CN109294974A - A kind of hybridized prussian carp myeloid tissue cell line and its construction method are applied with it - Google Patents
A kind of hybridized prussian carp myeloid tissue cell line and its construction method are applied with it Download PDFInfo
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Abstract
The invention discloses a kind of hybridized prussian carp myeloid tissue cell lines and its construction method to apply with it, cell line classification entitled hybridized prussian carp myeloid tissue cell line (Spinal cord tissue cell lines of Carassius auratus gibelio, CSC), deposit number is CCTCC NO:C2018211.Above-mentioned cell line can be applied in the vaccine of the separation of carp herpesvirusⅡtype, culture, detection and preparation carp herpesvirusⅡtype.The present invention provides necessary technology platform for the separation identification of CyHV-2 and its research of complete biological characteristics, has established important basis for the prevention and control of crucian Hematopoietic Necrosis disease.
Description
Technical field
The invention belongs to aquatile cell and technical field of aquiculture disease prevention, specifically, being related to a kind of different
Pond crucian carp myeloid tissue cell line and its construction method is educated to apply with it.
Background technique
Carp herpesvirusⅡtype (Cyprinid herpesvirus II, CyHV-2) is otherwise known as simplex keratitis hematopoiesis
Organ necrosis disease viral (Herpesviral haematopoietic necrosis virus, HVHNV) or goldfish blood forming organ
Necrosis virus (Goldfish haematopoietic necrosis virus, GFHNV), other two kinds of blisters with cyprinid fish
Exanthema virus CyHV-1 (Carp pox) and CyHV-3 (Koi herpesvirus, KHV) belong to Alloherpesviridae section,
Cypriniviruse belongs to.CyHV-2 is reported for the first time in nineteen ninety-five, and the goldfish cultivated between 1992~1993 years to western Japan is made
At huge economic loss, the illness goldfish death rate is up to 100%.Subsequent other countries and area also have the disease to break out in succession
Report, there is mortality in the goldfish juvenile fish of the circulating water cultivation of U.S.West Coast one in spring in 1997, and the death rate is up to
80% or more, after be proved to be and infected by CyHV-2.The whole world that the international trade of ornamental fish has greatly facilitated the disease passes
It broadcasts, the goldfish that subsequent China Taiwan, Australia, Britain cultivate breaks out the disease in succession.Hungary reports cultivation within 2011
Pond crucian carp has also discovered CyHV-2 infection.From 2009, in China, the main culture zone Jiangsu Province of crucian has been broken out is drawn by CyHV-2
The crucian Hematopoietic Necrosis's disease risen, by mid-September, 2018, in Jiangsu Province the ground such as the shining sun, great Feng, Baoying County, Gaoyou, East Platform
Disease occurring area is more than 100,000 mu, and the death rate on the morbidity pool mouthful is up to 90%, caused by economic loss reached several hundred million members.With this
Meanwhile in provinces such as Hubei, Hunan, Jiangxi, Zhejiang, also go out CyHV-2 in illness crucian vivo detection in succession.The viral infection
Property it is strong, lethality is high, to crucian cultivation industry cause weight huge economic loss, seriously threaten the sound development of the industry.
Cell culture isolation technics is most accurate virus diagnostic method, usually World Organization for Animal Health (OIE)
The prefered method of the fishes virus detection of recommendation.But existing research discovery CyHV-2 is difficult to separate common cell in fishes virus
It is proliferated in system.Fat head carp cell (fathead minnow cells, FHM), carp epithelium oncocyte (epithelioma
Popuasum cuprini, EPC), grass carp gonad cell (grass carp ovary, CO), grass carp nephrocyte (grass carp
Kidney, CIK) it is insensitive to CyHV-2, only fancy carp fin cell (koi fin1, KF-1) can generate cytopathic effect
(CPE), but virus is after KF-1 cell was uploaded to for the 3rd~5 generation, and CPE disappears and can't detect viral nucleic acid.Although having been established
Hybridized prussian carp brain tissue cell system and sensitive to CyHV-2, but since inventor does not supply externally, also it is a lack of CyHV-2 sensitivity
Cell line, limit the research to CyHV-2, therefore establish the cell line sensitive to CyHV-2 and study the life of the cell line
Object characteristic carries out continuous passage to CyHV-2 and expands culture to further investigate the characteristic of virus, has great importance.
Summary of the invention
It is applied in view of this, the present invention provides a kind of hybridized prussian carp myeloid tissue cell lines and its construction method with it,
The cell line can be used for separating, detect and cultivate carp herpesvirusⅡtype；For carrying out serial passages in vitro to CyHV-2, expanding
Big culture is to further investigate the characteristic of the virus.
In order to solve the above-mentioned technical problem, the invention discloses a kind of hybridized prussian carp myeloid tissue cell line (Spinal
Cord tissue cell lines of Carassius auratus gibelio, CSC), deposit number is CCTCC NO:
C2018211。
The invention also discloses a kind of construction methods of above-mentioned hybridized prussian carp myeloid tissue cell line, including following step
It is rapid:
The processing of step 1, myeloid tissue: under aseptic condition take out hybridized prussian carp myeloid tissue, aseptic process be 30~
60mm3Tissue block, be put into the culture dish of the culture solution containing L15；
Step 2, originally culture: the tissue block in step 1 is equably put into T25 Tissue Culture Flask, tissue block is put
On one side upward, 3ml culture solution is added in culture bottle slowly culture bottle side comes overnight, tissue block is made to infiltrate culture solution, then
Upward by the one side of organized block, during which irregularly operation is once up to organizing block edge to grow cell, just by Tissue Culture Flask
Culture is set, replacement culture solution is primary within every 2~3 days；
Step 3, secondary culture: after originally culture hybridized prussian carp myeloid tissue cell grows up to single layer, trypsase is added and disappears
Change liquid and stand digestion 1~2 minute, hanged cell with culture solution, carries out secondary culture in such a way that 1 bottle passes 2 bottles, again to cell
After forming single layer, secondary culture next time is carried out according to the secondary culture method of above-mentioned cell, obtains a kind of hybridized prussian carp ridge
The cell line CSC of myeloid tissue.
Optionally, the hybridized prussian carp myeloid tissue cell L15 culture solution in the step 1 is to contain 10~20%V/V
Fetal calf serum, 100U/ml penicillin, 100 μ g/ml streptomysins, the culture solution of pH value 7.0~7.4.
Optionally, the tryptic digestive juice of the cell passage in the step 3 is 0.25%W/V trypsase-
EDTA digestive juice.
Optionally, the cell culture in step 2 and step 3, passage temperature be 23~25 DEG C, pH value 7.0~7.4.
The invention also discloses a kind of above-mentioned hybridized prussian carp myeloid tissue cell lines in the separation of carp herpesvirusⅡtype, training
Application in supporting and detecting.
Optionally, the carp herpesvirusⅡtype is carp herpesvirusⅡtype separation strains.
The invention also discloses a kind of above-mentioned hybridized prussian carp myeloid tissue cell lines in preparation carp herpesvirusⅡtype
Application in vaccine.
Optionally, the carp herpesvirusⅡtype is carp herpesvirusⅡtype separation strains.
Compared with prior art, the present invention can be obtained including following technical effect:
1) existing research discovery CyHV-2 is difficult to separate in common cell line in fishes virus and is proliferated.Fat head carp is thin
Born of the same parents (fathead minnow cells, FHM), grass carp gonad cell (grass carp ovary, CO), carp epithelium oncocyte
(epithelioma popuasum cuprini, EPC), grass carp nephrocyte (grass carp kidney, CIK) are to CyHV-
2 is insensitive, and only fancy carp fin cell (koi fin1, KF-1) can generate cytopathic effect (CPE), but virus is on KF-1 cell
After reaching for the 3rd~5 generation, CPE disappears and can't detect viral nucleic acid.And the present invention is susceptible to CyHV-2 different using being separately cultured
The myeloid tissue's cell for educating pond crucian carp establishes the hybridized prussian carp myeloid tissue cell line sensitive to CyHV-2.
2) present invention detects verifying to the carp herpesvirusⅡtype DNA for carrying out continuous passage on CSC cell, as a result confirms
Viral nucleic acid still can be detected in the 10th generation of CSC cell continuous passage；Cytopathic effect (CPE) is stable and obvious；To occur
The cell of pathological effect does ultra-thin electron microscopic section observation, and transmission electron microscope results are shown, there are the mature diseases of CyHV-2 in CSC cell
Virion and its reproduction process, it was demonstrated that CyHV-2 has good biological activity in CSC cell.
3) present invention establishes CyHV-2 sensitive cell line based on the separation, detection and culture of carp herpesvirusⅡtype
CSC provides necessary technology platform for the separation identification of CyHV-2 and its research of complete biological characteristics, makes for crucian
Important basis has been established in the prevention and control of blood organ necrosis disease.
4) application of the cell line CSC that the present invention establishes, the cell line further include but are not limited to: for being separately cultured carp
Application in herpesvirusⅡtype, detection and vaccine research and development, carries out carp herpesvirusⅡtype route of infection, sense on cell and level
Contaminate mechanism, pathogenic mechanism etc..
Certainly, it implements any of the products of the present invention it is not absolutely required to while reaching all the above technical effect.
Detailed description of the invention
The drawings described herein are used to provide a further understanding of the present invention, constitutes a part of the invention, this hair
Bright illustrative embodiments and their description are used to explain the present invention, and are not constituted improper limitations of the present invention.In the accompanying drawings:
Fig. 1 is different generation hybridized prussian carps myeloid tissue of the invention cell CSC schematic diagram；Wherein, a is primary hybridized prussian carp
Myeloid tissue's cell, b are the 3rd generation hybridized prussian carp myeloid tissue cell line；
Fig. 2 is the chromosome of the 26th generation hybridized prussian carp myeloid tissue cell line of the invention；
Fig. 3 is schematic diagram after hybridized prussian carp myeloid tissue of the present invention cell line infection CyHV-2；Wherein, a is normal cell,
B is the 4th day cell after infection, and c is the 6th day cell after infection.
Fig. 4 is the PCR testing result schematic diagram of different culture generation CyHV-2 of the invention；Wherein, M:100bp Marker；
1: 2nd generation CSC cell culture CyHV-2 cell toxicant；2: the 3 generation CSC cell culture CyHV-2 cell toxicants；3: the 4 generation CSC cells
Cultivate CyHV-2 cell toxicant；4: the 5 generation CSC cell culture CyHV-2 cell toxicants；5: the 6 generation CSC cell culture CyHV-2 cells
Poison；6: the 7 generation CSC cell culture CyHV-2 cell toxicants；7:CSC cell controls；8: negative control；9:CyHV-2 positive control；
Fig. 5 is the cell Electronic Speculum ultra-thin section schematic diagram of the 7th generation CyHV-2 cell toxicant infection CSC of the invention.
Specific embodiment
Carry out the embodiment that the present invention will be described in detail below in conjunction with embodiment, whereby to the present invention how application technology hand
Section solves technical problem and reaches the realization process of technical effect to fully understand and implement.
Agents useful for same of the embodiment of the present invention is purchased from biochemical shop if not otherwise specified；Experimental technique used, it is such as not special
It does not mentionlet alone bright, is routine techniques.
Material involved in the specific embodiment of the invention and reagent:
1) fish and strain are tested
Hybridized prussian carp, weight about 100~140g, body are about 17~20cm, try from Zhejiang Institute of Fresh Water Aquatic Products
Test base.It is temporarily supported 1 week before experiment in indoor.Carp herpesvirusⅡtype is separated by this laboratory and is saved.
2) main agents and consumptive material
L15 cell culture medium, penicillin/streptomycin, phosphate buffer (PBS), trypsase-EDTA, dimethyl sulfoxide
(DMSO), colchicine is purchased from Sigma company；Fetal calf serum is purchased from GIBICO company；DNAzol nucleic acid extracting reagent is
Invitrogen product；PCR is TaKaRa Products with rTaq enzyme, dNTPs.Tissue Culture Flask, pipette, cell cryopreservation tube
It is purchased from Corning company；Reagent needed for preparing transmission electron microscope ultra-thin section and consumptive material are purchased from Beijing Zhong Xing Berry company.
3) key instrument equipment
II grades of Biohazard Safety Equipments (Hfsafe-4B2)；Inverted microscope (Nikon)；Constant incubator (Sanyo, MIR-
153)；CCD camera (Nikon NIS Elements F530)；Low speed refrigerated centrifuge (EPPENDORF, 5424R)；Ultralow temperature
Refrigerator (MDF-382E)；Liquid nitrogen container (TYD-35-125)；Ultramicrotome (UC7, Leica)；Transmission electron microscope (H-
7650, Hitachi).
Embodiment 1
The foundation of hybridized prussian carp myeloid tissue cell line, its step are as follows:
(1) processing of myeloid tissue: taking out hybridized prussian carp myeloid tissue under aseptic condition, aseptic process is 30~60mm3
Tissue block, be put into the culture dish of the culture solution containing L15；
(2) originally culture: the tissue block in step (1) is equably put into T25 Tissue Culture Flask, the one of tissue block is put
Up, 3ml culture solution is added in culture bottle slowly culture bottle side comes overnight, so that tissue block is infiltrated culture solution, then will
The one side of organized block upward, during which irregularly just setting Tissue Culture Flask once up to organizing block edge to grow cell by operation
Culture, replacement culture solution is primary within every 2~3 days；The form of primary cell and passage cell is as shown in Figure 1.
(3) after originally culture hybridized prussian carp myeloid tissue cell grows up to single layer, the pancreas of 0.25%W/V secondary culture: is added
Protease-EDTA digestive juice stands digestion 1~2 minute, has hanged cell with culture solution, carries out passage training in such a way that 1 bottle passes 2 bottles
It supports, after cell forms single layer again, carries out secondary culture next time according to the secondary culture method of above-mentioned cell, obtain one
The cell line CSC of kind hybridized prussian carp myeloid tissue；
Wherein, the tryptic digestive juice of passage is 0.25%W/V trypsase-EDTA digestive juice, cell culture, biography
The temperature in generation is 23~25 DEG C, pH value 7.0~7.4；The hybridized prussian carp myeloid tissue cell culture fluid be containing 10~
20%V/V fetal calf serum, 100U/ml penicillin, 100 μ g/ml streptomysins, the L15 culture solution of pH value 7.0~7.4.
Above-mentioned construction method method is simple, easy.
The cell line has been committed to China typical culture collection center on September 28th, 2018 and has carried out preservation, and preservation is compiled
Number: CCTCC NO:C2018211, classification naming: hybridized prussian carp myeloid tissue cell line (Spinal cord tissue cell
Lines of Carassius auratus gibelio, CSC), address: Wuhan, China Wuhan University.
Embodiment 2
The biological characteristics of hybridized prussian carp myeloid tissue cell line CSC:
(1) morphology: cell type is fibroblast-like cells.
(2) growth characteristics: start after the CSC cell 30min of passage it is adherent, after 6h it is adherent completely；Population doubling time is
48h。
(3) stability: reaching for 42 generations before hybridized prussian carp myeloid tissue cell line CSC to the applying date, proliferation is stablized.
(4) it freezes and recovers:
Adherent quick after CSC cell recovery, growthform, situation and the cell not frozen are substantially similar, do not occur bright
Significant difference is other.Recovery cell Trypan Blue, counts through Cell counts, and the cell of about (78.56 ± 6.10) % is not colored, has
Cell activity.
(5) chromosome analysis
26th generation hybridized prussian carp myeloid tissue cell line CSC is in logarithmic growth phase, and the autumn of final concentration of 20 μ g/ml is added
Narcissus is plain, and cell is collected in digestion after 25 DEG C of incubation 4h, is pre-chilled with being added after the KCl solution Hypotonic treatment 25min of 0.075mol/L
Kano fixer, 1000rpm centrifugation 5min goes after supernatant to be fixed 3 times with the Kano fixer being pre-chilled again, each 15min.It is cold
It drips piece method and drips piece, dye 25min, micro- sem observation after drying with 5%Giemsa after drying.It is thin in 100 split coil methods of observation
In born of the same parents, the chromosome number of the 26th generation hybridized prussian carp myeloid tissue derived cell is 156 articles (Fig. 2), with triploid hybridized prussian carp
Karyological character coincide, i.e. whole chromosomes of hybridized prussian carp triploid 156, the chromosome without external source carp.
Embodiment 3
The application of hybridized prussian carp myeloid tissue cell line, process are as follows:
(1) acquisition and processing of carp herpesvirusⅡtype pathological material of disease are infected:
The just dead sick fish kidney, spleen and brain, myeloid tissue for infecting carp herpesvirusⅡtype of acquisition, shred and add etc.
Volume PBS is homogenized, and sterile tissue homogenate is prepared into through 0.22 μm of membrane filtration after 5000rpm4 DEG C of centrifugation 15min, after packing
Be stored in -80 DEG C it is spare；
(2) proliferation of the carp herpesvirusⅡtype in CSC:
CSC is cultivated to cell monolayer about 80%, discards culture medium after the cell maintenance medium of serum-free cleans 2 times, will
Above-mentioned processed pathological material of disease tissue homogenate supernatant 0.2ml is inoculated in CSC cell monolayer, 24 DEG C of absorption 1h, during which every 15~
20min weak vibrations culture bottle is once so as to uniform adsorption.After to be adsorbed, change serum-concentration be 2% L15 maintaining liquid after
Continuous 24 DEG C of cultures, observe cytopathy (CPE) day by day, until lesion is up to harvest virus after 80%, which is carp herpesviral II
The source of type separation strains.
(3) extraction of carp herpesvirusⅡtype cell toxicant nucleic acid and PCR detection:
Viral DNA will be extracted through DNAzol after CSC cell multigelation 2 times that obvious lesion occur.It is detected using PCR
The method of CyHV-2 detects the virus.PCR amplification primer
CyHV2-366F are as follows: GGACTTGCGAAGAGTTTGATTTCTAC；
CyHV2-366R are as follows: CCATAGTCACCATCGTCTCATC；
Take amplification condition are as follows: 94 DEG C initial denaturation 5 minutes, 94 DEG C be denaturalized 30 seconds, 60 DEG C anneal 30 seconds, 72 DEG C extend 30 seconds,
72 DEG C 5 minutes after 35 circulations.
It takes positive control dna template to do positive control simultaneously, CSC cell DNA and pure water is taken to do negative control respectively.Amplification
Product is identified through 2% (W/V) agarose gel electrophoresis.
(4) the Electronic Speculum observation of carp herpesvirusⅡtype infection CSC
The cytotoxic CSC cell of the 7th generation CyHV-2 will be infected after 2% glutaraldehyde is fixed, osmium tetroxide is fixed again, then is passed through
Ultra-thin section is carried out after dehydration embedding, after the double dyeing of acetic acid uranium-lead citrate, transmission electron microscope observing.
(5) result
The homogenate inoculation of carp herpesvirusⅡtype disease fish tissues with CSC cell monolayer 4d after occur cell rounding or vacuolation,
Pyknosis, formation plasomidum, space between cells become larger；After 6d CSC cell fusion and form multinucleate giant cell, cell monolayer falls off generation
, there is typical cytopathic effect (CPE) in seine phenomenon, and is passaged to the 10th generation CPE stabilization (Fig. 3), and normal CSC
Cell has no any variation.
The CyHV-2 cell culture DNA of different subculture is extracted, PCR detection is carried out, is obtained by two-wheeled PCR amplification
The segment of 357bp, it is (Fig. 4) in the same size with the amplified band of positive control, as a result it is determined as the positive.
The CyHV-2 virus of a large amount of maturations can be observed in the CSC cell that the 7th generation CyHV-2 is infected by transmission electron microscope observing
Particle illustrates that CyHV-2 has good biological activity (Fig. 5) in CSC cell, further demonstrates the present invention and is established
Hybridized prussian carp myeloid tissue cell line to virus sensibility, can be used for virus separation, culture and detection.Meanwhile it can make
For the cell model for studying carp herpesvirusⅡtype biological characteristics, the vaccine of carp herpesvirusⅡtype is prepared.
Above description has shown and described several preferred embodiments of invention, but as previously described, it should be understood that invention is not
It is confined to form disclosed herein, should not be regarded as an exclusion of other examples, and can be used for various other combinations, modification
And environment, and can be carried out within that scope of the inventive concept describe herein by the above teachings or related fields of technology or knowledge
Change.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of invention, then it all should be in the appended power of invention
In the protection scope that benefit requires.
Sequence table
<110>Chengdu Tianbang Biological Products Co., Ltd. of Zhejiang Institute of Fresh Water Aquatic Products
<120>a kind of hybridized prussian carp myeloid tissue cell line and its construction method are applied with it
<130> 2018
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<170> SIPOSequenceListing 1.0
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<213>artificial sequence (Artificial sequence)
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ggacttgcga agagtttgat ttctac 26
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<212> DNA
<213>artificial sequence (Artificial sequence)
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ccatagtcac catcgtctca tc 22
Claims (9)
1. a kind of hybridized prussian carp myeloid tissue cell line (Spinal cord tissue cell lines of Carassius
Auratus gibelio, CSC), which is characterized in that its deposit number is CCTCC NO:C2018211.
2. the construction method of hybridized prussian carp myeloid tissue described in claim 1 cell line, which is characterized in that including following step
It is rapid:
The processing of step 1, myeloid tissue: taking out hybridized prussian carp myeloid tissue under aseptic condition, aseptic process is 30~60mm3's
Tissue block is put into the culture dish of the culture solution containing L15；
Step 2, originally culture: the tissue block in step 1 is equably put into T25 Tissue Culture Flask, puts the one side of tissue block
Upward, 3ml culture solution is added in culture bottle slowly culture bottle side comes overnight, so that tissue block is infiltrated culture solution, then will have
Upward, during which irregularly Tissue Culture Flask is just being set training once up to organizing block edge to grow cell to the one side of tissue block by operation
It supports, replacement culture solution is primary within every 2~3 days；
Step 3, secondary culture: after originally culture hybridized prussian carp myeloid tissue cell grows up to single layer, tryptic digestive juice is added
Digestion 1~2 minute is stood, has hanged cell with culture solution, secondary culture is carried out in such a way that 1 bottle passes 2 bottles, is formed again to cell
After single layer, secondary culture next time is carried out according to the secondary culture method of above-mentioned cell, obtains a kind of hybridized prussian carp spinal cord group
The cell line CSC knitted.
3. construction method according to claim 2, which is characterized in that the hybridized prussian carp myeloid tissue in the step 1
Cell L15 culture solution be containing 10~20%V/V fetal calf serum, 100U/ml penicillin, 100 μ g/ml streptomysins, pH value 7.0~
7.4 culture solution.
4. construction method according to claim 2, which is characterized in that the tryptose of the cell passage in the step 3
Enzymic digestion liquid is 0.25%W/V trypsase-EDTA digestive juice.
5. construction method according to claim 2, which is characterized in that cell culture in step 2 and step 3, passage
Temperature is 23~25 DEG C, pH value 7.0~7.4.
6. hybridized prussian carp myeloid tissue cell line described in claim 1 separates, in culture and detection in carp herpesvirusⅡtype
Using.
7. application according to claim 7, which is characterized in that the carp herpesvirusⅡtype is carp herpesvirusⅡtype point
From strain.
8. application of the hybridized prussian carp myeloid tissue cell line described in claim 1 in the vaccine of preparation carp herpesvirusⅡtype.
9. application according to claim 7, which is characterized in that the carp herpesvirusⅡtype is carp herpesvirusⅡtype point
From strain.
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| CN111411087A (en) * | 2020-03-16 | 2020-07-14 | 中国水产科学研究院长江水产研究所 | Cyprinus carpioides herpesvirus II type low virulent strain and application thereof |
| CN111411087B (en) * | 2020-03-16 | 2021-03-23 | 中国水产科学研究院长江水产研究所 | Cyprinus carpioides herpesvirus II type low virulent strain and application thereof |
| CN113755438A (en) * | 2021-10-11 | 2021-12-07 | 浙江省淡水水产研究所 | Mandarin fish spinal cord tissue cell line and construction method and application thereof |
| CN113755438B (en) * | 2021-10-11 | 2023-08-08 | 浙江省淡水水产研究所 | Mandarin fish spinal cord tissue cell line and construction method and application thereof |
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