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A kind of single-chain antibody directly recognizing isoprocarb and its preparation method and application

A single-chain antibody and isoprocarb technology, which is applied in the field of direct recognition of isoprocarb single-chain antibody and its preparation, can solve the problems of expensive instruments and equipment, complicated pretreatment, high detection cost, etc., and achieve good antigen binding activity, good Application prospect, cost reduction effect

Active Publication Date: 2021-05-28
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The instrument detection methods all have defects such as expensive equipment and professional operators, complicated pre-treatment, long inspection cycle and high detection cost, which cannot meet the current rapid and accurate detection requirements for pesticide residues in agricultural products.

Method used

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  • A kind of single-chain antibody directly recognizing isoprocarb and its preparation method and application
  • A kind of single-chain antibody directly recognizing isoprocarb and its preparation method and application
  • A kind of single-chain antibody directly recognizing isoprocarb and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Construction Screening and Identification of scFv Recombinant Plasmid

[0054] 1. Experimental method

[0055] In the early stage, our laboratory has successfully synthesized anti-isoprocarb monoclonal antibody. The isoprocarb hapten was coupled with lactoferrin, mice were immunized, and a cell line secreting anti-carbofuran monoclonal antibody was obtained through fusion and cell selection.

[0056] 1. Extraction of total mRNA from hybridoma cells

[0057] S1. Consumables and utensils such as PCR tubes and centrifugal pipette tweezers related to the RNA experiment operation are completely soaked in 0.1% DEPC solution overnight. Pour the DEPC solution as much as possible the next day, and autoclave it for later use;

[0058] S2. Will culture well about 1×10 7 After the hybridoma cells are suspended, add them to a 15mL centrifuge tube, centrifuge at 1000r / min to pellet the cells, discard the supernatant, and use a pipette tip to suck up the residual liquid ...

Embodiment 2

[0116] Example 2 Extraction of soluble expression of scFv antibody

[0117] 1. Experimental method

[0118] The pComb3XSS phagemid carrier used in the present invention is a plasmid carrier containing a phage gene spacer, which combines the advantages of phage and plasmid vectors, and the phagemid has a succinic acid terminator (AmberStop Codon) between the multi-cloning site and gm , when the recombinant phage pComb3XSS-Fab transforms the inhibitory Escherichia coli XLl-Blue, the succinic acid terminator is read through to glutamic acid, and as a result, the antibody fusion protein will be expressed on the surface of the phage, that is, the surface display of the phage. When pComb3XSS-Fab was transformed into non-suppressor E. coli DH5α, the succinic acid terminator was identified as a stop codon, induced by the lactose analogue IPTG, and the antibody was expressed and secreted into the cytoplasm to become a soluble molecule.

[0119] Its expression and extraction steps are ...

Embodiment 3

[0130] Example 3 scFv soluble protein ci-ELISA detection

[0131] 1. Experimental method

[0132] The ci-ELISA detection steps are as follows:

[0133] S1. Coating: Dilute the coating material shown in (I) to a suitable 125ng / mL with the coating solution, add it to the well of the microtiter plate, 100μl / well, and put it in a water bath at 37°C overnight

[0134]

[0135] S2. Washing: Pour off the liquid in the wells, wash the plate twice with a plate washer, add 250 μl of washing solution to each well, and pat dry the liquid in the wells.

[0136] S3. Blocking: add 120 μl of blocking solution to each well, block at 37°C for 3 hours, pat dry the liquid in the well, and place it upside down in an oven at 37°C for 1 hour for later use.

[0137] S4. Adding samples and incubating: Dilute isoprocarb into a series of gradient standard solutions, dilute to 10000, 3333.33, 1111.11, 370.37, 123.46, 41.15, 13.72, 4.57, 1.52, 0.51ng / mL, add 50μl to each well, then Add 50 μl of reas...

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Abstract

The invention discloses a single-chain antibody that directly recognizes isoprocarb and its preparation method and application. The amino acid sequence of the single-chain antibody is shown in SEQ ID NO.5; the nucleotide sequence of its coding gene is shown in SEQ ID NO.6 shown. Using the ELiSA detection method of the single-chain antibody isoprocarb, the rapid detection and screening of a large number of samples can be realized, and the cost can be reduced; the single-chain antibody can be expressed in vitro through genetic engineering technology, and can be produced economically on a large scale in bacteria, avoiding monoclonal The risk of cell loss; the binding of the single-chain antibody to the antigen can be competitively inhibited by the free hapten isoprocarb, and has good antigen-binding activity. The genetically engineered antibody has good application prospects in the immunodetection of isoprocarb and the rapid detection and screening of a large number of samples.

Description

technical field [0001] The invention relates to the technical field of food safety detection, more specifically, to a single-chain antibody (scFv) that directly recognizes isoprocarb and its preparation method and application. Background technique [0002] Isoprocarb (IPC), also known as Miepusan, Yechansan, chemical name: 2-isopropylphenyl-N-methylcarbamate, belongs to a common contact carbamate Insecticides can paralyze insects to death by inhibiting the biological activity of acetylcholinesterase in insects. Such insecticides can also inhibit acetylcholinesterase when they enter the human body, causing acute poisoning and stimulating the central nervous system. Potentially dangerous, so many countries and regions have set strict limits on the residues of this pesticide in fruits and vegetables. According to the regulations of the Ministry of Agriculture of China, the maximum residue limit of isoprocarb in leafy vegetables is 0.1 μg / mL , the maximum residue limit allowed ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/44C12N15/13C12N15/70C12N1/21G01N33/577G01N33/543C12R1/19
CPCC07K16/44C07K2317/622C12N15/70G01N33/543G01N33/577G01N2430/10
Inventor 徐振林吴会玲傅慧君陈子键沈玉栋孙远明肖治理王弘
Owner SOUTH CHINA AGRI UNIV
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