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Screening and application of single-chain antibody against fumonisin

A fumonisin, single-chain antibody technology, applied in the field of genetic engineering, can solve the problems of unpublished nucleotide or amino acid sequence, high cost, time-consuming and laborious, etc., to improve stability or affinity, low production cost, expensive effect

Inactive Publication Date: 2013-10-30
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation of both polyclonal and monoclonal antibodies needs to involve animals, and the specificity of polyclonal antibodies is poor. Monoclonal antibody screening requires special cell culture equipment, and cell preservation requires special cryopreservation equipment. The production process is time-consuming, laborious, and costly. high
There are two documents about the preparation of anti-fumonisin single-chain antibodies reported abroad. Zhou et al. (1996) obtained single-chain antibodies by phage display screening of immune antibody gene libraries and hybridoma transformation (Zhou, H.R.; Pestka, J.J.; Hart, L.P. Molecular cloning and expression of recombinant phage antibody against fumonisin B1.J.Food Prot.1996, 59, 1208-1212.), but the antigens used for immunizing mice and screening antibodies are conjugated FB1-BSA and FB1 -OA, which is different from the antigen used in the present invention, the antibody screening method and process are quite different, and the article states that the obtained single-chain antibody has a low affinity, which needs to be further improved before it can be used for fumonisin detection; Lauer (2005) obtained anti-FB1 single-chain antibody by phage display screening antibody gene library can be used for fumonisin detection (Lauer, B.; Ottleben, I.; Jacobsen, H, J.; Reinard, T.Production of a single-chain variable fragment antibody against fumonisin B1.J.Agric.Food Chem.2005, 53, 899-904.), but its antibody gene source is a synthetic antibody gene library, which is completely different from the immune antibody gene library constructed in the present invention
More importantly, although the two foreign reports obtained single-chain antibodies against fumonisins, they did not publish their nucleotide or amino acid sequences, and there are no related detection products for sale in the domestic market.

Method used

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  • Screening and application of single-chain antibody against fumonisin
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  • Screening and application of single-chain antibody against fumonisin

Examples

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Embodiment 1

[0042] Example 1: Antigen Preparation

[0043] One-step coupling with glutaraldehyde (Azcona-Olivera, J.I.; Abouzied, M.M.; Plattner, R.D.; Norred, W.P.; Pestka, J.J. Generation of antibodies reactive with fumonisins B 1 , B 2 , and B 3 by using cholera toxin as the carrier-adjuvant.Appl.Environ.Microbiol.1992,58,169-173.) Fumonisin FB1 (purchased from Sigma Company) and keyhole limpet cyanin (KLH, purchased from Sigma Company ) coupling (KLH-NH 2 +OHC-(CH 2 ) 3 -CHO+FB1-NH 2 → KLH-N=HC-(CH 2 ) 3 -CH=N-FB1) as an immune antigen (FB1-KLH), the fumonisin FB1 was coupled with bovine serum albumin (BSA, purchased from Sigma) (BSA-NH 2 +OHC-(CH 2 ) 3 -CHO+FB1-NH 2 →BSA-N=HC-(CH 2 ) 3 -CH=N-FB1) was used as the screening antigen (FB1-BSA).

Embodiment 2

[0044] Example 2: Animal immunization

[0045] Two BALB / c mice (Experimental Animal Center, Wuhan Institute of Virology, Chinese Academy of Sciences) were immunized with the prepared antigen FB1-KLH 4 times with an interval of 10 days between each time. For the first time, 250 μl of immune antigen was mixed with an equal volume of Freund's complete adjuvant for immunization, and for the last three times, 250 μl of immune antigen was mixed with an equal volume of Freund's incomplete adjuvant for immunization. On the 5th day after the third immunization, the serum was collected from 1:10 3 Doubling diluted to 1:128×10 3 , with reference to the indirect ELISA method introduced by Lin Qiaoai and Dong Haiyan, "Experimental Guidance for Medical Immunology and Microbiology", Zhejiang University Press, 2006, to detect the level of antibodies in the serum of immunized mice. The test results showed that the serum of immunized mice produced higher antibody titers (dilution > 1:10 5 )....

Embodiment 3

[0046] Example 3: Spleen RNA Extraction, mRNA Purification and cDNA First-Strand Synthesis of Immunized Mice

[0047] 1) The immunized mice with higher antibody titers were given a booster immunization once more, and the spleens of the immunized mice were taken 7 days later.

[0048] 2) Using TRNzol-A + Total RNA extraction reagent (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., operated according to the instructions of the reagent) was used to extract total RNA from spleen.

[0049] 3) Purify the total RNA extracted in step 2) with an mRNA purification kit (purchased from QIAGEN, and operate according to the instructions of the kit).

[0050] 4) Use SuperScript TM III Reverse transcriptase (purchased from Invitrogen Company, operated according to the instructions of the enzyme) reverse-transcribed the purified mRNA obtained in step 3), and synthesized antibody heavy chain (IgG 1 and IgG 2a / 2b ) and light chain (kappa chain and lambda chain) variable r...

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Abstract

The invention belongs to the technical field of genetic engineering, and in particular relates to screening of a single-chain antibody against fumonisin and application thereof in immunoassay of fumonisin. The screening method comprises the following steps of: directly setting off from coupled antigen FB1-KLH(keyhole limpet hemocyanin) immunized mouse spleen cells, establishing a single-chain antibody gene library by a molecular cloning method and technology, then screening by a phage display technology, expressing ELISA(enzyme-linked immuno sorbent assay) test, sequencing, and finally obtaining the single-chain antibody against fumonisin named as FB-Mu 1H3 with high affinity and a coding gene thereof. The single-chain antibody can be directly applied to assay of fumonisin after being expressed and purified in a large quantity in escherichia coli, and the applications include assay of fumonisin pollution in field crops, feed, grain or food.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, specifically relates to the screening and application of an anti-fumonisin single-chain antibody, and is related to antibody screening technology. Background technique [0002] Fumonisin is a water-soluble mycotoxin discovered in the late 1980s by fungi such as Fusarium verticillioides and Fusarium proliferatum. Alcohol and tricarboxylic acid form diester compounds with similar structures. In 1988, Gelderblom first isolated fumonisin from the culture solution of Fusarium moniliforme. Subsequently, Laurenf et al. isolated fumonisin B1 (FB1) and fumonisin B2 (FB2) from fumonisins in 1989. Fumonisins are a group of related polar metabolites. So far, 11 kinds of fumonisins have been discovered, which are divided into groups A, B, C, and P. Among them, group B is the most widely distributed and the most toxic. . [0003] Fungi such as Fusarium verticillioides and Fusarium proliferatum,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/14C12N15/11G01N33/53
Inventor 廖玉才李和平胡祖权张静柏
Owner HUAZHONG AGRI UNIV
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