Preparation method and application of aspergillus flavus specific single-chain antibody

A technology of single-chain antibody and Aspergillus flavus, which is applied in the fields of botanical equipment and methods, microorganism-based methods, biochemical equipment and methods, etc., and can solve the problems of decreased antibody specificity and affinity, and increased production costs

Inactive Publication Date: 2014-02-05
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cross-linking sites generated by the chemical cross-linking process with random reaction characteristics may affect the binding of the antibody to the antigen, resulting in a decrease in antibody specificity and affinity. The optimization of the cross-linking reaction and the quality detection of the enzyme-labeled antibody will also lead to production cost increase

Method used

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  • Preparation method and application of aspergillus flavus specific single-chain antibody
  • Preparation method and application of aspergillus flavus specific single-chain antibody
  • Preparation method and application of aspergillus flavus specific single-chain antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1: Preparation of Aspergillus flavus cell wall proteins (SCWPs)

[0082] 1) Use a sterilized toothpick to pick the Aspergillus flavus strain wh-1 (collected from Hubei, Wuhan, China). The strain was morphologically identified as Aspergillus flavus, and its ability to produce aflatoxin was determined by LC-MS / MS Determination (Han Z, Ren Y, et a1. J. Agric. Food Chem. 2012, 60, 8233-8247), AFB 8 days after inoculation of peanut l And AFB 2 The contents were 775.39μg / kg and 26μg / kg; AFB 8 days after inoculation with corn l And AFB 2 The contents are 1056.8μg / kg and 55.04μg / kg), inoculated into PDA medium (composition: 20% (W / V) potato, 2% (W / V) glucose, 1.5% (W / V) agar) Cultivate at 28°C for 5 days, wash the conidia on the surface of the hypha with sterile water containing 0.05% Tween-20, and collect the spore liquid.

[0083] 2) Take 1ml (1×10 5 / M1) Inoculated in 160ml Czapek medium (composition: 3% (W / V) sucrose, 0.3% (W / V) NaNO 3 , 0.1%(W / V)K 2 HPO 4 ,0.05%(W / V)MgS...

Embodiment 2

[0103] Example 2: Immunization of hens with SCWPs

[0104] Laiheng hens (purchased from the chicken farm of Huazhong Agricultural University) were immunized with the prepared Aspergillus flavus SCWPs for 6 times, and the immunization method was intramuscular injection every 14 days. For the first time, 500 μl (200 μg) of SCWPs prepared in Example 1 were used as the antigen and mixed with 500 μl Freund's complete adjuvant for immunization, and the second to fifth booster immunizations: 500 μl antigen and 500 μl of Freund's incomplete adjuvant were mixed and immunized. One week after the third and fifth immunizations, blood was taken intravenously, and the serum was diluted 1:200 times, and the antibody titer in chicken serum was detected by indirect ELISA (refer to Lin Qiaoai, Dong Haiyan, editors, "Medical Immunology and Microbiology" Guidance for Learning Experiments, Zhejiang University Press, 2006). The results showed that the titer of the Aspergillus flavus-specific antibody...

Embodiment 3

[0105] Example 3: Extraction of total RNA from chicken spleen, purification of mRNA and synthesis of the first strand of cDNA

[0106] 1) Seven days after the last immunization, the spleen of the chicken with the highest antibody titer was taken, and the spleen cells were separated.

[0107] 2) Extract total RNA from chicken spleen using TRIzol total RNA extraction reagent (purchased from Invitrogen, operated in accordance with the instructions).

[0108] 3) Purify mRNA from the total RNA extracted in step 2) using an mRNA purification kit (purchased from QIAGEN, operated according to the instructions).

[0109] 4) Use SuperScript TM III Reverse Transcription Kit (purchased from Invitrogen, operated according to the instructions), using the mRNA obtained in step 3) as a template, using the primer oligo(dT) 12-18 (Commercial primers purchased from Invitrogen) Synthesize the first strand of cDNA.

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Abstract

The invention belongs to the field of detection of harmful biological molecules in food, and relates to a preparation method and application of an Aspergillus flavus specific single-chain antibody. The cell wall proteins of the Aspergillus flavus hypha are used for immunizing chickens and mice respectively; messenger RNA of chicken splenic lymphocytes after immunization are extracted; a chicken source single-chain antibody library is constructed; a phage display technology is employed to screen the library to obtain an Aspergillus specific single-chain antibody gene with high affinity to Aspergillus flavus. The antibody gene is subcloned into an expression vector of an alkaline phosphatase protein, and expressed and purified in Escherichia coli, so as to obtain a fusion protein and a hybridoma cell 2A8 secreting Aspergillus flavus specific monoclonal antibody. The monoclonal antibody as a capture antibody and AfSA4-AP fusion protein as a detection antibody are employed to establish a SandWich ELISA immunological detection system for detecting Aspergillus flavus contamination in crops and stored plant-derived products.

Description

Technical field [0001] The invention belongs to the technical field of molecular detection of food harmful microorganisms, and specifically relates to a preparation method of Aspergillus flavus specific single chain antibody and monoclonal antibody and its application in Aspergillus flavus immunological detection. Background technique [0002] Aspergillus flavus and A. parasiticus are widely distributed filamentous fungi that produce aflatoxin, which can infect peanuts (Arachis hypogaeaL.), corn (Zea maysL.), and cotton (Gossypium spp.) It causes diseases of various crops, endangers the production of grain and cash crops, and causes serious economic losses. Contamination by Aspergillus flavus and Aspergillus parasiticus is also prone to occur in grain storage, food and feed processing and transportation, leading to the accumulation of aflatoxin, which seriously threatens human and animal health and food safety. Aflatoxins mainly include AFB l , AFB 2 , AFG l , AFG 2 The four typ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/13C07K16/14C12N15/62C07K19/00C12N5/20C12P21/08G01N33/68C12Q1/04C12R1/91
Inventor 廖玉才薛升李和平
Owner HUAZHONG AGRI UNIV
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