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Colloidal gold rapid detection test paper for detecting transgenic protein g10-epsps and use method thereof

A technology of g10-epsps and colloidal gold, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of complex sample processing, high requirements for venues, instruments, and technical personnel, and no detection methods, etc., to achieve simple operation, On-site and sensitive detection, good anti-interference effect

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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  • Colloidal gold rapid detection test paper for detecting transgenic protein g10-epsps and use method thereof
  • Colloidal gold rapid detection test paper for detecting transgenic protein g10-epsps and use method thereof
  • Colloidal gold rapid detection test paper for detecting transgenic protein g10-epsps and use method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1, expression and purification of EPSPS protein

[0049] (1) Small-scale expression of protein

[0050] 1. Carrier construction

[0051] 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.

[0052]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.

[0053] 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

[0064] Embodiment 2, antibody preparation

[0065] (1) Monoclonal antibody preparation:

[0066] 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.

[0067] 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:

[0068]

[0069] 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

[0083] Embodiment 3, the preparation method of the colloidal gold-labeled mouse anti-g10-epsps monoclonal antibody specifically comprises the following steps:

[0084] 1. Preparation of 25nm colloidal gold:

[0085] 1.1 Preparation: Wash the 500ml beaker, 20ml small beaker, rotor, brown bottle, glass rod, etc. and put them into the acid tank (potassium dichromate: concentrated sulfuric acid: ultrapure water = 120g: 200ml: 1000ml) and soak for 24 hours. Take it out and rinse it with tap water for 3-4 times, then with ultrapure water for 3-4 times, and dry it in an oven at 37°C for later use.

[0086] 1.2 Configuration of 1% HAuCl4 aqueous solution: Weigh 1g of chloroauric acid powder into a brown bottle with a plastic weighing spoon, add 99ml of ultrapure water to fully dissolve, and store in the dark at 4°C.

[0087] Note: chloroauric acid powder is very easy to deliquescence, so it must be weighed quickly, and the remaining chloroauric acid powder should be sealed and stored...

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Abstract

The invention discloses a colloidal gold rapid test paper for detecting transgenic protein R1-EPSPS, which comprises a sample pad arranged on a bottom plate, a gold standard pad, a nitrocellulose membrane coated with a quality control line and a detection line, and a sample suction pad; Colloidal gold-labeled mouse anti-g10-epsps monoclonal antibody is coated on the gold standard pad, the detection line is coated with mouse anti-g10-epsps monoclonal antibody, and the quality control line is coated with anti-mouse IgG Secondary Antibodies. The invention also discloses a method for using the above-mentioned colloidal gold speed test paper: vertically insert the speed test paper into the sample to be tested; observe after 8 to 10 minutes; only C line appears, and the sample is judged to be negative; T line and C line appear simultaneously. line, the sample was judged to be positive. The hand-held colloidal gold rapid test paper of the invention can quickly, on-site and sensitively detect the transgenic protein g10-epsps in a sample.

Description

technical field [0001] The invention relates to the technical field of transgenic protein detection, in particular to a hand-held colloidal gold rapid test paper for detecting transgenic protein g10-epsps. Background technique [0002] The inventor's research group previously studied the herbicide-resistant EPSPS gene (g10-epsps) cloned from Pseudomonas bacterium. 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...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/573G01N33/558G01N33/532
CPCG01N33/577G01N33/532G01N33/558G01N33/573G01N2333/91182
Inventor 寿惠霞杜娟李林陆玲鸿武爱波徐伟
Owner ZHEJIANG UNIV
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