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Effective separation method of nibea albiflora intestinal epithelial cells

A technology of intestinal epithelial cells and separation method, which is applied in the field of effective separation of intestinal epithelial cells of Huanggu, can solve the problems of poor protease stability and cost reduction, and achieves good structural stability, reduced cost, and improved structural stability. Effect

Active Publication Date: 2021-06-25
MARINE FISHERIES RES INST OF ZHEJIANG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to overcome the problem of poor protease stability in the separation process of existing intestinal wall epithelial cells, the present invention discloses an effective separation method for intestinal epithelial cells of yellow croaker. The protease is immobilized on the carrier, which makes the immobilized protease have strong heat resistance and wider pH range, ensures the stable performance of the immobilized protease, facilitates the control of the protein digestion process, and makes the extraction and separation process of the whole intestinal epithelial cells more efficient Convenient and effective, and the immobilized protease can be used repeatedly to reduce costs

Method used

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  • Effective separation method of nibea albiflora intestinal epithelial cells
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  • Effective separation method of nibea albiflora intestinal epithelial cells

Examples

Experimental program
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Effect test

Embodiment 1

[0057] 1. The steps for preparing the immobilized protease:

[0058] A. Make a preliminary carrier structure: Zn(NO 3 ) 2 ·6H 2 O according to 1.02g: 70mL dispersed in ethanol, ultrasonic treatment for 25min to get Zn 2+Solution, mix trimesic acid, vinyl terephthalic acid and N,N-dimethylformamide according to the mass ratio of 1:1.1:3 to obtain a mixed solution, stir for 35 minutes to obtain a ligand solution, and then mix the mixed solution Quickly pour Zn 2+ In the solution, stir for 1.2h at 88°C; then add tetraethylenepentamine solution, raise the temperature to 135°C and stir for 2.3h, then cool down to room temperature to form an ordered framework structure; Zn 2+ The volume ratio of the solution, the mixed solution and the tetraethylenepentamine solution is 1:3.5:0.7; the ordered framework structure is added to the mixed solution of styrene monomer and lauroyl peroxide, the temperature is raised to 75°C, and the temperature is kept for 22h. The preliminary carrier ...

Embodiment 2

[0066] 1. The steps for preparing the immobilized protease:

[0067] A. Make a preliminary carrier structure: Zn(NO 3 ) 2 ·6H 2 O was dispersed in ethanol according to 1g:70mL, and ultrasonically treated for 30min to obtain Zn 2+ solution, mix trimesic acid, vinyl terephthalic acid and N,N-dimethylformamide according to the mass ratio of 1:1:4 to obtain a mixed solution, stir for 30 minutes to obtain a ligand solution, and then mix the mixed solution Quickly pour Zn 2+ In the solution, stir for 1 hour at 80°C; then add tetraethylenepentamine solution, raise the temperature to 130°C and stir for 2.5 hours, then cool down to room temperature to form an ordered framework structure; Zn 2+ The volume ratio of the solution, the mixed solution and the tetraethylenepentamine solution is 1:3:0.8; the ordered framework structure is added to the mixed solution of styrene monomer and lauroyl peroxide, the temperature is raised to 80°C, and the temperature is kept for 20, The prelimin...

Embodiment 3

[0075] 1. The steps for preparing the immobilized protease:

[0076] A. Make a preliminary carrier structure: Zn(NO 3 ) 2 ·6H 2 O according to 1.05g: 70mL dispersed in ethanol, ultrasonic treatment for 30min to get Zn 2+ Solution, mix trimesic acid, vinyl terephthalic acid and N,N-dimethylformamide according to the mass ratio of 1:1.2:2 to obtain a mixed solution, stir for 40 minutes to obtain a ligand solution, and then mix the mixed solution Quickly pour Zn 2+ In the solution, stir for 1 hour at 95°C; then add tetraethylenepentamine solution, raise the temperature to 140°C and stir for 2 hours, then cool down to room temperature to form an ordered framework structure; Zn 2+ The volume ratio of solution, mixed solution and tetraethylenepentamine solution is 1:4:0.6; add the ordered framework structure into the mixed solution of styrene monomer and lauroyl peroxide, raise the temperature to 70°C, and keep it warm for 24h. The preliminary carrier structure is obtained; the...

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Abstract

The invention relates to the technical field of cell separation, and discloses an effective separation method of nibea albiflora intestinal epithelial cells aiming at the problem of poor protease stability in the existing intestinal wall epithelial cell separation process. The effective separation method comprises the following steps: soaking nibea albiflora in seawater containing penicillin and streptomycin for fasting for 1-2 days; separating the intestinal tract, cutting off the intestinal wall, putting into a PBS (Phosphate Buffer Solution), and removing fat and peritoneum on the intestinal tract; cutting the intestinal wall into pieces to obtain intestinal tissue blocks, cleaning the intestinal tissue blocks with a DMEM / F12 culture solution, centrifuging the intestinal tissue blocks for 2-4 times; adding intestinal tract tissue blocks and immobilized protease digestive juice at the same time, and carrying out enzymolysis digestion; and stopping digestion, and adding a DMEM / F12 culture solution for later use after treatment. The protease carrier with high loading capacity is introduced, and the digestion protease is fixed on the carrier, so that the immobilized protease has higher heat resistance and wider pH value range, and the stable performance of the immobilized protease is ensured.

Description

technical field [0001] The invention relates to the technical field of cell separation, in particular to an effective method for separating intestinal epithelial cells of yellow croaker. Background technique [0002] Cells are the basic unit of the morphological structure and functional activities of organisms. A cell is different from any structural unit in the non-living world. Its most unique attribute is that it is an open system that can survive independently and perform self-regulation. It is in a dynamic balance under the condition of exchanging matter, energy and information with the outside world. . The cells of all organisms have the ability to respond adaptively to changes in environmental nutrient conditions. Intestinal cells are not only responsible for the digestion and absorption of nutrients by the fish body, but also have a strong immune regulation function, which is an important line of defense for fish to resist harmful substances and microorganisms in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N11/14C12N11/082
CPCC12N5/0679C12N5/0625C12N11/14C12N11/082C12N2509/00C12N2509/10Y02A40/81
Inventor 王立改汪波谭朋徐冬冬陈睿毅竺奇慧
Owner MARINE FISHERIES RES INST OF ZHEJIANG