Purification method of marine bivalve shellfish blood cell

A technology of bivalve mollusk and purification method, which is applied in the field of purification of marine biological immune cells, can solve the problems of blood cell impurity and less blood collection, and achieve the effect of simple operation, low equipment requirements and good shape of blood cells

Inactive Publication Date: 2013-04-17
EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The existing blood collection method is to find the pericardial cavity of the shellfish, and directly insert the pericardial cavity with a disposable syringe to draw blood, but there are many disadvantages in this blood collection method: first, the amount of blood taken is small; Contains a lot of mucus and impurities, the blood cells obtained are not pure

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] (1) Mix fresh cockle blood and anticoagulant at a volume ratio of 1:1 to obtain a mixed solution; the anticoagulant is 8g NaCl, 2.9g Na 2 HPO 4 , 0.2g KCl, 0.2g KH 2 PO 4 Dissolve in 1000ml of distilled water, then add 5g of EDTA, heat and stir at 60°C for two hours to dissolve;

[0032] (2) Filter the above mixture, settle naturally, and then remove the surface layer; then centrifuge, remove the supernatant, and finally wash twice with PBS, resuspend the blood cells, and obtain pure shellfish blood cells, which can be stored at -20°C Store for later use.

Embodiment 2

[0034] (1) Mix fresh clam blood and anticoagulant at a volume ratio of 1:1 to obtain a mixture; the anticoagulant is 8g NaCl, 2.9g Na 2 HPO 4 , 0.2g KCl, 0.2g KH 2 PO 4 Dissolve in 1000ml of distilled water, then add 5g of EDTA, heat and stir at 60°C for two hours to dissolve;

[0035] (2) Filter the above mixture through a double-layer 300-mesh sieve and store it in a 5ml centrifuge tube. Let the filtered mixture of shellfish blood and anticoagulant stand at 4°C for 12 hours, and then use a pipette gun to Absorb 4ml of the surface layer; then centrifuge at 4°C for 20min (rotating speed at 300×g), remove the supernatant, and finally wash twice with PBS (300×g, centrifuge at 4°C for 5min), resuspend the blood cells (about 10 6 cells / ml) to obtain pure shellfish blood cells, which can be stored at -20°C for later use.

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Abstract

The invention relates to a purification method of a marine bivalve shellfish blood cell, which comprises the steps that: (1) fresh marine bivalve shellfish blood is mixed with an anticoagulant to obtain mixed liquid; (2) the mixed liquid is filtered and naturally settled, and then a surface is removed; centrifugation is carried out, and supernatant is removed; and finally the liquid is washed by a phosphate buffer solution (PBS) for twice, and the blood cell is obtained after being suspended. The purification method of the marine bivalve shellfish blood cell is simple and feasible without adding any instrument and equipment, improves the prescription of the anticoagulant, purifies the blood, solves the problem that the blood cell has a plurality of impurities in a shellfish blood cell research process, and provides practical and effective technical means for the research on shellfish blood cells.

Description

technical field [0001] The invention belongs to the field of purification of marine biological immune cells, in particular to a method for purification of marine bivalve blood cells. Background technique [0002] The circulatory system of bivalve molluscs is open, the heart is surrounded by a thin and transparent pericardium, the venous sinuses contain a large amount of blood, there is a large vascular system on the mantle, and the dense vascular network is almost dense. It covers the entire mantle, so when the adductor muscle and the mantle are punctured, a large amount of blood seeps out and collects in the mantle cavity. The cells involved in cellular immunity of shellfish are hemocytes, which are the main barrier for shellfish to resist the invasion of foreign pathogenic microorganisms. Hemocytes play an immune defense role by participating in processes such as embedding, phagocytosis, nodule formation, cell clots, and wound repair. [0003] In recent years, with the ex...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/078
Inventor 李子牛来琦芳么宗利周凯应成琦
Owner EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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