Nano-antibody of carbofuran pesticide and preparation method and application of nano-antibody

A nanobody and detection method technology, applied in the biological field, can solve the problems of poor stability and easy inactivation of antibodies, and achieve the effects of good stability, high affinity and good effect

Active Publication Date: 2019-11-05
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although antibody-based immunoassay methods have the advantages of fast, sensitive, and high-throughput, antibodies often have poor stability and are prone to inactivation under extreme conditions.

Method used

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  • Nano-antibody of carbofuran pesticide and preparation method and application of nano-antibody
  • Nano-antibody of carbofuran pesticide and preparation method and application of nano-antibody
  • Nano-antibody of carbofuran pesticide and preparation method and application of nano-antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Construction of Anti-Carbofuran Pesticide Nanobody Immune Library

[0056]1. Experimental method

[0057] 1. Preparation of complete antigens BFNB-OVA and BFNB-KLH

[0058] Carbofuran pesticide hapten BFNB was coupled with ovalbumin OVA (albumin) and keyhole limpet heocyanin KLH (keyhole limpet heocyanin) to prepare complete antigens BFNB-OVA and BFNB-KLH.

[0059] The chemical formula of the carbofuran pesticide hapten BFNB is:

[0060]

[0061] 2. Immune Bactrian camels

[0062] Take 500 μg of BFNB-KLH emulsified with an equal volume of Freund's complete adjuvant, and perform multi-point immunization injections subcutaneously on the neck of Bactrian camels. Immunization was boosted every 2 weeks, and each time 500 μg of BFNB-KLH was emulsified with an equal volume of Freund's incomplete adjuvant for immunization, and blood was collected one week after each immunization. Serum titers were determined by an indirect competitive ELISA method.

[0063] 3....

Embodiment 2

[0080] Example 2 Construction of Anti-Carbofuran Pesticide Nanobody Immune Library

[0081] 1. Experimental method

[0082] 1. Use BFNB-OVA as the coating antigen, 100 μl per well of the microtiter plate, and incubate overnight in a 37°C water bath. The coating concentration gradient is 10, 5, 1, 0.2 μg / ml. After 12 hours, wash the plate twice with PBST, add 120 μl of 1% BSA to the wells of 10 and 1 μg / ml, add 120 μl of 1% fish collagen to the wells of 5 and 0.2 μg / ml, block for 3 hours, and dry at 37°C for later use .

[0083] 2. Take 100 μl phage library (about 10 11 pfu), add 2% BSA, OVA and KLH carrier protein respectively, and incubate at 37° C. for 1 h to remove the white antibody on the non-specific adsorption carrier. Then transfer to coated antigen wells, shake at room temperature for 1 hour, suck out unbound phage, wash the plate with PBST 5 times, 10 times, 15 times, 15 times. The phage antibodies adsorbed in the plate wells were eluted with 100 µl of eluent (2...

Embodiment 3

[0091] Example 3 Preparation of Budweiser Pesticide Nanobody and Establishment of Indirect Competitive ELISA Method

[0092] 1. Experimental method

[0093] 1. The Nb316-pComb3xss plasmid was extracted by an extraction kit, and then introduced into competent Escherichia coli BL21(DE3) by chemical transformation. Take a single clone for PCR identification and sequencing to confirm that the inserted fragment is the target fragment. Cultivate the BL21(DE3) colony containing the target fragment of the nanobody to logarithmic phase OD 600 The value is 0.6-0.8, add IPTG with a final concentration of 1mM, and induce expression at 37°C for 12h. The next day, the bacteria were obtained by centrifugation. Then the periplasmic cavity protein was extracted by sucrose osmotic pressure method, and the soluble Nanobody Nb316 in the periplasmic cavity was recovered after one-step Ni column purification.

[0094] 2. Using BFNB-OVA as the coating source, dilute to a working concentration of...

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Abstract

The invention discloses a nano-antibody of carbofuran pesticide and a preparation method and application of the nano-antibody. An amino acid sequence of the nano-antibody is shown as SEQ ID NO.1, genes of the nano-antibody are encoded, and a nucleotide sequence of nano-antibody is shown as SEQ ID NO.2. The nano-antibody is against the carbofuran pesticide and can be used for detecting the carbofuran pesticide, the detection result is accurate, the effect is good, the stability is good, and the detection effect is good under conditions of high temperature and an organic solvent. The nano-antibody can be widely used for the detection of carbofuran pesticide residues in agricultural products and can be taken as a precursor, modifying can be carried out by random or site-specific mutagenesis techniques to obtain mutants with better properties (affinity, specificity, stability and the like), the nano-antibody can be used for developing and further used in fields of food, medicine, agriculture and the like, and the application and promotion value is great.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically, to a nanobody of carbofuran pesticide and its preparation method and application. Background technique [0002] Carbamate compounds are used as insecticides, acaricides, herbicides and fungicides in pesticides, and have formed a large category of pesticides with many varieties, good efficacy and low toxicity. Carbamate pesticides are the main cause of acute pesticide poisoning, and they are also the key detection varieties of pesticide residues in vegetables nowadays. [0003] Carbofuran’s trade name is carbofuran, which is a carbamate broad-spectrum systemic insecticide, acaricide, and nematicide, which can be used to control more than 300 kinds of pests and nematodes in the soil and on the ground in various crops. It also has the effect of shortening the growth period of crops, promoting the growth and development of crops, and effectively increasing crop yield. It is widely us...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/44C12N15/13C12N15/70C12N1/21G01N33/53C12R1/19
CPCC07K16/44C07K2317/22C07K2317/33C07K2317/569C07K2317/92C07K2317/94C12N15/70G01N33/5308
Inventor 王弘张瑾如杨金易徐振林孙远明沈玉栋肖治理
Owner SOUTH CHINA AGRI UNIV
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