Citrinin-resisting single-chain antibody as well as preparation method and application thereof

A technology of penicillin and single-chain antibody is applied in the biological field to achieve the effects of simplifying the screening and preparation process, improving the recovery yield, good sensitivity and specificity

Active Publication Date: 2015-11-11
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, ribosome display technology is a technology for protein or polypeptide screening and evolution completely in vitro, which has the ability to construct high capacity (≥10 11 above), high-quality gene library, easy screening and expression of specific proteins, easy in vitro evolution and recombination, and ability to display and screen cytotoxic molecules. Small molecular proteins, peptides and antibodies have opened up a new way. At present, a variety of high-affinity antibodies have been obtained by applying this technology, but there is no report on the use of ribosome display technology to screen single-chain antibodies against citrinin.

Method used

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  • Citrinin-resisting single-chain antibody as well as preparation method and application thereof
  • Citrinin-resisting single-chain antibody as well as preparation method and application thereof
  • Citrinin-resisting single-chain antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Construction of ribosome display gene library

[0028] 1. Extraction of RNA and synthesis of cDNA: Take the spleen of mice without any immunization under sterile conditions, operate according to the instructions of the reagents, and use the Trizol method to extract the total RNA of the mouse spleen. According to the instructions of the ReverTraAce-α-reverse transcription kit, cDNA was synthesized by reverse transcription from the total RNA of mouse spleen.

[0029] 2. Design and synthesis of primers: respectively design the VH, Linker, VL, and Cκ gene fragments and the PCR primers required for the construction of the ribosome display gene library. The primer sequences are shown in Table 1.

[0030]3. Amplification of VH, Linker, VL, and CK gene fragments: Using the synthesized mouse cDNA as a template, PCR amplifies VH, Linker, VL, and CK gene fragments. The reaction conditions are respectively:

[0031] VH: 95°C for 3min; 95°C for 30s, 59°C for 1min, 72°...

Embodiment 2

[0044] Example 2: Ribosome display and affinity screening

[0045] 1. In vitro transcription and translation: using the constructed ribosome display gene library as a template, use the TNTQuickCoupledTranscription / TranslationSystems in vitro transcription and translation kit to prepare a 50 μL standard reaction system, add each component as shown in Table 2, and place it at 30°C React in a constant temperature water bath for 60 minutes to prepare antibody-ribosome-mRNA triplet complex (ARM).

[0046] 2. Affinity screening: The coating antigen CIT-OVA and the carrier protein OVA were prepared with the coating buffer to a final concentration of 0.1 mg / mL, respectively added to the microtiter plate, 100 μL per well, overnight at 4 °C. The liquid in the well was discarded, and washed 5 times with PBST, 3 min each time. 5% skimmed milk was used for blocking, 100 μL per well, and blocked for 2 h at 37 °C. Discard the liquid in the wells, wash with PBSTM 5 times, each time for 3 ...

Embodiment 3

[0057] Example 3: Expression and Identification of Single Chain Antibody

[0058] 1. Cloning and expression of single-chain antibody gene fragments: Take the PCR amplification products with higher abundance and carry out ligation transformation with pMD18-T vector, after overnight growth, select all single clone colonies for PCR identification, and the positive clones obtained after identification are sequenced and analyzed . 32 positive clones with complete gene sequence, no stop codon, and no lethal mutation were selected for prokaryotic expression with the expression vector pTIG-TRX.

[0059] 2. Identification of prokaryotic expression products of single-chain antibodies: Inoculate the positive recombinant plasmid into 100mL liquid LB medium (containing ampicillin, Amp) at a ratio of 1:100, and place it in a constant temperature shaker at 37°C and 250rpm. to the OD of the bacterial solution 600nm At 0.7 (about 3.5h), add IPTG inducer with a final concentration of 1mM, p...

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Abstract

The invention provides a single-chain antibody resisting citrinin. Artificial coupling antigen citrinin-BSA / BSA and citrinin-OVA / OVA antigen are combined to perform affinity screening for an antibody-ribosome-mRNA triplet complex, an RT-PCR method is used for recovering an affinity screened product, a ribosome display technology is used for screening a single-chain antibody, a screened gene fragment is expressed by virtue of pronucleus and then is identified by utilizing an ELISA method to obtain the citrinin-resisting single-chain antibody. The screening and preparation process of the antibody is greatly simplified, interference of a protein carrier is alleviated, and a probability for obtaining a specificity single-chain antibody is increased; the recovery yield is increased. Due to renal toxicity, teratogenicity and carcinogenicity, the citrinin poses a severe threat to the health of human beings and livestock, and the citrinin-resisting single-chain antibody can be used as a reagent for detecting citrinin pollution residue in an agricultural product and a food sample.

Description

technical field [0001] The present invention belongs to biotechnology. The invention relates to a single-chain antibody against citrinin and its preparation method and application. Background technique [0002] Citrinin (Citrinin, CIT) is a toxic secondary metabolite produced by fungi such as Penicillium and Aspergillus. It is widely found in various agricultural products and foods such as barley, red rice, oats, and flour. Because it has strong nephrotoxicity and carcinogenicity, it poses a huge threat to human health, so it is necessary to establish a detection method with high sensitivity, strong specificity, simple and fast to detect citrinin in agricultural products and food, Ensure the safety of agricultural products and food. [0003] The detection of citrinin toxin mainly includes thin layer chromatography and high performance liquid chromatography, etc., which can carry out qualitative and quantitative detection of toxin, but the equipment is expensive, the operat...

Claims

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Application Information

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IPC IPC(8): C07K16/14C12N15/13C12N15/70G01N33/68
Inventor 杜爱芳程海卫王素华赵现锋杨怡陈学秋陈一飞
Owner ZHEJIANG UNIV
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