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Physarum polycephalum 14-3-3 protein monoclonal antibodies and preparation method thereof

A monoclonal antibody and protein technology, applied in the field of immunology, can solve problems that have not been seen yet, and achieve good results for commercial use

Inactive Publication Date: 2014-03-12
SHENZHEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there have been no reports on monoclonal antibodies against 14-3-3 protein P14-3-3 and monoclonal antibodies against yeast 14-3-3 proteins Bmh1 and Bmh2

Method used

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  • Physarum polycephalum 14-3-3 protein monoclonal antibodies and preparation method thereof
  • Physarum polycephalum 14-3-3 protein monoclonal antibodies and preparation method thereof
  • Physarum polycephalum 14-3-3 protein monoclonal antibodies and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: Related experimental techniques, reagents and biological materials involved in the present invention

[0050] 1. Cultivation of Physarum polycephalum and preparation of bacteria crushing solution:

[0051] Liquid culture of Chrysomyces polycereus: Chorpheomyces polycereus M3CVII strain (ATCC No. 204388) was donated by Professor Eggehard Holler, Institute of Biophysics, University of Regensburg, Germany. Add 25ml MSD-A solution (3.54g Citric acid, 0.06g FeCl 2 4H 2 O, 0.2g KH 2 PO 4 , 0.6g MgSO 4 ·7H 2 O, 0.084g MnCl 2 4H 2 O, 0.04g ZnSO 4 ·7H 2 O, 0.6g CaCl 2 2H 2 O, 10g Tryptone, 1.5g Yeastextract, 10g Glucose, adjust the pH to 4.6 with 30% KOH, distill the volume to 1L with distilled water, store at room temperature after autoclaving. Solid MSD containing 2% agar powder) and 0.25ml MSD-B solution (0.05g Hematin dissolved in 100ml 1% NaOH solution, stored at 4°C after autoclaving), insert appropriate amount of bacteria, 300rpm, dark culture at...

Embodiment 2

[0074] Example 2: Screening of antigenic determinant peptides and expression and purification of antigenic determinant peptide fusion proteins

[0075] The hydrophilicity, surface accessibility and antigenic index of the P14-3-3 amino acid sequence (its coding sequence GenBank registration number is EF140724) were analyzed with DNAStar biological software. Greater than zero, surface accessibility greater than 1) Select peptides that meet the requirements of the epitope, a total of 1-MTHDESREDS-10, 18-EQAERYDEMV-27, 28-EAMKRVAKLD-37, 35-ENSLIAYKSA-44, 70-EQKEENKGNE were selected -79, 109-QHLIVSSTTG-118, 237-DMQAGGDEPK-246 and 245-PKPVEDEAPA-254 and other 8 potential epitope peptides, and with the assistance of Abmart Shanghai Branch, the company's unique SEAL (Surface Epitope Antibody Library) technology expressed and purified these potential antigenic determinant peptides in Escherichia coli.

Embodiment 3

[0076] Example 3: Immune antigen (completed with the assistance of Abmart Shanghai Branch)

[0077] Take the purified P14-3-3 epitope polypeptide fusion protein solution and mix it with equal amounts of protein, dilute it with PBS solution to a protein mixture solution with a final concentration of 100 μg / ml, take the protein solution and mix it with an equal amount of Freund’s complete adjuvant After emulsification, 8-12-week-old female BALB / c mice were subcutaneously injected into the back at multiple points, and the immunization dose was 40 μg / mouse. After 2 weeks, the antigen solution was emulsified with the same amount of Freund's incomplete adjuvant for the second routine immunization; after another 3 weeks, the third routine immunization was carried out; after another 3 weeks, blood was collected from the tail of the mice, and indirect The antibody titer produced by the mice was detected by ELISA; one month later (the 3rd day before fusion), the mixed antigenic determin...

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Abstract

The invention discloses three physarum polycephalum 14-3-3 protein (P14-3-3) monoclonal antibodies, and the three monoclonal antibodies are mAb alpha+, mAb beta+ and mAb gamma+; the microbial preservation number of hybridomas cell strains McAb alpha+, McAb beta+ and McAb gamma+ which respectively express and secrete the above three monoclonal antibodies is CCTCC NO: C2012131, C2012132 and C2012133, and the preservation position is China Center for Type Culture Collection; and the purpose is to provide an effective means for researching the structure and the function of 14-3-3 protein and the P14-3-3 expression variation with cell cycle and life cycle. The prepared antibodies all have the titer higher than 100 K and the affinity constant higher than 2.12*10<8>, are applicable to research the structure and the function of low-grade eukaryote 14-3-3 protein and the 14-3-3 protein expression variation rule with cell cycle and life cycle, and have extremely good commercial use.

Description

technical field [0001] The invention belongs to the field of immunology, in particular to a hybridoma cell strain secreting anti-14-3-3 protein monoclonal antibody, the secreted 14-3-3 protein monoclonal antibody and application thereof. Background technique [0002] 14-3-3 protein (14-3-3s) is a molecular chaperone protein ubiquitous in eukaryotes, but it has not been found in prokaryotes [1] . Previous reports have shown that there are nine 14-3-3s subtypes (α, β, γ, δ, ε, η, ξ, σ, and τ) encoded by 7 genes in vertebrates, where α and σ are Phosphorylated forms of β and ξ [1] . Arabidopsis has 13 subtypes of 14-3-3, which are divided into ε (μ, ε, π, ι, and ο) and non-ε (κ, λ, ψ, ν, υ, ω, and χ) two families [2] , soybean has 18 14-3-3s subtypes (a, b, c, d, e, f, g, h, i, j, k, l, m, n, o, p, q and r), It is the largest 14-3-3 family ever discovered [3] . Yeast contains two 14-3-3 subtypes, Bmh1 and Bmh2, and the genes encoding these two proteins are called rad24...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/14G01N33/68C12R1/91
Inventor 邢苗刘士德张建华陈乃权罗慧琳
Owner SHENZHEN UNIV
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