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
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[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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