises the following steps: Extracting total RNA from the sample to be tested; Using the extracted RNA as a template to obtain the first strand of cDNA by reverse transcription; Using the cDNA as a template to amplify IgH by PCR, and analyzing the PCR amplification product. The reaction system of PCR amplification of IgH is shown in Table 3, and the amplification reaction conditions are: 95℃ 1 min, 95℃ 30 s, 55℃ 30 s, 72℃ 30 s, 30 cycles, 72℃ 10 min.

[0026] The above examples are only used to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A primer for detecting a receptor library of mouse B cells immunized with a banded midge sensitizer, characterized in that, The primers consist of any of the primer pairs shown in (1) to (16) below: (1) The DNA molecule shown in SEQ ID NO.1 and the DNA molecule shown in SEQ ID NO.17; (2) The DNA molecule shown in SEQ ID NO.2 and the DNA molecule shown in SEQ ID NO.17; (3) The DNA molecule shown in SEQ ID NO.3 and the DNA molecule shown in SEQ ID NO.17; (4) The DNA molecule shown in SEQ ID NO.4 and the DNA molecule shown in SEQ ID NO.17; (5) The DNA molecule shown in SEQ ID NO.5 and the DNA molecule shown in SEQ ID NO.17; (6) The DNA molecule shown in SEQ ID NO. 6 and the DNA molecule shown in SEQ ID NO. 17; (7) The DNA molecule shown in SEQ ID NO.7 and the DNA molecule shown in SEQ ID NO.17; (8) The DNA molecule shown in SEQ ID NO. 8 and the DNA molecule shown in SEQ ID NO. 17; (9) The DNA molecule shown in SEQ ID NO.9 and the DNA molecule shown in SEQ ID NO.18; (10) The DNA molecule shown in SEQ ID NO.10 and the DNA molecule shown in SEQ ID NO.18; (11) The DNA molecule shown in SEQ ID NO.11 and the DNA molecule shown in SEQ ID NO.18; (12) The DNA molecule shown in SEQ ID NO.12 and the DNA molecule shown in SEQ ID NO.18; (13) The DNA molecule shown in SEQ ID NO.13 and the DNA molecule shown in SEQ ID NO.18; (14) The DNA molecule shown in SEQ ID NO.14 and the DNA molecule shown in SEQ ID NO.18; (15) The DNA molecule shown in SEQ ID NO.15 and the DNA molecule shown in SEQ ID NO.18; (16) The DNA molecule shown in SEQ ID NO.16 and the DNA molecule shown in SEQ ID NO.

18.

2. A kit for detecting a receptor library of mouse B cells immunized with banded midge sensitizer, characterized in that, It includes one or any combination of the primers described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The kit also includes the reverse transcription primer Oligo(dT). 18 At least one of the following: reverse transcriptase, dNTP mixture, DEPC-treated water, and reaction buffer.

4. The reagent kit according to claim 3, characterized in that, The kit also includes a standard of mouse B cell receptor library immunized with banded Culicoides sensitizer.

5. The use of the primer of claim 1 or the kit of claim 2 in detecting a mouse B cell receptor library immunized with the banded midge sensitizer.

6. The application according to claim 5, characterized in that, Detection of the receptor repertoire of mouse B cells immunized with banded midge sensitizers includes the following steps: Total RNA was extracted from the sample to be tested; Using the extracted RNA as a template, the first strand of cDNA was obtained by reverse transcription; Using the cDNA as a template, IgH was amplified by PCR, and the PCR amplification products were analyzed.

7. The application according to claim 6, characterized in that, The PCR amplification conditions for IgH were: 95℃ for 1 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s, 30 cycles; 72℃ for 10 min.

8. The application according to claim 6, characterized in that, Each 50 μL PCR amplification reaction system for IgH includes: 1000 ng cDNA template, 25 μL DreamTaq Green PCR Master Mix (2×), 1.6 μL IGHV upstream primer, 1.6 μL IGHV downstream primer, and DEPC-treated water to make up the difference.

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