阅读说明：本技术 一种鸡补体受体2(ChCR2)单克隆抗体及其应用 (Chicken complement receptor 2(ChCR2) monoclonal antibody and application thereof ) 是由 李永清 靳换 孔子萌 江波 许健 刘文晓 宋翠平 于 2020-08-06 设计创作，主要内容包括：本发明涉及一种抗鸡补体受体2(ChCR2)单克隆抗体及其应用,本发明以纯化后的ChCR2蛋白片段作为免疫原,免疫小鼠后提取免疫合格的的小鼠脾脏B淋巴细胞,通过细胞融合技术将淋巴细胞与小鼠骨髓瘤细胞进行融合,经过筛选和亚克隆技术,获得抗ChCR2单克隆抗体,本发明的抗ChCR2单克隆抗体可以用于检测ChCR2蛋白抗原的生物学检测试剂,并应用在研究鸡B细胞功能的相关检测产品中。(The invention relates to an anti-chicken complement receptor 2(ChCR2) monoclonal antibody and application thereof, wherein a purified ChCR2 protein fragment is used as an immunogen, a mouse spleen B lymphocyte qualified for immunization is extracted after the mouse is immunized, the lymphocyte and a mouse myeloma cell are fused by a cell fusion technology, and an anti-ChCR 2 monoclonal antibody is obtained by screening and subcloning technologies.)

1. The chicken complement receptor 2 monoclonal antibody hybridoma cell 3E9 with the preservation number as follows: CGMCC No. 19286.

2. A monoclonal antibody secreted by the hybridoma cell of claim 1.

3. The chicken complement receptor 2 monoclonal antibody according to claim 2, which specifically recognizes an epitope of chicken complement receptor 2, wherein the amino acid sequence of the epitope is shown as SEQ ID No. 1.

4. The monoclonal antibody of claim 2, wherein the monoclonal antibody is used for preparing a detection reagent for detecting chicken complement receptor 2 antigen.

5. The use according to claim 4, wherein the detection reagent is a reagent used in ELISA, western blot, IFA, co-immunoprecipitation, immunohistochemistry, and flow cytometry.

6. A detection reagent or kit comprising the monoclonal antibody of claim 2.

Technical Field

The invention belongs to the technical field of biological preparation, and particularly relates to a chicken complement receptor 2(ChCR2) monoclonal antibody and application thereof.

Background

Complement receptor 2 (CR 2), a glycoprotein with a transmembrane domain, is mainly expressed on the surface of mature B cells and follicular dendritic cells, and is a receptor for the C3 lytic fragment C3d (g), IFN- α, EBV-gp350/220, CD23, etc., and the loss of CR2 on B cells leads to the loss of T cell-dependent antibody responses. After HSV virus envelope proteins gB and gH/gL are respectively combined with CR2 and a related B cell receptor Ig2 alpha, the virus can rapidly enter B cells, and the B cell immune response and the humoral immunity are stimulated. The ligand of CR2 is the cleavage end product of complement, C3d, and thus the antigenic signal received by CR2 is actually linked through C3 d. The antigen is combined with CR2 through C3d to cause CD19/CR2 complex activation, and the activated CD19/CR2 complex is crosslinked with CD81 to synergistically amplify the intracellular BCR stimulation signal. BCR signaling is again manifested by downstream signaling and effector molecule activity, ultimately leading to B cell proliferation and functional alterations, and antigen-signaling communication with T cells, followed by secondary CTL responses. The human CR2 monoclonal antibody can block the invasion of virus by combining with CR2, thereby playing the role of immune protection. The research on the effect of the CR2 gene of the chicken on chicken diseases and immune systems has guiding significance for clinically evaluating the chicken immune system diseases. Therefore, how to prepare a monoclonal antibody capable of detecting the ChCR2 antigen is of great significance for researching the effect of the ChCR2 in the immune system of the chicken.

Disclosure of Invention

The invention provides an anti-chicken complement receptor 2(ChCR2) monoclonal antibody and application thereof, aiming at solving the existing ChCR2 antigen detection technology.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a hybridoma cell 3E9 for preparing a chicken complement receptor 2(ChCR2) monoclonal antibody, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (address: the institute of microbiology, China academy of sciences, No. 3, North Chen West Lu No.1 institute of China, Beijing, Inward Yang district, zip code 100101) in 10 days 01 and 10 days 2020, and the preservation numbers are as follows: CGMCC No. 19286.

In a specific embodiment, the invention also specifically discloses a preparation method of the ChCR2 monoclonal antibody hybridoma cell strain, which comprises the steps of immunizing a mouse with BALB/c in example 1 by using ChCR2(His-CR 2-delta TM) protein with a transmembrane region removed, fusing spleen cells of the mouse which is qualified for immunization with myeloma cells, screening positive hybridoma cells, cloning and purifying the positive hybridoma cells by a limiting dilution method, and finally obtaining the monoclonal antibody hybridoma cell strain which specifically secretes the single epitope of the anti-ChCR 2 protein.

The protein immunization is carried out by four times of protein immunization; the myeloma cell is SP2/0 myeloma cell.

In a specific embodiment, the invention also provides a chicken complement receptor 2 monoclonal antibody, which is secreted by the hybridoma cells described above.

The preparation method comprises the steps of injecting the hybridoma cells obtained in the above way into an abdominal cavity of a female mouse producing BLAB/c, collecting ascites when the abdomen of the mouse is obviously enlarged, and purifying a monoclonal antibody to obtain the monoclonal antibody against chicken complement receptor 2(ChCR 2). Wherein, the menstruation refers to the pregnant mother mouse which is born with the small mouse.

Preferably, the chicken complement receptor 2 monoclonal antibody specifically recognizes an epitope of chicken complement receptor 2, and the amino acid sequence of the epitope is shown in SEQ ID NO. 1.

The invention also provides application of the anti-chicken complement receptor 2 monoclonal antibody in preparing a related immunological detection reagent for detecting chicken complement receptor 2 antigen.

The detection reagent is used as the reagent for ELISA, western blot, IFA, co-immunoprecipitation, immunohistochemistry and flow cytometry.

The invention also provides a detection reagent or a kit containing the chicken complement receptor 2 monoclonal antibody.

The monoclonal antibody provided by the invention is applied to preparation of chicken complement receptor 2 antigen-related detection products, and the anti-ChCR 2 monoclonal antibody prepared by the invention specifically identifies ChCR2 protein in tissues and cells, so that the detection of the tissues and cells expressing the ChCR2 protein is realized. Human CR2 protein is expressed predominantly in B cells and follicular dendritic cells, whereas the distribution of ChCR2 protein in chicken is unknown.

The invention has the beneficial effects that:

the invention provides the anti-ChCR 2 protein monoclonal antibody for the first time, which can specifically recognize and combine the amino acid sequence of the specific site on the ChCR2 protein, and has high specificity and strong sensitivity. The detection tool containing the anti-ChCR 2 monoclonal antibody can be used for experimental techniques of ELISA, western blot, IFA, co-immunoprecipitation, immunohistochemistry and flow cytometry, and can also be used for preparing single-chain antibodies, immunotoxins, immune complexes, antibody medicaments and the like.

Drawings

FIG. 1 is a purified electrophoretogram of monoclonal antibody 3E 9;

FIG. 2 shows the results of the indirect immunofluorescence assay using monoclonal antibody 3E 9;

FIG. 3 is a graph of the WB assay identifying the binding of 3E9 to His-CR 2;

FIG. 4 is a schematic drawing of the truncation of ChCR2 and the identification of the 3E9 recognition epitope;

FIG. 5 is a graph of the response of monoclonal antibody 3E9 to ChCR2 antigen binding;

FIG. 6 shows the results of the identification of cross-reactivity between monoclonal antibody 3E9 and other antigens;

FIG. 7 shows the result of the monoclonal antibody 3E9 subclass identification.

Detailed Description

The following examples are intended to further illustrate the invention but should not be construed as limiting it. The invention discloses an anti-ChCR 2 monoclonal antibody and application thereof, and can be realized by appropriately improving process parameters by taking the contents of the monoclonal antibody as reference by a person skilled in the art. It is to be expressly understood that modifications and substitutions may be made thereto without departing from the spirit and scope of the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention. The methods, devices and materials used in the examples which follow, if not specifically indicated, are all conventional and commercially available methods, devices and materials used in the art.

The sources of materials and reagents used in the following examples are as follows: myeloma cell SP2/0 is stored for the laboratory; BLAB/c mice were purchased from Beijing Wittiulihua laboratory animal technology, Inc.; cell culture plates, cell culture flasks were purchased from Coaster, DMEM/HIGH GLUCOSE (1 ×), HT Supplement (100 ×), from Gibco, HAT Supplement (50 ×), PEG1500, freund's complete adjuvant, freund's incomplete adjuvant from Sigma, penicilin-Streptomycin-Amphotericin B from invitrogen; unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

The chicken complement receptor 2(ChCR2) monoclonal antibody hybridoma cell 3E9 disclosed by the invention has been preserved in the common microorganism center of China Committee for culture Collection of microorganisms (address: China academy of sciences, Japan, No.1, West Lu, No. 3, national institute of sciences, Japan, Taiyang, Beijing) in 10.01.2020, and the registration number is CGMCC No. 19286.