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Specific monoclonal antibodies of coding regions and constant regions of goose immunoglobulin, and applications thereof

A technology of immunoglobulin and monoclonal antibody, applied in the constant region of υ chain and its monoclonal antibody, μ chain, preparation and detection or purification of goose immunoglobulin reagents, α chain, goose immunoglobulin λ chain, The field of goose immunoglobulin coding region and its monoclonal antibody can solve problems such as difficulties in goose basic immune research and achieve reliable results

Active Publication Date: 2014-07-09
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no other related reports at home and abroad, except for Lv Xuefeng (2011, CN 102161993A) who amplified the whole goose light chain Igλ gene by RACE method, which also provides a basis for the development of goose basic immunization. The research work brings certain difficulties

Method used

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  • Specific monoclonal antibodies of coding regions and constant regions of goose immunoglobulin, and applications thereof
  • Specific monoclonal antibodies of coding regions and constant regions of goose immunoglobulin, and applications thereof
  • Specific monoclonal antibodies of coding regions and constant regions of goose immunoglobulin, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1 Cloning of goose immunoglobulin λ chain, α chain, μ chain, υ chain and its constant region cDNA sequence

[0101] 1. Extraction of total RNA from goose spleen

[0102] Fresh goose spleen was taken, immediately ground in liquid nitrogen, and RNA was extracted using Trizol reagent (Invitrogen). The specific operation is as follows: add 1ml Trizol reagent per 100mg tissue, and place at room temperature for 10min; add 0.2ml chloroform for every 1ml TRIzol used, shake vigorously for 15 seconds, place at room temperature for 15min, and centrifuge at 12000rpm for 15min at 4°C; transfer the aqueous phase to a new tube, and use Precipitate the RNA in the aqueous phase with 2.5 times the volume of isopropanol at -20°C, then centrifuge at 12,000 rpm for 15 minutes at 4°C; wash the RNA pellet with 1ml of 75% ethanol (add at least 1ml of 75% ethanol for every 1ml of TRIzol used), at 4°C Centrifuge at 10,000 rpm for 5 minutes, discard the supernatant; place at room temper...

Embodiment 2

[0143] Embodiment 2 Preparation of anti-goose immunoglobulin constant region monoclonal antibody

[0144] 1. Preparation of immunogen

[0145] The specific operation of protein A affinity chromatography to purify goose serum immunoglobulin is as follows: take out the column filled with Protein A filler (GenScript, Nanjing), drain the liquid, and wash with deionized water for 2 to 3 times (the liquid added to the column is Filtered through a 0.45μm filter), and washed with Wash Buffer (0.15M NaCl, 20mM Na 2 HPO 4 , pH 8.0) equilibrate the column for 2-3 times; add serum diluted with 2 times of Wash Buffer, rotate the column continuously to fully combine the target protein with the filler, and collect the effluent; add 1ml of Wash Buffer, wash 7-8 times, and collect the effluent Add 1ml of Elution Buffer (citric acid 0.1M, pH 2.8), elute 7 to 8 times, collect the effluent, and immediately neutralize it to pH 7.4 with 1M Tris-base solution; SDS-PAGE detects the protein purifica...

Embodiment 4

[0159] Monoclonal antibody detection specific antibody after embodiment 4 enzyme labeling

[0160] HRP-labeled anti-goose immunoglobulin lambda chain constant region monoclonal antibody (secreted by hybridoma cell line 1B11) detection specific antibody:

[0161] Take 0.1mg BSA as immunogen to immunize adult geese by intramuscular injection, collect blood once every 4 days, separate serum, use conventional indirect ELISA method to detect specific antibodies produced in goose body, use BSA as antigen to coat microtiter plate, 5 % skim milk was used as blocking solution, 1:10 goose serum was used as primary antibody, and HRP-labeled 1B11 monoclonal antibody was used as secondary antibody to determine the growth and decline of specific antibodies in the body of goose immunized with BSA ( Figure 16 a). Similarly, the indirect ELISA method was used to measure goose serum immunized with gosling plague virus (GPV) virus-like particles (VLP) preserved in our laboratory, and the GPV v...

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Abstract

The present invention discloses specific monoclonal antibodies of coding regions and constant regions of goose immunoglobulin, and applications thereof. The coding regions of the goose immunoglobulin comprise all coding region sequence of a lambda chain, coding sequences of variable regions, a D fragment and a J fragment in a heavy chain, constant region coding sequences of an alpha chain, and parts of coding sequences of constant regions of a mu chain and a upsilon chain. The constant regions of the goose immunoglobulin of the present invention can be used for preparations of antibodies of constant regions of the lambda chain, the mu chain, the alpha chain and the upsilon chain of the goose immunoglobulin, and further can be used for identification of the variety and the type of the goose immunoglobulin binding with the anti-goose immunoglobulin monoclonal antibody. The present invention further provides a method for preparing the anti-goose immunoglobulin monoclonal antibody by adopting the goose serum immunoglobulin as the immunogen, the anti-goose immunoglobulin monoclonal antibody prepared by the method, and hybridoma cell strain secreting the monoclonal antibody, wherein the goose serum immunoglobulin is purified by adopting a Protein A affinity chromatography method. The anti-goose immunoglobulin constant region antibody of the present invention can be used for detection or purification of goose immunoglobulin.

Description

technical field [0001] The present invention relates to goose immunoglobulin coding region and its monoclonal antibody and application thereof, particularly relates to goose immunoglobulin λ chain, α chain, μ chain, υ chain constant region and its monoclonal antibody, and the monoclonal antibody in preparation Application in detecting or purifying goose immunoglobulin reagents. The invention belongs to the field of biotechnology. Background technique [0002] my country is a big poultry-raising country in the world, and it is also a major goose breeding and consumer country in the world. The sustainable, stable and healthy development of goose farming is not only directly related to the healthy and stable development of animal husbandry in the construction of new rural areas in our country, but also to people. lifestyle and health level. [0003] Immunoglobulins IgM, IgG, and IgA are important effector molecules of humoral immunity, and changes in their content can be used ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C12N15/13G01N33/68C07K16/42G01N33/577C12N5/20
Inventor 王君伟郭永丽高明春马波
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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