CN102321578B - Purification method of marine bivalve shellfish blood cell - Google Patents
Purification method of marine bivalve shellfish blood cell Download PDFInfo
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- CN102321578B CN102321578B CN 201110195020 CN201110195020A CN102321578B CN 102321578 B CN102321578 B CN 102321578B CN 201110195020 CN201110195020 CN 201110195020 CN 201110195020 A CN201110195020 A CN 201110195020A CN 102321578 B CN102321578 B CN 102321578B
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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
The invention belongs to the purifying field of marine organisms immunocyte, particularly the purification process of a kind of ocean bivalve shellfish hemocyte.
Background technology
The recycle system of bivalve shellfish belongs to open, heart is surrounded by a thin and transparent pericardial sac film, contain a large amount of blood in the venous sinus, at mantle a large vascular system is arranged, fine and close vasoganglion almost is covered with on whole mantle, so after puncturing closed shell flesh and mantle, a large amount of blood ooze out and collect in the mantle cavity.The cell that shellfish participates in cellular immunization is hemocyte, and it is the main barrier of resisting the invasion and attack of exotic disease pathogenic microorganism in the body of shellfish, hemocyte by participate in embedding, engulf, tubercle forms, the processes such as cell clot and wound repair play immunologic defence function.
In recent years, along with the expansion of shellfish culture scale, shellfish disease is day by day serious, has caused heavy economic losses for the shellfishery of China.So study shellfish immunology, the particularly research of shellfish hemocyte, become the focus of research to solve the disease problem that occurs in the shellfish culture, these researchs need to obtain the shellfish hemolymph of lot of pure.
Existing blood-sampling method is the pericardial sac that finds shellfish, directly insert pericardial sac with disposable syringe and extract blood, but there are a lot of drawbacks in this blood-sampling method: one, and amount for taking blood is few; Its two, institute gets and contains a lot of mucus and impurity in the blood, the hemocyte that obtains is impure.
Summary of the invention
Technical problem to be solved by this invention provides the purification process of a kind of ocean bivalve shellfish hemocyte, and the method is simple to operate, and is low for equipment requirements, may obtain the lot of pure hemocyte.
The purification process of a kind of ocean of the present invention bivalve shellfish hemocyte comprises:
(1) fresh ocean bivalve shellfish blood is mixed with antithrombotics, get mixed solution;
(2) above-mentioned mixed solution is filtered, then natural subsidence removes the top layer; Carry out again centrifugally, remove supernatant liquor, wash twice with PBS at last, resuspended hemocyte, and get final product.
Ocean bivalve shellfish described in the step (1) is the ocean bivalve shellfishs such as philippine clam whelp, clam, clam, the razor clam of hanging, mud blood clam, moerella irideseens or scallop.
The prescription of antithrombotics is with NaCl 8g, Na described in the step (1)
2HPO
42.9g, KCl 0.2g, KH
2PO
40.2g be dissolved in 1000ml distilled water, then add EDTA 5g, 60 ℃ of lower heated and stirred and get final product.
Ocean bivalve shellfish blood described in the step (1) is mixed into according to volume ratio 1:1 with antithrombotics and mixes.
Being filtered into described in the step (2) filtered mixed solution through double-deck 300 mesh sieve thin,tough silk.
The condition of the natural subsidence described in the step (2) is for to leave standstill 12h in 4 ℃.
Removal top layer described in the step (2) is for to suck top layer 4ml with liquid-transfering gun.
Centrifugal described in the step (2) is in 4 ℃ of centrifugal 20min, and rotating speed is under this rotating speed of 300 * g(, and most of shellfish hemocyte all can sedimentation in chip-proof situation).
PBS washes at twice o'clock in 4 ℃ of centrifugal 5min in the step (2), and rotating speed is that 300 * g(low temperature can guarantee that the hemocyte endoenzyme is alive, behind the centrifugal 5min, and the basic sedimentation of shellfish hemocyte, fragment and impurity are suspended among the pbs); Hemocyte concentration is about 10 in the resuspended hemocyte
6Cells/ml.
The fresh ocean bivalve shellfish blood that adopts among the present invention can be used the several different methods collection, and wherein a kind of preferred acquisition mode is:
A: support temporarily: according to the life habit of different bivalve shellfishs, in the filtering sea of its optimal temperature and salinity, support temporarily 24h;
B: sterilize and open shell: with shellfish shell, single-edge blade with 75% alcohol disinfecting after again with sterilization seawater flushing three times, determine the position of closed shell flesh, insert between two shells with single-edge blade, draw gently the closed shell flesh of shellfish, but make the shell free-open-close;
C: blood sampling: scratch in the mantle cavity that rear blood collects in shell, draw shellfish blood with disposable dropper, avoid being drawn onto other impurity, continue to draw, until reserve without a large amount of blood.
Present method is simple, need not increase plant and instrument, can carry out blood cell collection to the ocean bivalve shellfish, for the research of shellfish hemocyte provides effective technique means.
The present invention has improved the prescription of antithrombotics, and blood is carried out purifying, has solved the many problems of hemocyte impurity in the shellfish hemocyte research process, and the hemocyte that obtains lot of pure is had unusual effect.
Beneficial effect:
Compared with prior art, the invention has the advantages that:
(1) the inventive method is simple to operate, and is low for equipment requirements, may obtain the lot of pure hemocyte
(2) the present invention is applicable to all ocean bivalve shellfishs, is preferably philippine clam whelp, clam, clam, the razor clam of hanging, mud blood clam, moerella irideseens, scallop;
(3) among the present invention with the mixed solution of the shellfish blood that is drawn to and antithrombotics after filtration, leave standstill, centrifugal, resuspended after, make resulting hemocyte purer;
(4) the present invention adopts the antithrombotics prescription of improvement, makes the hemocyte of acquisition agglutination reaction not occur, and the blood cell shape that obtains is good.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims limited range equally.
Embodiment 1
(1) fresh mud blood clam blood is mixed according to volume ratio 1:1 with antithrombotics, get mixed solution; Wherein antithrombotics is with 8g NaCl, 2.9g Na
2HPO
4, 0.2g KCl, 0.2g KH
2PO
4Be dissolved in 1000ml distilled water, add afterwards 5g EDTA, the dissolving in two hours of 60 ℃ of heated and stirred makes;
(2) above-mentioned mixed solution is filtered, then natural subsidence removes the top layer; Carry out centrifugally again, remove supernatant liquor, wash twice with PBS at last, resuspended hemocyte namely obtains pure shellfish hemocyte, can deposit for subsequent use in-20 ℃.
Embodiment 2
(1) fresh clam blood is mixed according to volume ratio 1:1 with antithrombotics, get mixed solution; Wherein antithrombotics is with 8g NaCl, 2.9g Na
2HPO
4, 0.2g KCl, 0.2g KH
2PO
4Be dissolved in 1000ml distilled water, add afterwards 5g EDTA, the dissolving in two hours of 60 ℃ of heated and stirred makes;
(2) above-mentioned mixed solution is deposited in the 5ml centrifuge tube after double-deck 300 mesh sieve thin,tough silk filter, 4 ℃ of the mixed solutions of the shellfish blood after filtering and antithrombotics are left standstill 12h, with liquid-transfering gun top layer 4ml is sucked afterwards; Be 300 * g) at 4 ℃ of centrifugal 20min(rotating speeds again, remove supernatant liquor, wash (300 * g, 4 ℃ of centrifugal 5min), resuspended hemocyte (about 10 twice with PBS at last
6Cells/ml), namely obtain pure shellfish hemocyte, can deposit for subsequent use in-20 ℃.
Claims (8)
1. the purification process of an ocean bivalve shellfish hemocyte comprises:
(1) fresh ocean bivalve shellfish blood is mixed with antithrombotics, get mixed solution; Wherein, the prescription of antithrombotics is with NaCl 8g, Na
2HPO
42.9g, KCl 0.2g, KH
2PO
40.2g be dissolved among the distilled water 1000ml, then add EDTA 5g, 60 ℃ of lower heated and stirred and get final product;
(2) above-mentioned mixed solution is filtered, then natural subsidence removes the top layer; Carry out again centrifugally, remove supernatant liquor, wash twice with PBS at last, resuspended hemocyte, and get final product.
2. the purification process of a kind of ocean according to claim 1 bivalve shellfish hemocyte, it is characterized in that: the ocean bivalve shellfish described in the step (1) is philippine clam whelp, clam, clam, the razor clam of hanging, mud blood clam, moerella irideseens or scallop.
3. the purification process of a kind of ocean according to claim 1 bivalve shellfish hemocyte, it is characterized in that: the ocean bivalve shellfish blood described in the step (1) is mixed into according to volume ratio 1:1 with antithrombotics and mixes.
4. the purification process of a kind of ocean according to claim 1 bivalve shellfish hemocyte, it is characterized in that: being filtered into through double-deck 300 mesh sieve thin,tough silk described in the step (2) filtered.
5. the purification process of a kind of ocean according to claim 1 bivalve shellfish hemocyte, it is characterized in that: the condition of the natural subsidence described in the step (2) is for to leave standstill 12h in 4 ℃.
6. the purification process of a kind of ocean according to claim 1 bivalve shellfish hemocyte, it is characterized in that: the removal top layer described in the step (2) is for to suck top layer 4ml with liquid-transfering gun.
7. the purification process of a kind of ocean according to claim 1 bivalve shellfish hemocyte is characterized in that: centrifugal in 4 ℃ of centrifugal 20min described in the step (2), rotating speed is 300 * g.
8. the purification process of a kind of ocean according to claim 1 bivalve shellfish hemocyte, it is characterized in that: PBS washes at twice o'clock in 4 ℃ of centrifugal 5min in the step (2), and rotating speed is 300 * g; Hemocyte concentration is 10 in the resuspended hemocyte
6Cells/ml.
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| CN 201110195020 CN102321578B (en) | 2011-07-13 | 2011-07-13 | Purification method of marine bivalve shellfish blood cell |
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| CN 201110195020 CN102321578B (en) | 2011-07-13 | 2011-07-13 | Purification method of marine bivalve shellfish blood cell |
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| CN102321578A CN102321578A (en) | 2012-01-18 |
| CN102321578B true CN102321578B (en) | 2013-04-17 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103994963A (en) * | 2014-04-29 | 2014-08-20 | 中国水产科学研究院东海水产研究所 | Method used for detecting ostrea plicatula eosinophilic and basophilic granulocyte phagocytic ability |
| CN105476646B (en) * | 2016-01-06 | 2019-04-09 | 上海海洋大学 | A kind of extracting method of efficient bivalve shellfish hemolymph |
| CN107817259B (en) * | 2017-10-30 | 2020-05-08 | 中国水产科学研究院黄海水产研究所 | Method for collecting blood cells of shellfish in Scapharidae of seawater and observing microstructure |
| CN107988140B (en) * | 2018-01-25 | 2021-02-02 | 鲁东大学 | Abalone blood cell anticoagulant protective agent and preparation method thereof |
| CN108396008B (en) * | 2018-01-25 | 2021-03-23 | 鲁东大学 | Abalone blood cell processing method |
| CN110794024B (en) * | 2019-11-30 | 2022-05-10 | 中国海洋大学 | Standardized determination method for shellfish blood physiological indexes |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101846685A (en) * | 2010-06-03 | 2010-09-29 | 中国海洋大学 | Enzyme-linked immunoassay kit for chlamys ferreri blood cells and preparation method thereof |
| CN101899425A (en) * | 2010-06-03 | 2010-12-01 | 中国海洋大学 | Method for separating and purifying scallop phenol oxidase |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101846685A (en) * | 2010-06-03 | 2010-09-29 | 中国海洋大学 | Enzyme-linked immunoassay kit for chlamys ferreri blood cells and preparation method thereof |
| CN101899425A (en) * | 2010-06-03 | 2010-12-01 | 中国海洋大学 | Method for separating and purifying scallop phenol oxidase |
Non-Patent Citations (1)
| Title |
|---|
| 刘东武，等.菲律宾蛤仔、中国蛤蜊、文蛤和紫石房蛤血细胞的分类研究.《水产科学》.2005,第24卷(第10期),5－7. * |
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Address after: 200090 Shanghai military road, Yangpu District, No. 300 Applicant after: East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences Address before: 200090 No. 100, military road, Shanghai, Yangpu District Applicant before: East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences |
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