Monoclonal antibody for paralichthys olivaceus resistant T cell surface marker molecule CD4-1 as well as preparation method and application thereof

A monoclonal antibody, cell surface technology, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, microorganism-based methods, biochemical equipment and methods, etc., to achieve novel design effects

Active Publication Date: 2018-07-31
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of favorable tools to label CD4 + T lymphocytes, from protein level to fish CD4 + Classification and Identification of T Lymphocytes and About Fish CD4 + The immunological functions of T cells are rarely reported

Method used

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  • Monoclonal antibody for paralichthys olivaceus resistant T cell surface marker molecule CD4-1 as well as preparation method and application thereof
  • Monoclonal antibody for paralichthys olivaceus resistant T cell surface marker molecule CD4-1 as well as preparation method and application thereof
  • Monoclonal antibody for paralichthys olivaceus resistant T cell surface marker molecule CD4-1 as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Preparation of CD4-1 antigenic peptide and monoclonal antibody of flounder T cell surface marker

[0040] 1. Preparation of Antigenic Peptides

[0041] (1) The amino acid sequence of flounder CD4-1 was downloaded from NCBI GenBank database, the transmembrane sequence of flounder CD4-1 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 CD4-1 protein. The results showed that the transmembrane region of flounder CD4-1 was in the 415-437 region, in which the 1-414 region was the extracellular region, and the 438-464 region was the intracellular region ( figure 1 A). There are four extracellular Ig-like domains in flounder CD4-1 molecule, of which amino acids 19-118, 200-320 are variable domains, and amino acids 122-193, 325-408 are constant...

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 CD4-1 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 for a paralichthys olivaceus resistant T cell surface marker molecule CD4-1. The monoclonal antibody is named as a hybridoma cell JF-CD4-1-2A10, the preservation number is CCTCC NO: C201879, the preservation organization is China Center For Type Culture Collection, the address is Wuhan university of Wuhan city in Hubei province, and the preservation date is March 29th, 2018, and is secreted by the hybridoma cell. An indirect enzyme-linked immunosorbent assay reaction experiment on the monoclonal antibody shows that the monoclonal antibody can be specially combined with paralichthys olivaceus CD4-1 polypeptide, indirect immune fluorescence and flow cytometry prove that the monoclonal antibody can specially identify the paralichthys olivaceus lymphatic cell surface CD4-1 molecule, and an immunoblotting result shows that the monoclonal antibody disclosed by the invention can specially identify paralichthys olivaceus CD4-1 recombinant protein and natural paralichthys olivaceus CD4-1 protein of which the molecular weight is 52 kDa. According to the monoclonal antibody disclosed by the invention, the monoclonal antibody for the paralichthys olivaceus resistant T cell surface marker molecule CD4-1 is prepared, and an important tool is provided for detecting paralichthys olivaceus CD4-1<+T> lymphatic cells.

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 CD4-1 and the preparation method thereof belong to the technical field of fish molecular immunology. Background technique [0002] CD4 molecule is a single-chain transmembrane glycoprotein, which is a member of the Ig superfamily and is mainly expressed on the surface of mature T lymphocytes. CD4 + Cells, as an important class of T lymphocytes, participate in humoral immunity and cellular immunity, and play a vital role in resisting pathogen invasion and other related immune defenses. CD4 + T cell TCR recognizes exogenous antigenic peptide-MHC on the surface of APC After the molecule-like complex is activated, Th cells produce a variety of cytokines, chemotaxis and activate other immune cells to clear pathogens. The CD4 molecule is also a receptor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N5/20G01N33/68C12R1/91
CPCC07K16/2812G01N33/56972G01N33/6872G01N2333/70514
Inventor 邢婧田洪飞战文斌唐小千绳秀珍
Owner OCEAN UNIV OF CHINA
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