Diazepam lipocalin imitating antibody
A technology of imitating antibodies and antibodies, applied in the direction of peptides, DNA/RNA fragments, specific peptides, etc., which can solve the problems of no reports of diazepam-mimicking antibodies and few small-molecule antibodies.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
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 S30Extract System) 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 / L magnesium acetate) , each ...
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...
PUM
Property | Measurement | Unit |
---|---|---|
molecular weight | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com