Immune magnetosome for detecting Bt insecticidal protein in mice tissue and preparation method thereof
A technology for insecticidal proteins and magnetosomes, which is applied in measuring devices, biological testing, material inspection products, etc., can solve the problems of quantitative detection application errors, detection limit can only reach μg level, etc., and achieve high repeatability and preparation method Meticulous and rigorous, the effect of high detection sensitivity
Inactive Publication Date: 2012-04-18
WUHAN UNIV OF TECH
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Problems solved by technology
[0004] The actual impact of transgenic Bt crops on non-target small animals such as rodents has rarely been reported at home and abroad.
At present, the widely used Bt insecticidal protein detection ELISA kit (produced by EnviroLogix, USA) is generally only used for qualitative research, and there are large errors in quantitative detection applications, and the detection limit can only reach the μg level
However, there is no report on the detection method for Bt insecticidal protein residues in mouse tissues such as liver, kidney, pancreas and intestinal tract. Detection Technology and Products
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[0020] The present invention is illustrated below with specific examples.
[0021] 1. The specific steps for preparing magnetosomes are as follows:
[0022] (1) After thawing and thawing the frozen magnetotactic bacterial cells with a wet weight of 24.5g, 25g or 25.5g, suspend them in 100ml of 10mM Tris-HCl, pH 8.0 buffer solution, ultrasonically oscillate for 10min, and ultrasonically oscillate on a Branson model 450, 20kHz; under the condition of 80W, the magnetotactic bacteria is Magnetospirillum magneticum AMB-1, purchased from ATCC, No. 700264;
[0023] (2) The suspension was centrifuged for 15min (8,000×g), and the obtained precipitate was resuspended with 100ml of 10mM Tris-HCl, pH 8.0 buffer solution, and then sonicated again; the sonication was performed under the condition of Branson model 450, 20kHz; 80W, Treat for 10 minutes;
[0024] (3) Collect the supernatant after centrifugation into a beaker, place it on a 2.5cm×12cm bar magnet for 1 hour, absorb the non-mag...
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The invention relates to an immune magnetosome for detecting Bt insecticidal protein in mice tissue and a preparation method thereof. The immune magnetosome is coated by anti-Bt insecticidal protein polyclonal antibody with the particle size being 50nm. The preparation method of the immune magnetosome comprises the following steps of: performing ultrasonic disruption and magnetic adsorption to obtain purified magnetosome of magnetotactic bacteria, mixing adjuvant and Bt insecticidal protein, immunizing mice, performing separation and purification to obtain mouse anti Bt insecticidal protein polyclonal antibody and coupling the antibody and the magnetosome by a carboxy-amino chemical coupling method to obtain the immune magnetosome for detecting Bt insecticidal protein in mice tissue. By chemiluminescence EIA detection, the immune magnetosome provided by the invention has a luminous unit of 83kcounts / microgram magnetosome. The correlation of luminous intensity and Bt protein concentration shows that as the protein concentration goes up, the luminous intensity goes down with a range being 1-103ng / ml and with a minimum detectable concentration being 1ng / ml. The method for the practical detection of Bt insecticidal protein content in mice tissue has advantages of high sensitivity, stable result and simple operation, and shows a good application prospect.
Description
technical field [0001] The invention relates to a preparation method of an immune magnetosome for detecting Bt insecticidal protein in mouse tissue. Background technique [0002] Magnetotactic bacteria are a kind of bacteria with magnetotactic behavior, which contain nano-magnetic particles-magnetosomes coated by membranes. Magnetosomes are single magnetic domain crystals with permanent magnetism, the size is tens of nanometers, and most of them are magnetite (Fe 3 o 4 ) properties, a few pyrite (Fe 3 S 4 )nature. Usually, multiple magnetosomes are arranged in chains in bacteria to form a biological magnetic compass, which orients magnetotactic bacteria in the geomagnetic field, thereby simplifying their search for a suitable microaerobic environment for growth. Compared with artificial magnetic nanomaterials, some characteristics of magnetosomes are more obvious. For example, under the strict control of the biomineralization mechanism, the crystal size is highly unifor...
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IPC IPC(8): G01N33/68G01N33/531
Inventor 吴刚潘卫东
Owner WUHAN UNIV OF TECH
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