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Coding gene of anti-Cry1Ac toxin single-chain variable fragments (scFv) and immuno-polymerase chain reaction (PCR) detection method

A technology that encodes genes and single-chain antibodies, applied in anti-bacterial immunoglobulin, genetic engineering, plant genetic improvement, etc., to achieve the effect of reducing costs, high detection signal, and avoiding the process of reporting DNA markers

Inactive Publication Date: 2014-04-16
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The invention provides an anti-Cry1Ac single-chain antibody gene and a method for detecting Cry1Ac by immuno-PCR using a phage antibody, which solves the problem of labeling DNA reporter molecules in the process of immuno-PCR with traditional antibodies

Method used

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  • Coding gene of anti-Cry1Ac toxin single-chain variable fragments (scFv) and immuno-polymerase chain reaction (PCR) detection method
  • Coding gene of anti-Cry1Ac toxin single-chain variable fragments (scFv) and immuno-polymerase chain reaction (PCR) detection method

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Experimental program
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Effect test

Embodiment 1

[0033] In the affinity screening process of the present invention, the single-chain antibody gene contained in the human semi-synthetic antibody library (Tomlinson J) used is inserted into the phagemid pIT2, and the single-chain antibody is fused with the pIII protein when expressed in the phage On the capsid protein, it is a fusion type single chain antibody.

[0034] Coat the bottom of the cell culture flask with 4 mL of 100 μg / mL irrelevant protein at 4 °C overnight. The next day, after blocking with 2% MPBS, add the phage antibody library solution, incubate at room temperature for 2 h, transfer the supernatant to a cell culture flask coated with Cry1Ac and seal it, incubate at room temperature for 2 h, and inoculate with 0.5% Tween-20 Wash away unbound or low-binding recombinant phages with PBS (10 washes in the first round, and 10 more washes in each subsequent round). For those with higher binding capacity, 200 μL of 100 μg / mL Cry1Ac solution was first added, and eluted...

Embodiment 2

[0036] Single colonies were randomly picked from the counting plates of the third and fourth rounds of screening and inoculated into 200 μL / well 2×TY-AG medium 96 microwell plates, cultured at 250 rpm at 37 °C overnight, and the next day from Pipette 2 μL of bacterial solution from each well and transfer to a 96-well plate supplemented with 200 μL / well of 2×TY-AG medium, and incubate at 37 °C for 2 h at 250 rpm. After culturing the reseeded microwell plate for 2 h, add 25 μL of 2×TY-AG medium and add 10 9 pfu helper phage, continue to culture at 37 ℃ 250 rpm for 1 h, and finally at 1 800 g Centrifuge for 10 min and discard the supernatant. The bacterial pellet was resuspended in 200 μL 2×TY-AK medium, and cultured overnight at 30 °C and 250 rpm. The next day, at 1800 g After centrifugation for 10 min under the same conditions, the supernatant was taken for Phage-ELISA as follows:

[0037] Coating: 100 μL per well, 5 μg / mL Cry1Ac coated 96-microwell plate overnight.

[...

Embodiment 3

[0115] 1) Treat PCR tubes with 0.8% glutaraldehyde at 65 °C for 2 h and then air dry. Add 50 μL of the coating antigen Cry1Ac, dilute to 0.5-32 ng / mL with CBS and store overnight at 4 °C.

[0116] 2) Wash 3 times with PBST (0.05% Tween20), add 150 μL of blocking solution (3% MPBS, 0.1% SDS) and incubate at 37°C for 2 h.

[0117] 3) Wash with PBST (0.05% Tween20) 3 times. join 10 8 100 μL of pfu / mL phage antibody supernatant was incubated at 37 °C for 1 h.

[0118] 4) Wash 5 times with PBST (0.05% Tween20), and then wash 4 times with ultrapure water. Add primers and reaction system according to the following PCR steps, and perform PCR experiment. The primers are FAM-LMB3 and pHEN.

[0119] Bacteria solution (template) 2 μL MgCl 2 (25mM) 2 μL 10× buffer 5 μL dNTP mix (10 mM) 1 μL FAM-LMB3 (100 μM) 0.1 μL pHEN (100 μM) 0.1 μL Taq (5 U / μL) 0.5 μL sterile water 37.5 μL total capacity 50 μL

[0120] Th...

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Abstract

The invention relates to a coding sequence of anti-Cry1Ac scFv and an immuno-PCR detection method for detecting Cry1Ac through the phage scFv. The scFv are obtained through elutriation in a natural phage antibody base according to the method. The method is characterized in that the specificity of the scFv is determined by a CDR region sequence in a heavy chain and light chain variable region sequence, immuno-PCR results indicate that a fluorescence detection system can be used for quantitative detection of the Cry1Ac, and the detection range is 0.2-100ng / mL. The method is fast, simple and high in flexibility; a step for marking reporter DNA molecules during an existing immuno-PCR process is avoided, the experimental operation is simplified, and potential practical values are provided.

Description

technical field [0001] The invention relates to a coding gene of an anti-Cry1Ac toxin single-chain antibody, and the application of immuno-PCR detection of Cry1Ac toxin by using the translated and expressed active polypeptide combined with the gene. Background technique [0002] Bacillus thuringiensis ( Bacillus thuringiensis , referred to as Bt) is an aerobic Gram-positive bacterium widely distributed in nature. The Cry1Ac toxin secreted by it has a good poisoning effect on Lepidoptera pests and has become a widely used biopesticide. With the development of transgenic technology, it has been used to control agricultural pests on a larger scale. In order to satisfy consumers' right to know and right to choose, as well as the needs of international trade, the research on environmental safety assessment and labeling management of Bt genetically modified plants urgently needs to be supported by the development of rapid and effective detection technology. [0003] At this stag...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/13C07K16/12G01N33/68C12Q1/68
Inventor 王耘刘贤金张存政刘媛王淮庆
Owner JIANGSU ACAD OF AGRI SCI
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