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Hybridoma cell line YQQD8 and general monoclonal antibodies produced by same to capsaicine, dihydrocapsaicin and nonivamide

A hybridoma cell line, a technology for synthesizing capsaicin, used in microorganism-based methods, microorganisms, biological testing, etc.

Active Publication Date: 2016-06-01
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few reports on antibodies against capsaicinoids at home and abroad, especially monoclonal antibodies to capsaicinoids.

Method used

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  • Hybridoma cell line YQQD8 and general monoclonal antibodies produced by same to capsaicine, dihydrocapsaicin and nonivamide
  • Hybridoma cell line YQQD8 and general monoclonal antibodies produced by same to capsaicine, dihydrocapsaicin and nonivamide
  • Hybridoma cell line YQQD8 and general monoclonal antibodies produced by same to capsaicine, dihydrocapsaicin and nonivamide

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The preparation of embodiment 1 capsaicinoid general artificial immune antigen

[0022] Preparation of general artificial hapten of capsaicinoids:

[0023]Weigh 0.28 g of vanillin amine hydrochloride and dissolve it in 6 ml of tetrahydrofuran, add 0.15 g of triethylamine dropwise with stirring, and stir at room temperature for 30 min. Accurately weigh 0.15 g (0.0015 mol) of succinic anhydride and add to the above reaction solution, and stir overnight at room temperature. Add 3 mL of ethyl acetate to the reaction solution and stir at room temperature for 2 min. After filtration, the resulting precipitate is the hapten 4-[(4-hydroxy-3-methoxy)benzylamino]-4-carbonylcarboxylic acid (4-[ (4-hydroxy-3-methoxybenzyl)amino]-4-oxobutanoic acid), the molecular formula is C 12 h 15 NO 5 .

[0024] The results of NMR identification are: 1 HNMR(400MHz,DMSO)δ12.17(s,1H),8.87(s,1H),8.31(d,J=6.0Hz,1H),4.21(d,J=5.8Hz,2H),3.80(s, 3H), 2.52(t, J=6.9Hz, 2H), 2.42(t, J=6.7Hz, 2H). ...

Embodiment 2

[0032] Embodiment 2: Preparation of animal immunization and hybridoma cell line YQQD8

[0033] 1. Animal immunization

[0034] Five 7-week-old BALB / c mice were purchased and immunized with the above-mentioned capsaicin-like artificial antigen-immune antigen. For the first immunization, the immune antigen was quickly mixed with an equal volume of Quick Antibody-Mouse5W (purchased from Beijing Boaolong Biotechnology Co., Ltd.), and then injected into the calf muscle of the mouse's hind leg to immunize the mouse. On the 21st day and 42nd day, the immunization was boosted in the same way. The dose of immune antigen used in the three immunizations was the same, 12.5 μg per mouse. Eight days after each immunization, blood was collected from the tail vein, serum was separated, and the serum titer of mice was monitored by indirect ELISA method. Eight days after the third immunization, blood was collected from the tail vein, and the serum was separated. The titer of the mouse serum ...

Embodiment 3

[0039] Embodiment 3: anti-capsaicin, dihydrocapsaicin, synthetic capsaicin universal monoclonal antibody hybridoma cell line YQQD8 antibody variable region sequence determination

[0040] (1) Extraction of total RNA: use the total RNA extraction kit of Tiangen Company and follow the instructions to extract the total RNA that can produce the hybridoma cell line YQQD8;

[0041] (2) Synthesis of cDNA: use the total RNA obtained in step 1 as a template, and oligo(dT)15 as a primer, perform reverse transcription according to the instructions of SuperScriptTM-2II reverse transcriptase, and synthesize the first strand of cDNA; primer oligo(dT)15 Purchased from Invitrogen;

[0042] (3) Cloning of variable region genes by PCR method: design primers according to the conserved sites of mouse antibody gene sequences in GENEBANK, and use cDNA as a template to amplify antibody light and heavy chain variable region genes. The PCR program was: 94°C for 30s, 55°C for 45s, 72°C for 1min, 30 cy...

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Abstract

The invention relates to a hybridoma cell line YQQD8 and general monoclonal antibodies produced by the same to capsaicine, dihydrocapsaicin and nonivamide. With the collection number CCTCC NO.C201534, the cell line YQQD8 has been collected in China Center for Type Culture Collection (CCTCC) in Wuhan University in Wuhan city of China on April 23th, 2015 and is classified and named as the hybridoma cell line YQQD8. Titer of mouse hydroperitoneum antibody of YQQD8, tested by a conventional non-competitive enzyme linked immunosorbent assay (ELISA) method, can be up to 2.56*106. The general monoclonal antibodies to capsaicine, dihydrocapsaicin and nonivamide, produced by the YQQD8, can be applied to identify capsaicine, dihydrocapsaicin and nonivamide effectively; 50% inhibition concentrations (IC50) of the YQQD8 to capsaicine, dihydrocapsaicin and nonivamide are sequentially 8.5ng / mL, 5.0ng / mL and 13.5ng / mL respectively; and cross reactivity to capsaicine, dihydrocapsaicin and nonivamide ranges from 62.9% to 170%. The hybridoma cell line YQQD8 can be applied to quantitative measurement of total content of capsaicine, dihydrocapsaicin and nonivamide.

Description

technical field [0001] The invention relates to a hybridoma cell line YQQD8 and the universal monoclonal antibody against capsaicin, dihydrocapsaicin and synthetic capsaicin produced by the hybridoma cell line YQQD8. Background technique [0002] Capsaicin compounds are the main chemical substances that make peppers pungent, and their components mainly include capsaicin, dihydrocapsaicin, homodihydrocapsaicin, nordihydrocapsaicin, homocapsaicin and other compounds. Wherein capsaicin and dihydrocapsaicin account for about 90% of the total amount of capsaicinoids, and provide about 90% of the hot stimulation. In addition to giving food a spicy taste and being used as a condiment, capsaicinoids also have anti-inflammatory and analgesic, anti-bacterial, anti-tumor effects, can promote fat burning, lower blood lipids, inhibit gastric acid secretion, and have protective effects on gastric mucosal damage. Use value. In the field of pesticides, capsaicinoids are considered to be i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/44G01N33/68G01N33/577C12R1/91
Inventor 李培武杨青青张奇马飞张文柯艳妮张良晓丁晓霞张兆威
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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