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Transgenic protein g10-epsps quantitative detection method and used kit

A technology of g10-epsps and 1.g10-epsps, applied in the field of acquisition and quantitative detection of transgenic protein EPSPS, can solve the problems of complex sample processing, high requirements for venues, instruments, and technical personnel, and no detection methods, etc., to achieve The effect of accurate quantitative detection method, low cost and short operation time

Inactive Publication Date: 2016-08-17
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the routine detection method of transgenic protein is PCR, but the PCR method has relatively high requirements on the site, equipment, and technical personnel, and the sample processing is complicated, so it is not suitable for a large number of screening tests.
Some transgenic proteins can already be detected by quick test strips and ELISA kits, but g10-epsps is a newly introduced transgenic protein, and there is no detection method for this aspect at present

Method used

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  • Transgenic protein g10-epsps quantitative detection method and used kit
  • Transgenic protein g10-epsps quantitative detection method and used kit
  • Transgenic protein g10-epsps quantitative detection method and used kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1, expression and purification of EPSPS protein

[0061] (1) Small-scale expression of protein

[0062] 1. Carrier Construction

[0063] According to the protein sequence (g10-epsps), the gene sequence was synthesized and constructed into the vector pET30a to obtain the pET30a-EPSPS positive plasmid.

[0064] 2. Strain activation: The g10-epsps-E positive plasmid was constructed using the commercial ET expression vector backbone sold by EMD Biosciences, transformed into BL21(DE3), and coated with LB solid medium (Kana concentration 50 μg / mL). On the next day, single-clonal colonies were picked and inserted into 5 mL of LB liquid medium (Kana concentration 50 μg / mL), and cultured at 37°C for 12h-14h.

[0065] 3. Small test expression: the next day, the bacteria were inserted into 5mL LB liquid medium (Kana concentration 50μg / mL) at 1:50 (v:v), cultivated at 37°C until OD=0.4-0.6, and absorbed 1mL of the bacteria solution After centrifugation, it was used as...

Embodiment 2

[0076] Embodiment 2, antibody preparation

[0077] (1) Monoclonal antibody preparation:

[0078] 1. Immunogen preparation: Mix and emulsify the expressed and purified protein with an equal volume of Freund's adjuvant and YOULONG adjuvant into a water-in-oil state for immunization of mice.

[0079] 2. Immunization strategy: immunize 4 Balb / c mice with the protein, subcutaneously immunize 3 times with an interval of 4 weeks, and finally detect by ELISA, the antiserum titers are as follows:

[0080]

[0081] 3. Cell fusion: Two weeks after the last immunization, the antigen (specifically expressed and purified protein of g10-epsps protein) was injected intraperitoneally for booster immunization, and cell fusion was performed three days later. The mice were killed by neck dislocation, sterilized by soaking in 70% ethanol for 30 minutes, and the abdominal cavity was cut open on an ultra-clean table, the spleen was taken out, ground, passed through an 80-mesh sieve, and splenocy...

Embodiment 3

[0095] Embodiment 3, the assembly of kit of the present invention:

[0096] 1. Preparation of buffer

[0097] (1) Coating buffer, 0.1M carbonate buffer CB (Na 2 CO 3 -NaHCO 3 Buffer), the pH value is 9.6;

[0098] (2) diluent, 0.01mM phosphate buffer saline PBS, pH value is 7.4;

[0099] (3) washing liquid, PBS containing 0.2% Tween-20;

[0100] That is, add 0.2 g of Tween-20 to 100 ml of PBS, the PBS is 0.01 mM phosphate buffer saline PBS, the pH value is 7.4;

[0101] (4) Blocking solution, CB containing 1% BSA;

[0102] That is, add 1 g of BSA to 100 ml of CB, which is 0.1 M Na 2 CO 3 -NaHCO 3 Buffer, pH 9.6;

[0103] (5) Stop solution, 2M H 2 SO 4 .

[0104] 2. Preparation of ELISA plate

[0105] (1) Coated

[0106] Pipette a certain amount of EPSPS antibody (mouse anti-g10-epsps monoclonal antibody obtained in Example 2) into the required volume of coating buffer to make the concentration about 2 μg / mL, and configure it as a coating working solution. Add 1...

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Abstract

The invention discloses a g10-epsps quantitative detection kit including the following components: I, an antibody pre-coated ELISA plate; II, a sample diluent which is a phosphate buffer solution; III, a washing liquid which is a phosphate buffer solution containing Tween-20; IV, a reaction stop liquid which is a H2SO4 solution; V, an enzyme labeled antibody which is a mouse anti g10-epsps monoclonal antibody labeled by horseradish peroxidase; VI, a color developing agent; and VII, a g10-epsps standard substance. The invention also discloses a method for quantitative detection of a transgenic protein g10-epsps in a plant by using the kit, wherein the method includes the following steps: extracting a protein from a to-be-tested sample, to obtain a protein extract liquid; using the g10-epsps quantitative detection kit for detecting the protein extract liquid; and making a concentration-absorbance standard curve by using g10-epsps protein standard substances, and calculating the g10-epsps protein concentration in the to-be-tested sample.

Description

technical field [0001] The invention relates to the technical field of transgenic plant detection, in particular to a method for obtaining and quantitatively detecting transgenic protein EPSPS. Background technique [0002] The inventor's research group previously studied the herbicide-resistant EPSPS gene cloned from Pseudomonas (g10). EPSP synthase (5-enolpyruvyl-shikimate-3-phosphatesynthase, EPSPS) is a key enzyme in the biosynthesis of aromatic amino acids—tryptophan, tyrosine, and phenylalanine in organisms; glyphosate is passed through Inhibiting the activity of EPSP synthetase to block the synthesis of aromatic amino acids eventually led to the death of the tested plants. However, the activity of EPSP synthase of some bacteria is not inhibited by glyphosate, so the expression of bacterial EPSPS gene can protect plants from herbicide damage. The herbicide resistance gene EPSPS is transferred into soybeans, aiming at cultivating transgenic soybeans with excellent gly...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/573G01N33/543G01N33/535
CPCG01N33/577G01N33/535G01N33/543G01N33/573G01N2333/91182
Inventor 寿惠霞杜娟李林陆玲鸿武爱波徐伟
Owner ZHEJIANG UNIV
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