CN105675882A - Method for evaluating inhibition of marine fish mucus lectin for paralembus digitiformis - Google Patents

Method for evaluating inhibition of marine fish mucus lectin for paralembus digitiformis Download PDF

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Publication number
CN105675882A
CN105675882A CN201610076868.9A CN201610076868A CN105675882A CN 105675882 A CN105675882 A CN 105675882A CN 201610076868 A CN201610076868 A CN 201610076868A CN 105675882 A CN105675882 A CN 105675882A
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albumen
ltl
shield
fitc
polypide
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黄智慧
马爱军
夏丹丹
商晓梅
曲江波
杨志
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Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
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Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4724—Lectins

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
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  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
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  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
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  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
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Abstract

The invention discloses a method for evaluating inhibition of marine fish mucus lectin for paralembus digitiformis, and belongs to the technical field of aquatic product disease control. The method comprises activity inhibition evaluation and combining evaluation of Lily-type lectin protein for paralembus digitiformis. By means of the method, the inhibition of the marine fish Lily-type lectin (LTL) protein for the activity of paralembus digitiformis can be easily and conveniently evaluated, and the combining phenomenon of the LTL protein and paralembus digitiformis can be clearly observed through a fluorescence microscope.

Description

A kind of assessment seawater fish mucus le ctin suppresses the ciliophoran method of shield
Technical field
The invention belongs to Aquatic product disease prevention techniques field, assess seawater fish mucus le ctin Lily-typelectin (LTL) more particularly to one and suppress the ciliophoran method of shield.
Background technology
In recent years, along with improving constantly of aquaculture intensive degree, continuing to increase of cultivation density, incidence rate and the hazardness of Fish parasitic disease improve constantly, and cause very big loss to fish production. A large amount of mucus of Fish body surface secretion extensively cover fish surface and constitute the first door with extraneous contact; change at protection fish body opposing breeding environment and resist extraneous causal organism invasion and attack and play vital effect, especially parasitic function of resisting has been subject to the extensive concern of Chinese scholars. Wherein mucus le ctin (lectin) is as the important natural immunity factor in Fish epidermal mucus, but studies less to the immune defence mechanism of its causal organism. According to Fish body surface mucus le ctin structure, identify four kinds of type agglutinins at present altogether, it may be assumed that half lactadherin, C type agglutinin, Lily type agglutinin and L-rhamnose binding lectin, show molecular diversity widely. Wherein the third type is a kind of specificity mannose-binding lectin (pufflectin) extracted from red fin east (Takifugurubripes), itself and monocotyledon agglutinin (MMB) have high homology, and Suzuki is by its called after Lily-typelectin (LTL).
Before the present invention makes, phytohemagglutinin study mechanism in eliminating fish parasites cause of disease is comparatively deep, and it provides basic data for parasite control; And as the mucus le ctin Lily-typelectin of Fish self secretion, have and monocotyledon agglutinin (MMB) high homology, it is carried out the research of parasite inhibiting mechanism and will improve the Research Thinking that fish body autoimmune provides new. And parasite vigor inhibition is not had correlational study by Lily-typelectin at present.
In the parasite disease of marine fish, it is noticed gradually addicted to the disease caused by the ciliate of tissue by protozoacide is especially a kind of in recent years, high by the mortality of its initiation, Epidemic Scope is wide, thus balantidiasis is considered as one of most important disease in industrialized culture Fish.
Agglutinin is carried out the assessment that shield ciliate vigor suppresses by the present invention, and the method at home and abroad there is no report; Research and development for seawater fish anti-ciliate medicine are played positive, important impetus by this appraisal procedure.
Summary of the invention
The technical problem to be solved in the present invention is in that to provide a kind of assessment seawater fish mucus le ctin to suppress the ciliophoran method of shield.It is to the ciliophoran inhibition of shield to evaluate seawater fish mucus le ctin.
The present invention completes according to following operational aspect:
A kind of assessment seawater fish mucus le ctin suppresses the ciliophoran method of shield, and it includes Lily-typelectin albumen and the ciliophoran vigor of shield is suppressed assessment and combines assessment;
Described vigor suppresses assessment: rocks shield ciliate polypide gently and collects liquid, draws shield ciliate polypide and collects liquid, transfers in EP pipe, and the LTL albumen adding variable concentrations in EP pipe is hatched in 26-28 DEG C. Matched group is tested without LTL albumen, and under the microscope, different time points observes the survival of insect when variable concentrations and dead quantity, under each concentration conditions, arranges three repeating groups, and its mortality rate takes its average;
Described combination is assessed 1. fluorescent marker protein FITC-LTL and is prepared: LTL albumen to be crosslinked is put into cross-linking reaction liquid and dialyses three times; Being dissolved in DMSO by FITC, concentration is 1mg/ml; In protein: FITC is slowly added in LTL protein solution by the ratio of FITC=1mg:150 μ g, rocks gently and makes it mix homogeneously with albumen, dark place is reacted; Add NH4Cl solution, to its final concentration 50mmol/L, terminates reaction; Cross-linking agent is dialysed more than four times in PBS, limpid to dialysis solution; After dialysis terminates, receiving albumen and add BSA, to the final concentration of 10mg/ml of BSA, the thimerosal solution added, to its final concentration 0.02% (mass percent), adds sodium sarcosinate to its final concentration 0.2% (mass percent);
2. FITC-LTL is combined with shield ciliate: taking shield ciliate polypide and collect liquid, with the 100mg/mlFITC-LTL of the labelling dilution proportion according to volume ratio 1:4, mixed liquor incubated at room is overnight; Mixed liquor filters and uses sterilizing seawater flushing three times, to get rid of unnecessary agglutinin, mixed liquor after drawing wash is placed in glass slide, fluorescence intensity on fluorescence microscopy Microscopic observation polypide, to prove whether seawater fish Lily-typelectin (LTL) albumen is combined with polypide.
Further, described cross-linking reaction liquid composition and final concentration of: 7.56g/LNaHCO3, 1.06g/LNa2CO3, 7.36g/LNaCl.
Further, the observation wavelength 450-490nm, filter 515nm of described fluorescence microscope.
Present invention beneficial effect compared with prior art:
First, by the method adopting the present invention, can be relatively simple seawater fish Lily-typelectin (LTL) albumen be had suppresses the inhibition of shield ciliate vigor to be estimated, and by fluorescence microscope LTL albumen visible in detail and shield ciliate fixation phenomenon;
Second, the preparation method of FITC-LTL improves the efficiency of FITC label L TL albumen, labeling effciency reaches more than 90%, protein loss is relatively low, owing to shield ciliate small volume only has 20-50 μm, in cohesive process, shield ciliate polypide collects the FITC-LTL dilution proportion according to volume ratio 1:4 of liquid and labelling, namely ensure the efficiency that shield ciliate is combined with labelled protein, ensure that again FITC-LTL still has simultaneously and be combined activity and inhibition with shield ciliate.
Accompanying drawing explanation
The FITC-SmLTL albumen that Fig. 1 provides for the embodiment of the present invention and shield ciliate fixation phenomenon: arrow is expressed as the shield ciliate being fluorescently labeled;
After the addition D-MANNOSE that Fig. 2 provides for the embodiment of the present invention, FITC-SmLTL albumen and shield ciliate fixation phenomenon: arrow is expressed as the shield ciliate being fluorescently labeled.
Detailed description of the invention
Describe the present invention in detail in conjunction with accompanying drawing by the examples below and suppress the application in the ciliophoran appraisal procedure of shield at turbot epidermal mucus agglutinin Lily-typelectin (SmLTL) albumen.
Embodiment one, shield ciliate polypide are collected liquid and are prepared
1. the cerebral tissue of clip health turbot and muscular tissue, mix according to the ratio of 1:1, adds the sterilizing sea water after membrane filtration, prepares meat mud with tissue refiner;
2. adding the sterilizing sea water after the filtration of 50 times, 70 DEG C of water-bath 2-3 prepared into shield ciliate live body culture medium after individual hour, and the culture medium prepared is placed under 4 DEG C of conditions and preserves for a long time.
3. clip infects the turbot body portion lesion tissue of shield balantidiasis, and sterile working shreds in culture dish, instills the sterilizing sea water after the filtration of 15-20 times of volume, is placed in 18-20 DEG C of constant incubator quiescent culture overnight;
4. under the microscope (100 ×), polypide free in liquid in the culture dish after standing is drawn with pipettor, instill in 50ml centrifuge tube, it is equipped with the 30ml shield ciliate culture medium prepared, it is placed in 18-20 DEG C of constant incubator and cultivates 20-24 hour, draw 1ml culture fluid under microscope and count.
5. draw 20ml ciliate culture fluid, instill the funnel equipped with filter paper and be filtered; After filtration, the filtration reversely dripping filter wash paper with the 10ml sterilizing sea water filtered is paid no attention to, and drips wash pools to the centrifuge tube of sterilizing, it is thus achieved that 10ml polypide collects liquid, and counts under the microscope, and its concentration is 3.1 × 102/ml。
Embodiment two, SmLTL albumen suppress assessment to ciliophoran
Described SmLTL albumen is made up of 118 aminoacid, and molecular weight is 13.47KDa, and isoelectric point, IP is 8.52, and its aminoacid sequence is as follows:
1MHHHHHHNRNSISTDQELRKGEFLMSVNGEFKAIFQDDGNFVIYKWSPIWDTKTCGKNPF
61RVLLQPDNNLVMYDKLSKPVWATGTHSNQANQRMRLTLTDGGRLVLDKDGGEIWGAGG
This experiment carries out restructuring and prepares SmLTL albumen.
The 1.SmLTL albumen suppression appraisal procedure to ciliate vigor: rock polypide gently and collect liquid, draw 1ml and collect liquid, transfer in EP pipe, add in EP pipe variable concentrations LTL albumen (200,100,50,25,12.5ug/ml) hatch under 28 °, matched group test without agglutinin; Under the microscope, different time point observes the survival of insect when variable concentrations and dead quantity, (3,6,12,24,48h). Under each concentration conditions, having three repeating groups, its mortality rate takes its average.
As shown in table 1: along with SmLTL protein concentration increases, shield ciliate vigor being had significant impact, concentration is more big, and mortality rate is more high.
The assessment in conjunction with vigor of 2.SmLTL albumen and ciliate
(1) SmLTL albumen (concentration 1mg/ml) to be crosslinked is put into cross-linking reaction liquid three (cross-linking reaction liquid making method: 7.56gNaHCO3 of dialysis by fluorescent marker protein FITC-SmLTL preparation, 1.06gNa2CO3,7.36gNaCl, adds water and is settled to 1L); Being dissolved in DMSO by FITC, concentration is 1mg/ml; In the ratio of S:F (SmLTL:FITC)=1mg:150 μ g, FITC is slowly added in LTL protein solution, rocks gently and make it mix homogeneously with albumen, 4 DEG C, dark place reaction 8h; Add the NH of 5mol/L4Cl to final concentration 50mmol/L, 4 DEG C terminate reaction 2h; Cross-linking agent is dialysed more than four times in PBS, limpid to dialysis solution; After dialysis terminates, receiving SmLTL albumen and press the concentration addition BSA of 10mg/ml, the thimerosal adding 20%, to final concentration 0.02%, adds sodium sarcosinate final concentration 0.2%, and subpackage also preserves in-20 DEG C of dark places.
(2) FITC-SmLTL is combined with shield ciliate: the polypide taking 400ul collects liquid, and with the FITC-LTL (100mg/ml) of the labelling dilution proportion according to 1:4, incubated at room is overnight; Matched group, adds 1MD-mannose in above-mentioned steps, and incubated at room is overnight; The sterilizing seawater flushing filtered three times, to get rid of unnecessary agglutinin, draws 20 μ l mixed liquors and is placed in glass slide, fluorescence microscopy Microscopic observation (wavelength 450-490nm, filter 515nm).
Result is as shown in Figure 1: shield ciliate body surface has stronger fluorescence signal as seen from Figure 1, it was shown that SmLTL albumen and shield ciliate have fixation phenomenon;
Fig. 2 is for after adding D-MANNOSE, FITC-SmLTL albumen and shield ciliate fixation phenomenon: can be shown by figure, shield ciliate body surface fluorescence intensity reduces, and reason is owing to D-MANNOSE and SmLTL have fixation phenomenon, then inhibit SmLTL albumen and the ciliophoran combination of shield.
Table 1SmTL is to ciliate vigor inhibition analysis

Claims (3)

1. an assessment seawater fish mucus le ctin suppresses the ciliophoran method of shield, it is characterised in that it includes Lily-typelectin albumen and the ciliophoran vigor of shield is suppressed assessment and combines assessment, and Lily-typelectin albumen is called for short LTL albumen;
Described vigor suppresses assessment: rocks shield ciliate polypide gently and collects liquid, draws shield ciliate polypide and collects liquid, transfers in EP pipe, and the LTL albumen adding variable concentrations in EP pipe is hatched in 26-28 DEG C. Matched group is tested without LTL albumen, and under the microscope, different time points observes the survival of insect when variable concentrations and dead quantity, under each concentration conditions, arranges three repeating groups, and its mortality rate takes its average;
Described combination is assessed 1. fluorescent marker protein FITC-LTL and is prepared: LTL albumen to be crosslinked is put into cross-linking reaction liquid and dialyses three times; Being dissolved in DMSO by FITC, concentration is 1mg/ml; In protein: FITC is slowly added in LTL protein solution by the ratio of FITC=1mg:150 μ g, rocks gently and makes it mix homogeneously with albumen, dark place is reacted; Add NH4Cl solution, to its final concentration 50mmol/L, terminates reaction; Cross-linking agent is dialysed more than four times in PBS, limpid to dialysis solution; After dialysis terminates, receiving albumen and add BSA, to the final concentration of 10mg/ml of BSA, the thimerosal solution added, to its final concentration 0.02% (mass percent), adds sodium sarcosinate to its final concentration 0.2% (mass percent);
2. FITC-LTL is combined with shield ciliate: taking shield ciliate polypide and collect liquid, with the 100mg/mlFITC-LTL of the labelling dilution proportion according to volume ratio 1:4, mixed liquor incubated at room is overnight; Mixed liquor filters and uses sterilizing seawater flushing three times, to get rid of unnecessary agglutinin, mixed liquor after drawing wash is placed in glass slide, the fluorescence intensity on fluorescence microscopy Microscopic observation polypide, to prove whether seawater fish Lily-typelectin albumen is combined with polypide.
2. a kind of assessment seawater fish mucus le ctin according to claim 1 suppresses the ciliophoran method of shield, it is characterised in that the composition of described cross-linking reaction liquid and final concentration of: 7.56g/LNaHCO3, 1.06g/LNa2CO3, 7.36g/LNaCl.
3. assess seawater fish mucus le ctin according to claim 1 one kind and suppress the ciliophoran method of shield, it is characterised in that the observation wavelength 450-490nm, filter 515nm of described fluorescence microscope.
CN201610076868.9A 2016-02-04 2016-02-04 Method for evaluating inhibition of marine fish mucus lectin for paralembus digitiformis Pending CN105675882A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115119781A (en) * 2022-08-17 2022-09-30 中国海洋大学 Biological prevention and treatment method for ciliate disease

Citations (2)

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CN101698673A (en) * 2008-10-19 2010-04-28 中国水产科学研究院黄海水产研究所 Prawn white spot syndrome virus VP37p polypeptide fragment and application thereof
CN102492623A (en) * 2011-12-13 2012-06-13 中国水产科学研究院黄海水产研究所 Method for preparing and culturing culture medium for supravital culture of seawater scuticociliatida ciliates

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
CN101698673A (en) * 2008-10-19 2010-04-28 中国水产科学研究院黄海水产研究所 Prawn white spot syndrome virus VP37p polypeptide fragment and application thereof
CN102492623A (en) * 2011-12-13 2012-06-13 中国水产科学研究院黄海水产研究所 Method for preparing and culturing culture medium for supravital culture of seawater scuticociliatida ciliates

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115119781A (en) * 2022-08-17 2022-09-30 中国海洋大学 Biological prevention and treatment method for ciliate disease

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Application publication date: 20160615