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Chicken interleukin-10 monoclonal antibodies and preparation method and application thereof

A technology of monoclonal antibody and interleukin, which is applied in hybridoma cell lines, the preparation and application of chicken interleukin-10 monoclonal antibody, and the application field of detection or purification of chicken interleukin-10 protein, which can solve the problem of poor specificity of monoclonal antibody, Problems such as restricting production and application and low potency

Inactive Publication Date: 2015-04-29
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, hybridoma cell lines used to secrete chicken interleukin-10 monoclonal antibody have defects such as low titer and poor stability, and the specificity of the secreted monoclonal antibody is poor, which seriously restricts production and application, and needs to be improved urgently

Method used

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  • Chicken interleukin-10 monoclonal antibodies and preparation method and application thereof
  • Chicken interleukin-10 monoclonal antibodies and preparation method and application thereof
  • Chicken interleukin-10 monoclonal antibodies and preparation method and application thereof

Examples

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experiment example 1

[0024] Experimental example 1 Preparation and identification of chicken interleukin-10 monoclonal antibody

[0025] 1. Experimental method

[0026] 1.1 Immunization of mice

[0027] (1) Prepare SDS-PAGE strips containing ChIL-10 recombinant protein. The specific operation is as follows: the ChIL-10 gene was digested with the prokaryotic expression vector pET-30a, ligated, transfected into Escherichia coli BL21 (DE3), and harvested after 5 hours of IPTG induction with a final concentration of 0.1mmol / L. After sonication, SDS-PAGE electrophoresis was performed. Accurately cut out the gel where the target band is located, which is the above-mentioned ChIL-10 recombinant protein strip

[0028] (2) Before immunization, add a small amount of PBS and grind it with a grinding rod, and add an appropriate amount of double antibody solution, preferably the colloidal particles can pass through the needle of a 1mL syringe, that is, the immunogen;

[0029] (3) Select 5 clean-grade BALB / ...

experiment example 2

[0107] Experimental Example 2 Using the monoclonal antibody 2F6 of the present invention to detect IL-10 protein in chicken peripheral blood lymphocytes stimulated by LPS

[0108] 1. experimental method

[0109] Isolate B19 and B21 haplotype chicken peripheral blood lymphocytes, culture in DMEM medium containing 10% FBS for 2 hours, add LPS at a concentration of 30 μg / mL to stimulate for 36 hours, extract protein, use 2F6 cell culture medium as primary antibody, Take IRDye TM 700DX-labeled goat anti-mouse IgG (1:4000 dilution) was used as the secondary antibody for western blot reaction.

[0110] 2. Experimental results

[0111] See the experimental results Figure 4 . A 17kDa protein was detected in peripheral blood lymphocytes of B19 and B21 haplotype chickens stimulated with LPS for 36 hours, which was consistent with the size of natural ChIL-10 protein.

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Abstract

The invention discloses chicken interleukin-10 (ChIL-10) monoclonal antibodies and a preparation method and application thereof. The preparation method comprises the following steps: taking prokaryotically expressed recombinant protein ChIL-10 as an immunogen and eukaryotic cell line-expressed ChIL-10 protein as a screening antigen, carrying out cell fusion, and screening hybridoma cells by an IFA (incomplete Freund's adjuvant) to obtain three cell strains for secreting the chicken interleukin-10 monoclonal antibodies, wherein the monoclonal antibodies secreted by the hybridoma cell strains 2F6 are highest in in titer, stability and specificity, and have a microbiological collection number of CGMCC (China General Microbiological Culture Collection Center) NO.7657. The invention further provides the application of the hybridoma cell strains 2F6 and the monoclonal antibodies secreted by the hybridoma cell strains 2F6 to detection or purification of the chicken interleukin-10 protein.

Description

technical field [0001] The present invention relates to a monoclonal antibody, in particular to a chicken interleukin-10 monoclonal antibody and a hybridoma cell line secreting the monoclonal antibody. The present invention further relates to the application of the monoclonal antibody in detecting or purifying chicken interleukin-10 protein, belonging to Preparation and application field of chicken interleukin-10 monoclonal antibody. Background technique [0002] Interleukin, referred to as interleukin, is a cytokine produced by white blood cells that mediates intercellular interactions and plays a regulatory role in cell activation, proliferation and differentiation. With the development of molecular biology, research on chicken interleukin genes and functions has gradually become a hot spot. A variety of interleukins have been isolated from chickens, which can be used in immunotherapeutic agents, immune enhancers, and construction of new genetic engineering vaccines. Inte...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/24G01N33/68G01N33/577C07K14/54C07K1/22
Inventor 韩凌霞廉传江文辉强司昌德林欢陈洪岩
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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