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A diazepam lipocalin mimetic antibody

A technology for simulating antibodies and antibodies, which can be applied to peptides, DNA/RNA fragments, specific peptides, etc., and can solve the problems of few small-molecule antibodies and no reports of diazepam-mimicking antibodies.

Inactive Publication Date: 2018-05-01
INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are relatively few studies on the screening of small molecule antibodies using ribosome display technology, and the screening of diazepam analog antibodies has not been reported

Method used

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  • A diazepam lipocalin mimetic antibody
  • A diazepam lipocalin mimetic antibody
  • A diazepam lipocalin mimetic antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Scalability identification of lipocalin mimic antibody library:

[0019] Using the biletriene-binding protein (BBP), a member of the lipocalin family, as the backbone, 16 mutation sites were randomly introduced to construct a lipocalin ribosome display library. Using the constructed lipocalin library as a template and using T7F and PDR as upstream and downstream primers for PCR amplification, the target band is about 900bp.

[0020] The T7 fragment was amplified from the pET32a plasmid by upstream and downstream primers T7F and RT7 as figure 1 As shown in (A), use primers PDF and PDR to amplify the T20-V109 sequence in the shell protein D of Lambda phage as the spacer sequence (ie, the P segment), such as figure 1 (B) shown. It can be seen from the figure that the target bands appear at 102bp and 300bp, which are consistent with the size of the T7 fragment and the P protein gene fragment, indicating that the amplification is successful. Through the upstream primer FB...

Embodiment 2

[0028] Screening of mimetic antibody against diazepam lipocalin

[0029] 1 In vitro transcription and translation

[0030] In vitro transcription and translation use Promega's E.coli S30 (Escherichia coli S30ExtractSystem) linear template system, which couples transcription and translation.

[0031] 2 solid phase screening

[0032] (1) Coating: After the ELISA plate was soaked and dried in DEPC water, take the coupled complete antigen DZP-OVA / DZP-BSA, dilute it to 10 μg / mL with carbonate buffer, add 200 μL to each well, Incubate at 4°C for 8h.

[0033] (2) The next day, pour off the coating solution, wash the coated wells with sterilized PBS 3 times, each time for 3 minutes; then add 200 μL of blocking solution (0.5% BSA or 1% OVA) to the coated wells ), closed for 1h. Pour off the blocking solution, wash with PBS twice, each time for 2min, and then wash twice with pre-cooled WBT (150mmol / L NaCl, 50mmol / L Tris-acetate pH7.5, 0.1%Tween, 50mmol / Lmagnesium acetate), Each tim...

Embodiment 3

[0047] Identification of diazepam-lipocalin mimetic antibody after five rounds of screening

[0048] The plasmid pMD18-T-BBP was extracted after culturing the completely correct sequenced strain, and then subjected to double enzyme digestion to obtain the BBP fragment containing the enzyme cleavage site. The enzyme cleavage reaction system was the same as before.

[0049] figure 2 It is shown that after each round of screening, EDTA dissociates the mRNA from the triplex complex, reverses with PM as a primer, and obtains cDNA, and then amplifies the mock antibody fragment with FBBP and Rtail as primers. After identification by 1.5% agarose gel electrophoresis, a target band can be seen at a size of about 500bp. After it was recovered and connected with the ribosome display element, a 900bp library for the next round of screening was obtained. figure 2 It is the identification map after five rounds of screening for the target small molecule diazepam from the mock antibody li...

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Abstract

The invention discloses a diazepam lipocalin mimetic antibody, and the DNA sequence expressing the antibody is the sequence shown in SEQ ID NO.1 in the sequence list. The diazepam lipocalin mimetic antibody of the invention lays the foundation for realizing the rapid detection of diazepam. In lipocalin, the embedding rate of the recognition site that can interact with the target molecule is as high as 95% in the protein, while the embedding rate of the binding site in the antibody molecule is only 66%. This is due to the large mutual contact surface within the lipocalin molecule that can bind to the target molecule (Ingo, 2003). Lipocalin has a simple structure, small molecular weight, a large recognition site that can bind to target molecules, and a stable skeleton, which has unique advantages compared with antibodies.

Description

technical field [0001] The invention relates to a diazepam mimetic antibody. Belonging to the field of biotechnology, the simulated antibody can be used to develop an immunoassay kit for rapid detection of diazepam residues. Background technique [0002] Diazepam (Diazepam, DZP), alias stability or benzodiazepine, chemical name 7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepam Azepine-2-one, molecular formula C 16 h 13 ClN 2 O, molecular weight 284.76. It belongs to the long-acting benzodiazepine anxiolytics, which have anti-anxiety, sedative, hypnotic, anticonvulsant, antiepileptic, skeletal muscle relaxation and memory elimination effects. Widely used in clinical medicine, long-term use has addiction and dependence. Diazepam is a prohibited product in the country and is only sold as a prescription drug. It has sedative and hypnotic effects. Criminals often use this feature of DZP to commit crimes and cause great social harm. Moreover, poisoning and acciden...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/44C12N15/11
Inventor 宁保安高志贤孙思明彭媛白家磊
Owner INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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