Anti-goat IgM (Immunoglobulin M) [mu] chain monoclonal antibody, hybridoma cell strain secreting antibody and purpose thereof

A hybridoma cell line and monoclonal antibody technology, which is applied in the field of anti-goat IgMμ chain monoclonal antibody and hybridoma cell line, can solve the problems of difficult to purchase IgMμ chain, cumbersome methods, low recovery rate, etc., and achieve a wide range of applications and The effect of social needs

Active Publication Date: 2017-09-19
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

IgMμ chains of different species are not easily available in the market
When preparing its monoclonal antibody, SDS-PAGE is generally used to cut the gel to obtain the μ chain for immunization. The method is cumbersome and the recovery rate is not high.

Method used

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  • Anti-goat IgM (Immunoglobulin M) [mu] chain monoclonal antibody, hybridoma cell strain secreting antibody and purpose thereof
  • Anti-goat IgM (Immunoglobulin M) [mu] chain monoclonal antibody, hybridoma cell strain secreting antibody and purpose thereof
  • Anti-goat IgM (Immunoglobulin M) [mu] chain monoclonal antibody, hybridoma cell strain secreting antibody and purpose thereof

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Experimental program
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preparation example Construction

[0022] The preparation of embodiment 1 sheep IgM recombinant protein

[0023] 1. Expression of sheep IgM recombinant protein (tH protein)

[0024] 1. Primer design

[0025] Referring to the GenBank secreted IgM precursor sequence (Accession No.: X59994.1), synthetic primers were designed.

[0026] Upstream primer: 5'- GAATTC ATGCAGGTGCAGCTGCAGGAGT-3'

[0027] Downstream primer: 5'- CTCGAG TCAGTAGCAGGTGCTGGCCGT-3'

[0028] Wherein, the underlined parts are the restriction sites of EcoR I and Xho I respectively.

[0029] 2. RT-PCR amplification

[0030] (1) Separation of lymphocytes: Aseptically take 10 mL of healthy goat anticoagulant blood and mix and dilute with the same amount of PBS, take 5 mL of diluted peripheral blood and slowly add the same amount of lymphocyte separation medium for density gradient centrifugation, horizontal centrifuge at 2300r / min at room temperature Centrifuge for 15 minutes; absorb the white lymphocyte layer in the middle, add PBS to wash, ...

Embodiment 2

[0068] The construction of embodiment 2 hybridoma cell lines

[0069] 1 material

[0070] 1.1 Antigen, cell line, animal

[0071] Antigen: the IgM recombinant protein prepared in Example 1;

[0072] Mouse myeloma cells (Sp2 / 0) cells: preserved by Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences;

[0073] BALB / c mice: provided by the Experimental Animal Factory of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0074] 1.2 Main reagents

[0075] RPMI1640 culture medium, fetal bovine serum (FBS) were purchased from GIBCO; 8-azaguanine, HT, HAT, PEG, Freund's complete adjuvant, Freund's incomplete adjuvant and blocking solution (Blocking buffer) were purchased from Sigma company; 96-well cell plate and enzyme label plate were purchased from Costar Company; horseradish peroxidase-labeled donkey anti-mouse IgG was purchased from Wuhan Sanying Biotechnology Co., Ltd.; enzyme label substrate TMB was purchased from P...

Embodiment 3

[0123] The preparation of embodiment 3 monoclonal antibody

[0124] 1 material

[0125] 1.1 Cell lines, animals

[0126] Cell lines: hybridoma cell lines 2D11, 3E4 and 5D1 prepared in Example 2;

[0127] BALB / c mice: provided by the Experimental Animal Factory of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0128] 1.2 Main reagents

[0129] RPMI1640 culture medium and fetal bovine serum (FBS) were purchased from GIBCO Company; Blocking buffer was purchased from Sigma Company; 96-well microtiter plate was purchased from Costar Company; horseradish peroxidase-labeled donkey anti-mouse IgG was purchased from Wuhan Sanying Biotechnology Co., Ltd.; the enzyme-labeled substrate TMB was purchased from Promega; Tween-20 was purchased from Solarbio; other reagents were domestic analytical grade.

[0130] 1.3 Solution and culture medium

[0131] Coating solution (0.05mol / L sodium carbonate-sodium bicarbonate buffer, pH9.6): NaHCO3 2.9g, Na 2 C...

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Abstract

The invention discloses an anti-goat IgM (Immunoglobulin M) [mu] chain monoclonal antibody, a hybridoma cell strain secreting the antibody and a purpose thereof. According to the anti-goat IgM (Immunoglobulin M) [mu] chain monoclonal antibody, secretory IgM protein is used as target protein; a complete open reading frame of a gene of an IgM [mu] chain is obtained through cloning; the open reading frame is sub-cloned to a prokaryotic expression vector; recombinant protein is purified by utilizing a nickel column; a female BALB/c mouse at an age of 6 to 8 weeks is immunized; when the titer of an antibody is 1 to 10,000 or above, the single-cell suspension of the splenocyte of the immunized mouse and a myeloma cell of a logarithmic growth phase are subjected to melt; the hybridoma cell strain secreting the anti-goat IgM [mu] chain monoclonal antibody is obtained by sieving; a hybridoma cell is injected into the abdominal cavity of a parous BALB/c mouse at the age of 6 to 8 weeks; abdominal dropsy is collected when the injection is carried out for approximately 7 days; the abdominal dropsy is purified by protein G/A agarose; a higher-purity anti-goat IgM [mu] chain monoclonal antibody is obtained. The monoclonal antibody provided by the invention is high in valence, can be used for detecting goat IgM, moreover, is hopeful for realizing the early screening and monitoring on multiple goat diseases, and has wide application range and social demand.

Description

technical field [0001] The invention relates to an anti-goat IgMμ chain monoclonal antibody, and also relates to a hybridoma cell line secreting the antibody and its application, belonging to the field of biotechnology. Background technique [0002] IgM is one of the immunoglobulins. Secretory IgM is a pentamer and is the Ig with the largest molecular weight (about 900KD). It mainly exists in the blood, accounting for 5% to 10% of the total serum immunoglobulins. IgM is the earliest antibody that appears in the humoral immune response after the initial stimulation of the body by an antigen, and is the "vanguard" of the body's anti-infection; it has strong cytotoxic activity and cytolytic activity, and is the first-line antibody against intravascular infection. IgM Antibodies are generally protective antibodies, which play a major role in preventing bacteremia. [0003] In biological evolution, IgM is also the earliest Ig. In late embryonic development, the fetus begins to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/42G01N33/577C12R1/91
CPCC07K16/4283
Inventor 蒙学莲朱学亮窦永喜才学鹏张志东
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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