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Diethylstilbestrol single-chain antibody screening method and purpose of diethylstilbestrol single-chain antibody

A single-chain antibody and diethylstilbestrol technology, which is applied in the field of screening of diethylstilbestrol single-chain antibodies, can solve rare problems and achieve the effects of weak immunogenicity, small molecular weight, and clear sequence

Inactive Publication Date: 2013-06-05
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

There are few studies using ribosome display technology to screen antibodies against small molecule haptens, especially screening from natural antibody libraries

Method used

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  • Diethylstilbestrol single-chain antibody screening method and purpose of diethylstilbestrol single-chain antibody
  • Diethylstilbestrol single-chain antibody screening method and purpose of diethylstilbestrol single-chain antibody
  • Diethylstilbestrol single-chain antibody screening method and purpose of diethylstilbestrol single-chain antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Construction of ribosome-displayed mouse single-chain antibody

[0042] 1. RNA extraction and cDNA synthesis

[0043] Take 6-week-old healthy BALB / c mice and 3 C57 / BL6 mice, half male and half male. Total RNA of splenocytes was extracted by TRIZOL method, and cDNA was synthesized by reverse transcription kit.

[0044] 2. Amplification of VH and VL genes

[0045] 1. Primer design:

[0046] VH / for amplification heavy chain VH variable region upstream primer:

[0047] VH / for 5'TATA TCC ATG GCCCAGGTSMARCTGCAG 3’

[0048] VH / back amplification heavy chain VH variable region downstream primers:

[0049] VH / back 5’TGA GGA GAC GGT GAC CGT GGT GCC TTG GCC CC 3’

[0050] VL / for amplification heavy chain VH variable region upstream primer:

[0051] VL / for 5’GAC ATC GAG CTC ACT CAG TCT CCA 3’

[0052] VL / back amplification heavy chain VH variable region downstream primers:

[0053] VL / back 5’CGCG GTTGCGGTCCG TTT BAK YTC CAR CTT KGT SCC 3’

[0054] One-letter n...

Embodiment 2

[0074] Example 2 Screening of diethylstilbestrol single-chain antibody

[0075] 1. Solid-phase affinity screening

[0076] All reagent bottles, reagents and microtiter plates in this step should be treated with DEPC water to ensure that there is no RNase contamination.

[0077] (1) Coating and screening wells: Dilute the DES-BSA coating to 20 μg / mL with sterilized carbonate buffer, add 100 μL to each well of the microtiter plate, and coat overnight at 4°C;

[0078] (2) Pour off the coating solution, and wash the closed well with sterilized PBS 3 times, 3 min each time. Then wash twice with cold WBT, 3min each time. Finally, fill the screening well with cold WBT and place it on ice for at least 20 minutes;

[0079] (3) Transfer the translation products placed on ice (in vitro translation using promega Escherichia coli (E.coli) S30 Extract System) to the prepared screening wells, and shake gently in the ice room for 1 hour. The in vitro translation product was fully combined...

Embodiment 3

[0091] Example 3 Functional expression, purification and identification of anti-diethylstilbestrol single chain antibody

[0092] 1. Functional expression of anti-diethylstilbestrol single chain antibody

[0093] 1. Amplification of the single-chain antibody gene after screening

[0094] The 26 single-chain antibody genes that have been sequenced and translated completely correctly were amplified with the newly designed primers containing restriction sites. The scfv gene with restriction sites was amplified from the pMD18-T-scfv cloning vector. Primers SF-Trx-EcoRI and SR-Xhol-1 contain EcoRI and XhoI restriction sites, respectively.

[0095] SF-Trx-EcoRI (introduce EcoI restriction site): CGCGAATTCTAAATGGCCCAGGT

[0096] SR-XhoI-1 (introduce XhoI restriction site): AATCTACTCGAGCGCGGTTGCGGTCCGTTT

[0097]

[0098]Reaction conditions: pre-denaturation at 94°C for 5min; denaturation at 94°C for 30s; annealing at 56°C for 30s; extension at 72°C for 1min20s; 10cycles; denat...

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PUM

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Abstract

The invention relates to a diethylstilbestrol single-chain antibody screening method and a purpose of a diethylstilbestrol single-chain antibody, and belongs to the technical field of biology. The single-chain antibody is screened from a ribosome showing mouse source natural antibody base and is subjected to soluble expression. The invention further relates to a diethylstilbestrol antibody screening method with a ribosome showing technology. The diethylstilbestrol single-chain antibody screened through the method can be used for pollution detection, development of immune quick test kits used for quick test of diethylstilbestrol residual and treatment of relevant diseases, and a foundation is laid for future detection and research on the diethylstilbestrol single-chain antibody.

Description

technical field [0001] The invention relates to a screening method for a diethylstilbestrol single-chain antibody, which belongs to the field of biotechnology and is used for preparing a high-efficiency antibody that specifically recognizes diethylstilbestrol, developing an immune rapid test kit that can quickly detect diethylstilbestrol residues, and treating related diseases. Background technique [0002] Diethylstilbestrol (DES) is the first orally active synthetic non-steroidal estrogen substance. At present, it is mainly used clinically to treat various diseases caused by hypoestrogenism and hormone imbalance, as well as various diseases caused by ovarian insufficiency or abnormal pituitary function. In veterinary clinics, DES is mainly used to treat anestrous infertility in sows, induce estrus in female rabbits, treat production stoppage of laying hens, and fatten goats. In addition, diethylstilbestrol has obvious effects in promoting the synthesis and metabolism of a...

Claims

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Application Information

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IPC IPC(8): C07K16/26C12N15/13C12N15/10C12N15/70G01N33/74
Inventor 宁保安高志贤彭媛白家磊柳明孙思明孙亚楠张亦红
Owner INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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