Ochratoxin A anti-idiotypic nano-antibody and preparation method thereof

A technology of ochratoxin and nano-antibody, which is applied in the field of molecular biology, can solve the problems of long production cycle, limited single output, small molecular weight of antibodies, etc., and achieve the effects of reducing production costs, large application value, and high storage capacity

Inactive Publication Date: 2019-03-29
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

[0005] Using polyclonal antibody technology to prepare anti-idiotypic antibody production technology is relatively simple, easy to operate, and low cost, but the later purification of serum is more cumbersome, and the single output is limited
However, the monoclonal antibody method has a long production cycle, complicated preparation process and high cost, and the cell fusion rate and positive rate are far lower than Ab1 class monoclonal antibodies. However, once this method is successful, the resulting hybridoma cell line can be Long-term stable storage enables the preparation of a large am

Method used

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  • Ochratoxin A anti-idiotypic nano-antibody and preparation method thereof
  • Ochratoxin A anti-idiotypic nano-antibody and preparation method thereof
  • Ochratoxin A anti-idiotypic nano-antibody and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Construction of Phage Display Nanobody Immune Library

[0027] 1. Alpaca immunity

[0028] Take 200 μg of anti-ochratoxin A monoclonal antibody (dissolved in PBS7.4), emulsify it with an equal volume of Freund’s incomplete adjuvant, and inject it into three-year-old male alpaca (Alpaca) subcutaneously at multiple points. Immunized once a week, a total of 8 times, blood testing began after the fourth immunization. Seven to 10 days after the fourth immunization, 10 mL of blood was collected from the jugular vein of an EDTA vacuum blood collection tube. At the same time, the blood collection tube was gently inverted to avoid blood coagulation, and then the blood was processed with the filter in the LeukoLOCK kit, and then sequentially washed with 3 mL of LPBS buffer and 3 mL of RNAlater Wash the filter with the buffer solution, and the required white blood cells will be trapped in the filter, and finally the filter should be sealed and stored at -80°C for RNA ex...

Embodiment 2

[0055] Example 2 Panning and identification of ochratoxin A anti-idiotype nanobody

[0056] (1) Panning of ochratoxin A anti-idiotypic nanobodies

[0057] Taking ochratoxin A monoclonal antibody as the target (the said ochratoxin A monoclonal antibody is secreted by the hybridoma cell line 1H2 with the preservation number CCTCC NO.C201329, and the hybridoma cell line 1H2 was released in March 2013 It was preserved in the China Center for Type Culture Collection (CCTCC) on the 7th, and has been disclosed in the patent "Hybridoma Cell Line 1H2, Anti-Ochratoxin A Monoclonal Antibody Produced by It and Its Application", application number: 201310115921.8), passed Reduce the concentration of the original coating and the concentration of ochratoxin A standard substance for competitive elution round by round, alternately use blocking reagents, and perform affinity enrichment panning to obtain antibodies against ochratoxin A monoclonal antibodies, namely anti-antibodies, and Known as...

Embodiment 3

[0082] Example 3 Expression and purification of ochratoxin A anti-idiotype nanobody

[0083] (1) Preparation of Top10F' competent cells:

[0084] (1) Pick an appropriate amount of Top10F' to streak on the LB-tetracycline plate, and culture overnight at 37°C;

[0085] (2) Pick the Top10F' monoclonal into 5mL LB medium, culture at 37°C, 250rpm until OD 600 0.6-0.8;

[0086] (3) Transfer the bacterial liquid to a pre-cooled centrifuge tube (about 1.3mL / tube), centrifuge at 4°C, 8000rpm, 8min;

[0087] (4) Put it on ice immediately after centrifugation, discard the supernatant, and add 1 mL of pre-cooled 0.1M CaCl 2 Resuspend the bacteria in the solution, pipette and mix well, and then ice-bath for 30 minutes;

[0088] (5) At 4°C, centrifuge at 8000rpm for 8min, put it back on ice quickly, suck up the liquid completely, and use 100μL pre-cooled 0.1MCaCl for precipitation 2 The solution is resuspended, which is Top10F'competent cells.

[0089] (2) Extraction and transformatio...

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Abstract

The invention belongs to the field of molecular biology, in particular to relates to an ochratoxin A anti-idiotypic nano-antibody and a preparation method thereof, wherein the amino acid sequence of ochratoxin A anti-idiotypic nano-antibody is shown in SED ID No:1. The antibody uses ochratoxin A monoclonal antibody to immunize alpaca, and constructs a bacteriophage display nanometer antibody library by extracting the total RNA of its white blood cells, and the library capacity is high and the diversity is good; taking ochratoxin A monoclonal antibody as the target, three round of affinity enrichment and eluting are used to reduce the coating concentration and competitive eluting concentration one by one, and the positive bacteriophage ochratoxin A anti-idiotypic nano-antibody gene is transformed into the expressed strain; after induction, purification and identification, the protein form nano-antibody VHH2-24. With excellent performance is obtained.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to an ochratoxin A anti-idiotype nanobody and a preparation method thereof. Background technique [0002] Ochratoxin A (ocbratoxinA, OTA) is a toxic fungal secondary metabolite, mainly produced by Aspergillus and Penicillium, which mainly pollutes food, vegetables, coffee, grapes, cocoa and other economic crops. The globalization of toxin-producing fungi and the continuous development of world trade in crop products have resulted in the widespread spread of ochratoxin A in the world, and has seriously endangered the health of animals and humans. It has strong nephrotoxicity and hepatotoxicity, and has teratogenicity, carcinogenicity, immunotoxicity and embryotoxicity. Many countries have set limit standards, so it is extremely important to establish a low-cost, fast and effective on-site detection method. [0003] At present, the methods used for the detection of ochrat...

Claims

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Application Information

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IPC IPC(8): C07K16/14C12N15/70
CPCC12N15/70C07K16/14
Inventor 李培武唐晓倩李慧姜俊张文张奇
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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