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Monoclonal antibody capable of resisting surface marker molecule CD4-2 of T cell of paralichthys olivaceus as well as preparation method and application of monoclonal antibody

A monoclonal antibody and cell surface technology, applied in anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, chemical instruments and methods, anti-animal/human immunoglobulin, etc., can solve fish T Problems such as cell grouping cannot be clearly defined to achieve a novel design effect

Active Publication Date: 2018-09-04
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the lack of detection antibodies for specific molecular markers of lymphocytes, the classification of fish T cells cannot be clearly defined, so the research on the functions and regulatory mechanisms of T lymphocytes and their subsets is only in its infancy

Method used

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  • Monoclonal antibody capable of resisting surface marker molecule CD4-2 of T cell of paralichthys olivaceus as well as preparation method and application of monoclonal antibody
  • Monoclonal antibody capable of resisting surface marker molecule CD4-2 of T cell of paralichthys olivaceus as well as preparation method and application of monoclonal antibody
  • Monoclonal antibody capable of resisting surface marker molecule CD4-2 of T cell of paralichthys olivaceus as well as preparation method and application of monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Preparation of CD4-2 Antigen Peptide, a Marker Molecule on the Surface of Flounder T Cells, and Development of Monoclonal Antibody

[0040] 1. Preparation of Antigenic Peptides

[0041] (1) The amino acid sequence of flounder CD4-2 was downloaded from NCBI GenBank database, the transmembrane sequence of flounder CD4-2 was analyzed by TMHMM software, and its structural domain was analyzed by SMART software. The purpose of predicting the transmembrane region and domain is to ensure that the designed antigenic peptide is located on the extracellular region and Ig-like domain of the flounder CD4-2 protein. The results showed that the transmembrane region of flounder CD4-2 was in the 3-22, 222-244 region, of which the 23-221 region was the extracellular region, and the 1-2, 245-302 region was the intracellular region ( figure 1 A). There are two extracellular Ig-like domains in the CD4-2 molecule of flounder, in which amino acids 18-107 are variable domains, an...

Embodiment 2

[0082] Example 2: Indirect enzyme-linked immunoassay identification of monoclonal antibody of the present invention

[0083] (1) Coating antigen: Dilute the unconjugated KLH CD4-2 polypeptide with carbonate coating solution (pH 9.6) to a concentration of 50µg / ml, add it to a 96-well microtiter plate (100µl / well), 4 ℃ coated overnight;

[0084] (2) Aspirate the coating solution, wash with PBST, 5min each time, three times;

[0085] (3) Add 200µl 3% bovine serum albumin (in PBS) to each well and block at 37°C for 1 hour;

[0086] (4) Wash three times with the method (2);

[0087] (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 at 37°C for 1 hour;

[0088] (6) Wash three times with method (2);

[0089] (7) Add alkaline phosphatase-labeled goat anti-mouse Ig (diluted 1:4000) as the secondary antibody to the microtiter plate at 50 µl per well, and incubate at 37°C fo...

Embodiment 3

[0093] Example 3: Identification of monoclonal antibodies of the present invention by indirect immunofluorescence method

[0094] (1) Percoll discontinuous density gradient centrifugation of flounder peripheral blood leukocytes was resuspended in 0.01M PBS, and the concentration was adjusted to 1×10 7 pieces / ml.

[0095] (2) Drop the blood cell suspension on a clean glass slide, 10 µl per drop, settle in a wet box at room temperature for 1 hour, take it out and fix it in acetone for 20 minutes, take it out and air dry.

[0096] (3) Use the hybridoma supernatant (hybridoma culture medium) as the primary antibody, and the negative control as the myeloma cell supernatant, add it to the blood cell sample on the glass slide, and incubate in a 37°C humid box for 45 minutes.

[0097] (4) Take out the slide and wash it three times with 0.01M PBS for 5 minutes each time.

[0098] (5) Use fluorescein isothiocyanate-labeled goat anti-mouse antibody as the secondary antibody, add it to ...

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Abstract

The invention discloses a monoclonal antibody capable of resisting a surface marker molecule CD4-2 of a T cell of paralichthys olivaceus. The monoclonal antibody is secreted by a hybridoma cell whichis named by hybridoma cell JF-CD4-2-1D8 and collected in the China Center for Type Culture Collection, with the collection number of CCTCC NO: C201880, in Wuhan University in Wuhan, Hubei, on March 29. 2018. An experimental result of indirect enzyme-linked immunosorbent assay of the monoclonal antibody shows that the monoclonal antibody can be specifically combined with CD4-2 polypeptide; an immunoblotting result shows that the monoclonal antibody disclosed by the invention can specifically identify the paralichthys olivaceus CD4-2 recombinant protein with the molecular weight of 25kDa and theparalichthys olivaceus natural CD4-2 protein with the molecular weight of about 33 kDa; indirect immunofluorence and flow cytometry show that the monoclonal antibody can specifically identify CD4-2 molecules on the surface of lymphocyte of the paralichthys olivaceus. As the monoclonal antibody capable of resisting the surface marker molecule CD4-2 of the T cell of the paralichthys olivaceus, an important tool is provided for detection of paralichthys olivaceus CD4-2+T lymphocyte; the monoclonal antibody has an important practical significance for deep understanding of immunologic mechanisms of fish cells and development of an efficient and environment-friendly fish vaccine.

Description

technical field [0001] The invention relates to a monoclonal antibody and a preparation method thereof, in particular to an anti-flounder ( Paralichthys olivaceus ) The monoclonal antibody of T cell surface marker molecule CD4-2 and its preparation method belong to the technical field of fish molecular immunology. Background technique [0002] The CD4 molecule is a single-chain transmembrane glycoprotein, which belongs to the membrane-anchored protein of the immunoglobulin family. It is mainly expressed on the surface of mature T lymphocytes, and also has a small amount of expression on monocytes-macrophages and nerve cells. As a co-receptor of T lymphocytes, CD4 can improve the stability of TCR / antigen-MHC complex, regulate the differentiation and apoptosis of lymphocytes, and act as a receptor for IL-16 to regulate CD4 + Growth and chemotaxis of T cells. CD4 + Cells, as an important class of T lymphocytes, participate in humoral immunity and cellular immunity, and p...

Claims

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Application Information

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IPC IPC(8): C07K16/28G01N33/577G01N33/569
CPCC07K16/2812C07K2317/33C07K2317/34G01N33/56966G01N33/577G01N2333/70514G01N2469/10
Inventor 邢婧田洪飞战文斌唐小千
Owner OCEAN UNIV OF CHINA
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