Anti-paralichthys olivaceus immunoglobulin D monoclonal antibody as well as application and preparation method thereof

A monoclonal antibody and immunoglobulin technology, applied in the direction of immunoglobulin, antibodies, chemical instruments and methods, etc., can solve the problems of IgD deficiency, low content, limited to gene detection level, etc., and achieve the effect of novel design

Active Publication Date: 2015-01-28
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, IgD with monomeric structure is regarded as the most mysterious immunoglobulin. Its content in the body is very low, and antibody activity is difficult to be detected. Its gene and protein structure vary greatly among different species. Compared with other As far as several immunoglobulins are concerned, there is a lack of research on their immunological functional properties
[0003] In recent years, studies have shown that IgD may play an important role in fish system immune surveillance and immune response regulation, but due to the lack of powerful detection tools for IgD, most of the research is limited to the level of gene detection. The current research on IgD at the protein level is very lacking

Method used

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  • Anti-paralichthys olivaceus immunoglobulin D monoclonal antibody as well as application and preparation method thereof
  • Anti-paralichthys olivaceus immunoglobulin D monoclonal antibody as well as application and preparation method thereof
  • Anti-paralichthys olivaceus immunoglobulin D monoclonal antibody as well as application and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Construction of a plasmid expressing the IgD heavy chain δ1-δ4 region of flounder and protein expression

[0025] (1) Peripheral blood cells of flounder were extracted with anticoagulant (6.7 mg heparin sodium and 0.5 g BSA per 50 ml of single cell suspension RPMI-1640) at a ratio of 1:1, and the percoll discontinuous density gradient centrifugation was used to obtain the flounder Peripheral blood leukocytes, the total RNA of leukocytes was extracted by Trizol method.

[0026] (2) According to the principle of primer design and the published full-length sequence of a flounder IgD heavy chain coding gene (GeneBank: AB052658), using the primer design software Primer5.0, combined with the characteristics of the multiple cloning site of the pET28(a) plasmid, choose Nhe I and Hind The restriction site of III was used as the insertion position of the target gene, and the expression primers for the δ1-δ4 region of the IgD heavy chain of flounder were designed:...

Embodiment 2

[0035] Example 2: Preparation of mouse anti-flounder IgD heavy chain δ1-δ4 region monoclonal antibody

[0036] 1. Immunity

[0037] Purified recombinant flounder IgD heavy chain protein was used as antigen. The dose of each immunization is 0.1ml, and the immunization is divided into 4 times. The interval between the first 2 immunizations is 2 weeks, and the interval between the next 3 immunizations is 1 week. The first two times are intraperitoneal injections, and the last two times are tail vein injections:

[0038] (1) Basic immunization: the purified recombinant protein is mixed with Freund's complete adjuvant in equal volume (V / V) as the antigen;

[0039] (2) Booster immunization: the purified recombinant protein was mixed with the same amount (V / V) of Freund's incomplete adjuvant as the antigen;

[0040] (3) Second booster immunization: purified recombinant flounder IgD heavy chain protein as antigen;

[0041] (4) Expansion immunization three days before fusion: puri...

Embodiment 3

[0072] Example 3: Indirect enzyme-linked immunoassay identification of the monoclonal antibody of the present invention:

[0073] (1) Coating antigen: Dilute the purified recombinant flounder IgD heavy chain δ1-δ4 region protein with carbonate coating solution (pH 9.6) 1:10, add it to a 96-well microtiter plate (50 μl / well), Coating overnight at 4°C;

[0074] (2) Aspirate the coating solution, wash with PBST, 5min each time, three times;

[0075] (3) Add 200 μl 3% bovine serum albumin (with PBS) to each well and block at 37°C for 1 hour;

[0076] (4) Wash three times with the method (2);

[0077] (5) Add the culture supernatant of the hybridoma cells screened and cloned above as the primary antibody to the microtiter plate at 50 μl per well, and incubate in a 37°C incubator for 1 hour;

[0078] (6) Wash three times with method (2);

[0079] (7) Add alkaline phosphatase-labeled goat anti-mouse Ig (diluted 1:4000) as the secondary antibody to the microtiter plate at 50 μl pe...

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Abstract

The invention discloses an anti-paralichthys olivaceus immunoglobulin D monoclonal antibody. The anti-paralichthys olivaceus immunoglobulin D monoclonal antibody is secreted by hybridoma cells of a hybridoma cell strain JF-IgD, with the collection No.: CCTCCNO: C2014159, the collection unit: China Center for Type Culture Collection, the collection address: Wuhan University in Wuhan, Hubei of China, and the collection date: September 2, 2014. Indirect enzyme-linked immune reaction experiment results show that the anti-paralichthys olivaceus immunoglobulin D monoclonal antibody can be specifically bound with recombinant flounder IgD heavy chain proteins; immunoblotting results show that the anti-paralichthys olivaceus immunoglobulin D monoclonal antibody can specifically recognize recombinant protein encoded by flounder IgD heavy chain genes delta1-delta 4 and flounder natural IgD heavy chain proteins with the molecular weight of 120kDa. The anti-paralichthys olivaceus IgD monoclonal antibody is prepared, and an important tool is provided for detecting flounder IgD protein molecules and IgD surface positive (IgD +) lymphocytes.

Description

technical field [0001] The present invention relates to a preparation method of a monoclonal antibody, in particular to an anti-flounder ( Paralichthys olivaceus ) The monoclonal antibody of immunoglobulin D (IgD) and its preparation method belong to the technical field of fish molecular immunology. Background technique [0002] Immunoglobulin (Ig) is an effector response molecule secreted by vertebrate B lymphocytes after antigen stimulation, activation, proliferation and maturation, and plays an important role in humoral immune defense. Mammalian immunoglobulin heavy chains can be divided into five types: γ, α, ε, μ, and δ according to their constant region amino acid composition and antigenicity differences, which respectively constitute IgG, IgA, IgE, IgM, and IgD. Compared with mammals, fish immunoglobulin research started late, and people believed that fish only had IgM immunoglobulin for a long time. With the development of molecular biology research, in recent years...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/42G01N33/68G01N33/577A61K39/395A61P37/02
Inventor 唐小千战文斌绳秀珍邢婧
Owner OCEAN UNIV OF CHINA
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