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Separation method of corn endosperm protein body

A separation method, the technology of corn endosperm, applied in the field of separation of corn endosperm protein body, can solve the problem of lack of separation of high-purity active endosperm protein body

Active Publication Date: 2020-04-03
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still a lack of technology for isolating high-purity active endosperm protein bodies

Method used

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  • Separation method of corn endosperm protein body
  • Separation method of corn endosperm protein body
  • Separation method of corn endosperm protein body

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1) Take W22 corn kernels 20 days after pollination, grind them into powder with liquid nitrogen, resuspend 10 g in solution A, let stand on ice for 15 minutes, and centrifuge at 300 g for 30 minutes at 4°C to remove impurities such as cell debris;

[0034] 2) The supernatant was subjected to a coarse discontinuous sucrose density gradient (1.0 M / L→1.5 M / L→2.0 M / L), ultracentrifuged at 35,100 rpm for 1 h at 4°C, and fraction 2 at the 1.5 M→2.0 M sucrose interface was collected Repeat the density gradient once after diluting with Solution B (10 mM Tris-HCl pH 8.5, 10 mM KCl);

[0035]3) After diluting the component 2-fold solution B at the 1.5 M→2.0 M sucrose interface, pass through a fine discontinuous sucrose density gradient (40% w / w → 45% → 50% → 55% → 60%), at 4°C , 35100 rpm ultracentrifugation for 1 h, take 50%-55% of the component between 2 times solution B and dilute it, repeat the density gradient once;

[0036] 4) After diluting the 50%-55% component 2 times s...

Embodiment 2

[0039] 1) Take B73 corn kernels 25 days after pollination, grind them to powder with liquid nitrogen, take 10 g and resuspend in solution A, let stand on ice for 15 minutes, centrifuge at 300 g for 30 minutes at 4°C to remove impurities such as cell debris;

[0040] 2) The supernatant was subjected to a coarse discontinuous sucrose density gradient (1.0 M / L→1.5 M / L→2.0 M / L), ultracentrifuged at 35,100 rpm for 1 h at 4°C, and fraction 2 at the 1.5 M→2.0 M sucrose interface was collected Repeat the density gradient once after diluting with Solution B (10 mM Tris-HCl pH 8.5, 10 mM KCl);

[0041] 3) After diluting the component 2-fold solution B at the 1.5 M→2.0 M sucrose interface, pass through a fine discontinuous sucrose density gradient (40% w / w → 45% → 50% → 55% → 60%), at 4°C , 35100 rpm ultracentrifugation for 1 h, take 50%-55% of the component between 2 times solution B and dilute it, repeat the density gradient once;

[0042] 4) After diluting the 50%-55% component 2 tim...

Embodiment 3

[0045] 1) After continuous sucrose density gradient centrifugation, collect samples sequentially from top to bottom, 600 μl each, and use methanol-chloroform extraction to remove high-density sucrose in the sample;

[0046] 2) Take the protein sample treated above for SDS-PAGE, and transfer the protein to a nitrocellulose membrane;

[0047] 3) Western Blot with antibodies to protein body, endoplasmic reticulum, mitochondria and peroxisome Marker protein to detect the protein samples obtained from the above treatment;

[0048] 4) Incubate the primary antibody for two hours, wash the membrane with TBST, 6 times, 5 minutes each time;

[0049] 5) Incubate the secondary antibody for two hours, wash the membrane with TBST, 6 times, 5 minutes each time, and develop it on the slide. For the results, see image 3 .

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Abstract

The invention relates to a method for separating a corn endosperm protein body. The method comprises the following steps: grinding fresh corn grains pollinated for 20-25 days with liquid nitrogen into a powder, resuspending the powder in a solution (comprising 10 mM Tris-HCl with the pH of 8.5, 10 mM KCl, 5 mM MgCl2, 7.2% sucrose, 1 mM DTT, 1 mM PMSF and a Roche mixed protease inhibitor), and centrifuging 300 g of the mixed solution for 30 minutes to remove cell debris; centrifuging the supernatant with a coarse-level discontinuous sucrose density gradient (1.0 M / L->1.5 M / L->2.0 M / L) for two rounds, taking a component of a 1.5 M->2.0 M sucrose interface, diluting the component with a solution B (comprising 10 mM Tris-HCl with the pH of 8.5, and 10 mM KCl) with the amount two times that of the component, and centrifuging the mixed solution with a fine-level discontinuous sucrose density gradient (40% w / w->45% w / w->50% w / w->55% w / w->60% w / w) for two rounds; taking a component between 50% and 55%, diluting the component with the solution B with the amount 2 times that of the component, and carrying out 30-60% continuous density gradient centrifugation; and collecting a fourteenth-layer component, diluting the fourteenth-layer component with the solution B with the amount 10 times that of the fourteenth-layer component, and centrifuging for 15 min at the speed of 13000 rpm, to obtain the high-pure active protein body. The method has extremely high extraction purity, maintains the biological activity, and provides an important technical means for production of active substances by using a protein body bioreactor.

Description

technical field [0001] The invention relates to a method for separating corn endosperm protein bodies. [0002] technical background [0003] corn( Zea mays ) is an annual gramineous herbaceous plant, which is an important food crop and an important source of feed, and is also the food crop with the highest total yield in the world. Corn is not only people's rations and "the king of feed", but also an important industrial raw material, which can be processed into more than 3,000 kinds of industrial products. As far as the utilization of corn is concerned, it has generally gone through three stages as human rations, livestock feed and industrial production raw materials. The proportion of ration consumption in the total consumption of corn is about 5%. Corn is one of the three major grain varieties, which plays a significant role in solving the problem of food and clothing for human beings. Today, corn is still an indispensable food on the table of people all over the world...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C07K1/14
CPCC07K14/415
Inventor 王桂凤王佳佳孙晓亮韩亮王刚宋任涛
Owner SHANGHAI UNIV
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