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Anti-amantadine (Anti-AMD) single-chain antibody scFv, and preparation method and application thereof

A single-chain antibody and amantadine technology, which is applied in the field of amantadine AMD single-chain antibody scFv and its preparation, and can solve problems such as unreported, unreported patents, etc.

Active Publication Date: 2020-03-10
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no report on the research on amantadine AMD single-chain antibody scFv at home and abroad, and there is no report on the application of related patents at home and abroad.

Method used

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  • Anti-amantadine (Anti-AMD) single-chain antibody scFv, and preparation method and application thereof
  • Anti-amantadine (Anti-AMD) single-chain antibody scFv, and preparation method and application thereof
  • Anti-amantadine (Anti-AMD) single-chain antibody scFv, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1. Obtainment of anti-amantadine monoclonal antibody 3F2 and cloning of its heavy chain and light chain variable region gene sequences

[0073] 1. The acquisition of anti-amantadine monoclonal antibody 3F2

[0074] 1. Establishment of hybridoma cell line 3F2

[0075] The method in the reference "New Hapten Synthesis, Antibody Production, and IndirectCompetitive Enzyme-Linked Immnunosorbent Assay for Amantadine in ChickenMuscle" was used to synthesize the whole amantadine antigen, and the synthetic amantadine whole antigen was used as the immunogen to immunize mice, and the mice were immunized three times After immunization, the mouse serum was taken to detect the antibody titer by indirect ELISA method, and those who passed the titer were given booster immunization for cell fusion. When the fused cell clones grew to 1 / 4-1 / 3, the cells were tested by indirect ELISA The culture supernatant was tested, and a positive hybridoma cell line stably secreting anti-amant...

Embodiment 2

[0089] Example 2. Preparation of amantadine AMD single-chain antibody scFv

[0090] 1. Construction of recombinant expression vector

[0091] The DNA molecule shown in SEQ ID No.2 is inserted between the Sfi I restriction sites of the vector pJB33 to obtain a recombinant expression vector. The recombinant expression vector expresses the single-chain antibody scFv shown in SEQ ID No.1.

[0092] 2. Construction of recombinant bacteria

[0093] The recombinant expression vector prepared in step 1 was electroporated into RV308 competent cells (purchased from American Standard Biology Collection, ATCC number 31608) to obtain recombinant bacteria. The shock conversion conditions are as follows: voltage 2.25kv; resistance 200Ω; capacitance 25μF; time 0.002s.

[0094] 3. Expression and purification of amantadine AMD single-chain antibody scFv

[0095] 1. Cultivation of recombinant bacteria

[0096] The recombinant bacteria prepared in step 2 were inoculated into 900 μL of 2×YT-G ...

Embodiment 3

[0115] Example 3. Sensitivity and specificity determination of amantadine AMD single-chain antibody scFv

[0116] 1. Synthesis of Coatogen

[0117] 1. Synthesis of coated original AMD-BSA

[0118] (1) Dissolve 500mg AMD in 25mL anhydrous pyridine, add 10mg 4-dimethylaminopyridine (DMAP) and 705mg p-carboxybenzenesulfonyl chloride respectively, stir overnight at room temperature, add a small amount of deionized water to terminate the reaction, and evaporate the solution to dryness. Add 10 mL of deionized water and 1M NaOH to adjust the pH to 9.0 to completely dissolve the residue, and then use glacial acetic acid to adjust the pH to 5.0. At this time, a large amount of white precipitate precipitates, filter, and dry the filter cake to obtain the hapten AMA-CSBA.

[0119] (2) Weigh 16.2 mg of the hapten AMA-CSBA prepared in step (1) and dissolve it in 1 mL of N,N-dimethylformamide (DMF) solution, add 17.2 mg of dicyclohexylcarbodiimide (DCC) React with 20.6 mg of hydroxysuccin...

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Abstract

The invention discloses an anti-amantadine AMD single-chain antibody, and a preparation method and application thereof. According to the invention, a heavy-chain variable region gene and a light-chainvariable region gene of the antibody can be obtained from a hybridoma cell strain 3F2 of a monoclonal antibody secreting amantadine (AMD) through a genetic engineering mean; and after the two genes are recombined, a prokaryotic expression plasmid vector is constructed, and escherichia coli is transformed to express scFv antibody active fragments capable of specifically recognizing the AMD. Experiments prove that the recombinant single-chain antibody scFv prepared by the method has specific recognition and binding activity withamantadine. Residual detection of amantadine can be realized through utilization of recognition and binding activity of the recombinant single-chain antibody on amantadine, and the constructed transformation vector plasmid is easy for storage and mass-produce.

Description

technical field [0001] The present invention relates to an amantadine AMD single-chain antibody scFv and a preparation method thereof, in particular to the protein sequence of the single-chain antibody and its coding gene sequence, a vector containing the coding gene and a method for preparing the single-chain antibody , belonging to the field of biological products. Background technique [0002] Amantadine and other antiviral drugs are used in the prevention and treatment of influenza A virus in animal husbandry, but the abuse of amantadine is likely to cause drug resistance, promote strain mutation and endanger human health, and has a series of neurotoxicity, etc. Side effects, such as causing nervousness, anxiety, nightmares and other symptoms. Announcement No. 560 of the Ministry of Agriculture of my country in 2005 clearly stipulates that antiviral drugs such as amantadine and rimantadine are prohibited from being used in veterinary medicine. Livestock and poultry. Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/44C12N15/70G01N33/53
CPCC07K16/44C07K2317/56C07K2317/622C12N15/70G01N33/53
Inventor 江海洋谢三磊温凯沈建忠王战辉于雪芝夏曦李建成史为民
Owner CHINA AGRI UNIV
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