CN109294974B - Carassius auratus gibelio spinal cord tissue cell line as well as construction method and application thereof - Google Patents
Carassius auratus gibelio spinal cord tissue cell line as well as construction method and application thereof Download PDFInfo
- Publication number
- CN109294974B CN109294974B CN201811231823.XA CN201811231823A CN109294974B CN 109294974 B CN109294974 B CN 109294974B CN 201811231823 A CN201811231823 A CN 201811231823A CN 109294974 B CN109294974 B CN 109294974B
- Authority
- CN
- China
- Prior art keywords
- spinal cord
- culture
- carassius auratus
- cord tissue
- cell line
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0669—Bone marrow stromal cells; Whole bone marrow
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
- A61P31/22—Antivirals for DNA viruses for herpes viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16051—Methods of production or purification of viral material
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Developmental Biology & Embryology (AREA)
- Oncology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Communicable Diseases (AREA)
- Cell Biology (AREA)
- Rheumatology (AREA)
- Hematology (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a Carassius auratus gibelio Spinal cord tissue cell line, a construction method and application thereof, wherein the cell line is classified as a Carassius auratus gibelio Spinal cord tissue cell line (Spinal cord tissue lines of Carassius auratus gibelio, CSC) with the preservation number of CCTCC NO: C2018211. the cell line can be applied to separation, culture, detection and preparation of the carp herpesvirus II vaccine. The invention provides a necessary technical platform for the separation and identification of CyHV-2 and the research of the complete biological characteristics thereof, and lays an important foundation for the prevention and control of crucian hematopoietic organ necrosis.
Description
Technical Field
The invention belongs to the technical field of disease prevention and control of aquatic organism cells and aquaculture, and particularly relates to a carassius auratus gibelio spinal cord tissue cell line and a construction method and application thereof.
Background
The type II herpesvirus of Cyprini (CyHV-2) is also known as herpesvirus hematopoietic necrosis virus (HVHNV) or Goldfish hematopoietic necrosis virus (GFHNV), and is in the Allopherevirus family, Cyprinivirus genus with the other two herpesviruses of Cyprinidae, CyHV-1(Carp pox) and CyHV-3(Koi herpesvirus, KHV). CyHV-2 was first reported in 1995, and caused huge economic loss to Japanese cultivated goldfish in 1992-1993, and the death rate of diseased goldfish reaches 100%. Later, other countries and regions have reported the outbreak, and the death rate of the juvenile goldfish cultured by circulating water in the west coast of the United states in 1997 is over 80 percent, and the juvenile goldfish is proved to be infected by CyHV-2. The international trade of the ornamental fish greatly promotes the global spread of the disease, and then goldfishes cultured in Taiwan, Australia and the great Britain in China are successively exposed to the disease. The bred silver crucian carps are reported to be infected by CyHV-2 in Hungarian in 2011. In 2009, hematopoietic necrosis of crucian caused by CyHV-2 is outbreak in Jiangsu province of main crucian culture areas in China, and as long as 9 middle-aged decades in 2018, the area of occurrence of diseases such as sunrise, Dafeng, Baohui, Gaoyou and east Taiwan in Jiangsu province exceeds 10 ten thousand mu, the death rate of disease pool mouths reaches 90%, and the economic loss caused by the diseases reaches hundreds of millions of yuan. Meanwhile, CyHV-2 is detected in the diseased crucian carp in the provinces of Hubei, Hunan, Jiangxi, Zhejiang and the like. The virus has strong infectivity and high lethality rate, causes great economic loss to the crucian breeding industry, and seriously threatens the healthy development of the industry.
Cell culture isolation techniques are the most accurate diagnostic methods for viral diseases and are generally the first choice for fish virus detection recommended by the world animal health Organization (OIE). However, it has been found that CyHV-2 is difficult to propagate in cell lines commonly used for fish virus isolation. Fat head carp cells (FHM), carp epithelial tumor cells (EPC), grass carp ovarian Cells (CO) and grass carp kidney Cells (CIK) are insensitive to CyHV-2, only koi fin1 (KF-1) can produce cytopathic effect (CPE), but after the virus is uploaded to KF-1 cells for 3-5 generations, CPE disappears and virus nucleic acid cannot be detected. Although a carassius auratus gibelio brain tissue cell line is established and is sensitive to CyHV-2, the inventor does not supply outside and lacks a CyHV-2 sensitive cell line to limit the research on the CyHV-2, so that the establishment of the CyHV-2 sensitive cell line and the research on the biological characteristics of the cell line have important significance in carrying out continuous subculture and expansion culture on the CyHV-2 to further research on the characteristics of viruses.
Disclosure of Invention
In view of the above, the invention provides a carassius auratus gibelio spinal cord tissue cell line, a construction method and an application thereof, wherein the cell line can be used for separating, detecting and culturing a carp herpesvirus II type; the method is used for in vitro continuous passage and expansion culture of CyHV-2 so as to deeply research the characteristics of the virus.
In order to solve the technical problems, the invention discloses a Carassius auratus gibelio Spinal cord tissue cell line (Spinal cord tissue lines of Carassius auratus gibelio, CSC) with a preservation number of CCTCC NO: C2018211.
the invention also discloses a construction method of the carassius auratus gibelio spinal cord tissue cell line, which comprises the following steps:
Optionally, the culture solution of carassius auratus gibelio spinal cord tissue cells L15 in the step 1 is a culture solution containing 10-20% of V/V fetal calf serum, 100U/ml of penicillin, 100 μ g/ml of streptomycin and having a pH value of 7.0-7.4.
Optionally, the trypsin digestion solution for cell passage in step 3 is 0.25% W/V trypsin-EDTA digestion solution.
Optionally, the temperature for cell culture and passage in the step 2 and the step 3 is 23-25 ℃, and the pH value is 7.0-7.4.
The invention also discloses application of the carassius auratus gibelio spinal cord tissue cell line in separation, culture and detection of the carp herpesvirus II.
Optionally, the herpesvirus of carp type ii is a herpesvirus of carp type ii isolate.
The invention also discloses an application of the carassius auratus gibelio spinal cord tissue cell line in preparation of a carp herpesvirus II type vaccine.
Optionally, the herpesvirus carpi type ii is a herpesvirus carpi type ii isolate.
Compared with the prior art, the invention can obtain the following technical effects:
1) CyHV-2 has been found to be difficult to propagate in cell lines commonly used for fish virus isolation. Fat carp cells (FHM), grass carp ovarian Cells (CO), carp epithelial carcinoma cells (EPC), and grass carp kidney Cells (CIK) are insensitive to CyHV-2, and only koi fin1 (KF-1) can produce cytopathic effect (CPE), but after the virus is uploaded to KF-1 cells for 3-5 generations, CPE disappears and virus nucleic acid cannot be detected. The invention adopts the separation culture of the spinal cord tissue cells of the carassius auratus gibelio susceptible to CyHV-2, and establishes the spinal cord tissue cell line of the carassius auratus gibelio susceptible to CyHV-2.
2) The invention detects and verifies the cyprinid herpesvirus II type DNA which is continuously passaged on the CSC cell, and the result proves that the virus nucleic acid can be still detected at the 10 th generation of the CSC cell after continuous passage; cytopathic effect (CPE) was stable and significant; the cells with pathological effect are observed by an ultrathin electron microscope section, and the results of a transmission electron microscope show that CyHV-2 mature virus particles and the replication process thereof exist in the CSC cells, so that the CyHV-2 has good biological activity in the CSC cells.
3) The invention establishes the CyHV-2 sensitive cell line CSC based on the separation, detection and culture of the carp herpesvirus II, provides a necessary technical platform for the separation and identification of CyHV-2 and the research of the complete biological characteristics of the CyHV-2, and lays an important foundation for the prevention and control of the crucian hematopoietic organ necrosis.
4) The present invention establishes a cell line CSC, and uses of the cell line include, but are not limited to: the method is applied to separation culture of the carp herpes virus II, detection and vaccine research and development, and develops the carp herpes virus II infection way, the infection mechanism, the pathogenic mechanism and the like on a cell and level.
Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is a schematic view of CSC of different generation carassius auratus gibelio spinal cord tissue cells; wherein, a is a primary carassius auratus gibelio spinal cord tissue cell, b is a 3 rd generation carassius auratus gibelio spinal cord tissue cell line;
FIG. 2 is the chromosome of the 26 th generation carassius auratus gibelio spinal cord tissue cell line of the present invention;
FIG. 3 is a schematic diagram of the carassius auratus gibelio spinal cord tissue cell line infected with CyHV-2; wherein, a is normal cell, b is cell at 4 days after infection, and c is cell at 6 days after infection.
FIG. 4 is a diagram showing the results of PCR detection of different generations of CyHV-2 in culture according to the present invention; wherein, M: 100bp Marker; 1: culturing CyHV-2 cytotoxicity on the 2 nd generation CSC cells; 2: culturing CyHV-2 cytotoxicity on 3 rd generation CSC cells; 3: culturing CyHV-2 cytotoxicity on the 4 th generation of CSC cells; 4: culturing CyHV-2 cytotoxicity on 5 th generation CSC cells; 5: culturing CyHV-2 cytotoxicity by 6 th generation CSC cells; 6: culturing CyHV-2 cytotoxicity on 7 th generation CSC cells; 7: CSC cell control; 8: negative control; 9: CyHV-2 positive control;
FIG. 5 is a schematic diagram of a thin section of a 7 th generation CyHV-2 cytotoxic CSC of the present invention using electron microscopy.
Detailed Description
The following embodiments are described in detail with reference to the accompanying drawings, so that the implementation process of the present invention for solving the technical problems and achieving the technical effects by applying technical means can be fully understood and implemented.
Reagents used in the examples of the present invention, unless otherwise specified, were purchased from biochemical stores; the experimental techniques used, if not specifically indicated, are conventional.
Materials and reagents involved in specific embodiments of the invention:
1) experimental fish and strain
The carassius auratus gibelio has the weight of about 100-140 g and the body length of about 17-20 cm, and is from the experimental base of fresh water aquatic product research institute in Zhejiang province. The experiment was temporarily kept indoors for 1 week. The carp herpesvirus type II is separated and stored in the laboratory.
2) Main reagent and consumable
L15 cell culture medium, penicillin/streptomycin, Phosphate Buffered Saline (PBS), trypsin-EDTA, dimethyl sulfoxide (DMSO), colchicine were purchased from Sigma; fetal bovine serum was purchased from GIBICO; the DNAzol nucleic acid extraction reagent is an Invitrogen product; rTaq enzyme and dNTPs for PCR were TaKaRa products. Cell culture flasks, pipettes, and cell cryopreservatives were purchased from Corning; reagents and consumables required for preparing ultrathin sections for transmission electron microscopy were purchased from Zhongxing Bairui, Beijing.
3) Main instrument equipment
A class II biosafety cabinet (Hfsafe-4B 2); inverted microscope (Nikon); thermostated incubator (Sanyo, MIR-153); a CCD camera (Nikon NIS Elements F530); low speed refrigerated centrifuge (EPPENDORF, 5424R); ultra-low temperature refrigerators (MDF-382E); a liquid nitrogen tank (TYD-35-125); microtomes (UC7, Leica); transmission electron microscope (H-7650, Hitachi).
Example 1
The method for establishing the carassius auratus gibelio spinal cord tissue cell line comprises the following steps:
(1) treatment of spinal cord tissue: taking out carassius auratus gibelio spinal cord tissue under aseptic condition, and carrying out aseptic treatment to 30-60 mm 3 The tissue block of (4) is placed in a culture dish containing L15 culture solution;
(2) primary culture: uniformly placing the tissue blocks obtained in the step (1) into a T25 cell culture bottle, enabling one surface of the tissue blocks to face upwards, adding 3ml of culture solution into the culture bottle, standing overnight, slowly turning over the side of the culture bottle to enable the tissue blocks to infiltrate the culture solution, enabling one surface with the tissue blocks to face upwards, performing irregular operation once until cells grow out of the edges of the tissue blocks, placing the cell culture bottle upright for culture, and replacing the culture solution once every 2-3 days; the morphology of primary cells and passaged cells is shown in FIG. 1.
(3) Subculturing: after the carassius auratus gibelio spinal cord tissue cells are primarily cultured to grow into a monolayer, 0.25% W/V trypsin-EDTA digestive liquid is added for standing and digesting for 1-2 minutes, the cells are suspended by the culture liquid, subculture is carried out in a mode of transferring 1 bottle to 2 bottles, after the cells form the monolayer again, next subculture is carried out according to the subculture method of the cells, and a cell line CSC of the carassius auratus gibelio spinal cord tissue is obtained;
wherein the trypsin digestive juice used for transfer is 0.25% W/V trypsin-EDTA digestive juice, the temperature for cell culture and passage is 23-25 ℃, and the pH value is 7.0-7.4; the carassius auratus gibelio spinal cord tissue cell culture solution is L15 culture solution containing 10-20% of V/V fetal calf serum, 100U/ml penicillin, 100 mu g/ml streptomycin and 7.0-7.4 of pH value.
The construction method is simple and easy to implement.
The cell line is submitted to China center for type culture Collection for preservation in 2018, 9 and 28 months, and the preservation number is as follows: CCTCC NO: c2018211, classification name: carassius auratus gibelio Spinal cord tissue cell line (CSC), address: wuhan university in Wuhan, China.
Example 2
Biological characteristics of the carassius auratus gibelio spinal cord tissue cell line CSC:
(1) morphology: the cell type is a fibroblast-like cell.
(2) Growth characteristics: the CSC cells after passage begin to adhere to the wall after 30min, and the adherence is complete after 6 h; the population doubling time was 48 h.
(3) Stability: the carassius auratus gibelio spinal cord tissue cell line CSC is transferred to 42 generations before application date, and the proliferation is stable.
(4) Freezing and recovering:
after the CSC cells are recovered, the CSC cells are attached to the wall quickly, the growth form and the condition are basically similar to those of the cells which are not frozen, and no obvious difference occurs. The resuscitated cells were stained with trypan blue, and about (78.56 ± 6.10)% of the cells were not stained and had cell activity by cell count.
(5) Chromosome analysis
The 26 th generation carassius auratus gibelio spinal cord tissue cell line CSC is in logarithmic growth phase, colchicine with the final concentration of 20 mug/ml is added, the cells are digested and collected after incubation for 4h at 25 ℃, pre-cooled carnot stationary liquid is added after hypotonic treatment is carried out for 25min by 0.075mol/L KCl solution, centrifugation is carried out for 5min at 1000rpm, supernatant is removed, and then the pre-cooled carnot stationary liquid is used for fixing for 3 times, 15min each time. Dropping by cold drop method, drying, dyeing with 5% Giemsa for 25min, drying, and observing with microscope. Among the observed 100 dividing cells, the number of chromosomes of the 26 th generation carassius auratus gibelio tissue derived cells is 156 (fig. 2), and the number of chromosomes is matched with the chromosome characteristics of the triploid carassius auratus gibelio, namely all the 156 chromosomes of the triploid carassius auratus gibelio do not contain the chromosomes of the exogenous carp.
Example 3
The application of the carassius auratus gibelio spinal cord tissue cell line comprises the following steps:
(1) collecting and treating cyprinid herpesvirus II infected pathological materials:
collecting kidney, spleen, brain and spinal cord tissues of dead fish infected with carp herpesvirus II, shearing, adding PBS (phosphate buffer solution) with equal volume for homogenizing, centrifuging at 5000rpm and 4 ℃ for 15min, filtering with 0.22 mu m filter membrane to prepare sterile tissue homogenate, subpackaging, and storing at-80 ℃ for later use;
(2) proliferation of carp herpesvirus type ii in CSC:
after the CSC is cultured to about 80 percent of cell monolayer, the culture medium is discarded, the CSC is washed for 2 times by serum-free cell maintenance liquid, 0.2ml of supernatant of the homogenate of the pathological tissue is inoculated to the CSC cell monolayer and is adsorbed for 1h at 24 ℃, and the culture bottle is slightly shaken once every 15 to 20min during the period so as to be uniformly adsorbed. After adsorption, the L15 maintenance solution with the serum concentration of 2% is changed to be cultured at 24 ℃, cytopathic effect (CPE) is observed day by day, and the virus is harvested after the pathological effect reaches 80%, and the virus is the source of the carp herpes virus II type isolate.
(3) Extracting and PCR detecting nucleic acid of herpes cyprinid virus type II cell virus:
and (3) repeatedly freezing and thawing the CSC cells with obvious lesions for 2 times, and then extracting virus DNA by DNAzol. The virus was detected by PCR for CyHV-2. Primer for PCR amplification
CyHV2-366F is: GGACTTGCGAAGAGTTTGATTTCTAC;
CyHV2-366R is: CCATAGTCACCATCGTCTCATC, respectively;
the amplification conditions were: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30 sec, annealing at 60 ℃ for 30 sec, extension at 72 ℃ for 30 sec, and extension at 72 ℃ for 5min after 35 cycles.
Meanwhile, a positive control DNA template is taken as a positive control, and CSC cell DNA and pure water are respectively taken as negative controls. The amplification product was identified by 2% (W/V) agarose gel electrophoresis.
(4) Electron microscope observation of carp herpesvirus II type infected CSC
Fixing CSC cells infected with 7 th generation CyHV-2 cell poison by 2% glutaraldehyde, fixing osmium tetroxide, dehydrating and embedding, performing ultrathin section, performing uranium acetate-lead citrate double staining, and observing by a transmission electron microscope.
(5) Results
After homogenating and inoculating the carp herpesvirus II type diseased fish tissue and CSC cell monolayer for 4d, cell rounding or vacuolation, solid shrinkage, cell syncytium formation and cell gap enlargement occur; after 6d, the CSC cells are fused and form multinucleated giant cells, and the cell monolayer is shed to generate a net-drawing phenomenon, a typical cytopathic effect (CPE) appears, and the CPE is stable after passage to 10 th generation (figure 3), while normal CSC cells do not see any change.
CyHV-2 cell culture DNA of different culture generations is extracted, PCR detection is carried out, 357bp fragments are obtained through two rounds of PCR amplification, the sizes of the fragments are consistent with those of amplified bands of positive control (figure 4), and the results are judged to be positive.
A transmission electron microscope is used for observing CSC cells infected with CyHV-2 of the 7 th generation, a large number of mature CyHV-2 virus particles can be observed, the CyHV-2 has good biological activity in the CSC cells (figure 5), and the sensitivity of the established carassius auratus gibelio spinal cord tissue cell line to viruses is further proved, so that the carassius auratus gibelio spinal cord tissue cell line can be used for separating, culturing and detecting the viruses. Meanwhile, the method can be used as a cell model for researching the biological characteristics of the carp herpesvirus II to prepare the vaccine of the carp herpesvirus II.
While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
<110> Zhejiang province fresh water aquatic product research institute Dudu Tianbang biological products Co., Ltd
<120> carassius auratus gibelio spinal cord tissue cell line as well as construction method and application thereof
<130> 2018
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 1
ggacttgcga agagtttgat ttctac 26
<210> 2
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
ccatagtcac catcgtctca tc 22
Claims (9)
1. A carassius auratus gibelio Spinal cord tissue cell line (CSC) is characterized in that the preservation number is CCTCC NO: C2018211.
2. the method for constructing the carassius auratus gibelio spinal cord tissue cell line as claimed in claim 1, is characterized by comprising the following steps:
step 1, treatment of spinal cord tissue: taking out carassius auratus gibelio spinal cord tissue under aseptic condition, carrying out aseptic treatment to obtain tissue blocks of 30-60 mm & lt 3 & gt, and putting the tissue blocks into a culture dish containing L15 culture solution;
step 2, primary culture: uniformly placing the tissue blocks in the step 1 into a T25 cell culture bottle, enabling one surface of the tissue blocks to be upward, adding 3ml of culture solution into the culture bottle, standing overnight, slowly turning over the culture bottle to enable the tissue blocks to infiltrate the culture solution, enabling one surface with the tissue blocks to be upward, performing irregular operation once until cells grow out from the edges of the tissue blocks, placing the cell culture bottle upright for culture, and replacing the culture solution once every 2-3 days;
step 3, subculturing: after the carassius auratus gibelio spinal cord tissue cells in primary culture grow into a monolayer, adding trypsin digestive juice, standing and digesting for 1-2 minutes, suspending the cells by using a culture solution, carrying out subculture in a mode of transferring 2 bottles by 1 bottle, and carrying out next subculture according to the subculture method of the cells after the cells form the monolayer again to obtain the cell line CSC of the carassius auratus gibelio spinal cord tissue.
3. The construction method according to claim 2, wherein the culture solution of carassius auratus gibelio spinal cord tissue cells L15 in step 1 is a culture solution containing 10-20% V/V fetal bovine serum, 100U/ml penicillin, 100 μ g/ml streptomycin and having a pH value of 7.0-7.4.
4. The method of claim 2, wherein the trypsin digestion solution for cell passage in step 3 is 0.25% W/V trypsin-EDTA digestion solution.
5. The construction method according to claim 2, wherein the temperature for cell culture and passage in the steps 2 and 3 is 23-25 ℃ and the pH value is 7.0-7.4.
6. The use of the carassius auratus gibelio spinal cord tissue cell line of claim 1 in separation, culture and detection of carp herpesvirus type ii.
7. The use according to claim 6, wherein the herpesvirus carpi type II is a herpesvirus carpi type II isolate.
8. The use of the carassius auratus gibelio spinal cord tissue cell line of claim 1 in the preparation of a vaccine for herpesvirus carpi type ii.
9. The use according to claim 8, wherein the herpesvirus carpi type ii is a herpesvirus carpi type ii isolate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811231823.XA CN109294974B (en) | 2018-10-22 | 2018-10-22 | Carassius auratus gibelio spinal cord tissue cell line as well as construction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811231823.XA CN109294974B (en) | 2018-10-22 | 2018-10-22 | Carassius auratus gibelio spinal cord tissue cell line as well as construction method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109294974A CN109294974A (en) | 2019-02-01 |
| CN109294974B true CN109294974B (en) | 2022-09-13 |
Family
ID=65158398
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811231823.XA Active CN109294974B (en) | 2018-10-22 | 2018-10-22 | Carassius auratus gibelio spinal cord tissue cell line as well as construction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109294974B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110305844B (en) * | 2019-03-06 | 2022-12-23 | 成都史纪生物制药有限公司 | Crucian spinal cord tissue cell line and application thereof |
| CN110893235B (en) * | 2019-11-13 | 2023-10-24 | 浙江省淡水水产研究所 | Inactivated vaccine for crucian hematopoietic necrosis and preparation method 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 |
| 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 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667176A (en) * | 2013-12-09 | 2014-03-26 | 中国水产科学研究院长江水产研究所 | Carassius auratus gibelio brain tissue cell line sensitive to cyprinid herpesvirus II, and establishing method and application thereof |
| CN104762252A (en) * | 2015-04-27 | 2015-07-08 | 上海海洋大学 | Grass goldfish pelvic fin cell line constructing method |
| CN105543162A (en) * | 2015-11-27 | 2016-05-04 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Crucian brain tissue cell line and application thereof |
| CN108546672A (en) * | 2018-04-20 | 2018-09-18 | 上海海洋大学 | A kind of construction method of hybridized prussian carp caudal fin cell system and its application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MX2010002122A (en) * | 2007-08-28 | 2010-07-06 | Univ Liege | A recombinant koi herpesvirus (khv) or cyprinid herpesvirus 3 (cyhv-3) and a vaccine for the prevention of a disease caused by khv/cyhv-3 in cyprinus carpio carpio or cyprinus carpio koi. |
-
2018
- 2018-10-22 CN CN201811231823.XA patent/CN109294974B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667176A (en) * | 2013-12-09 | 2014-03-26 | 中国水产科学研究院长江水产研究所 | Carassius auratus gibelio brain tissue cell line sensitive to cyprinid herpesvirus II, and establishing method and application thereof |
| CN104762252A (en) * | 2015-04-27 | 2015-07-08 | 上海海洋大学 | Grass goldfish pelvic fin cell line constructing method |
| CN105543162A (en) * | 2015-11-27 | 2016-05-04 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Crucian brain tissue cell line and application thereof |
| CN108546672A (en) * | 2018-04-20 | 2018-09-18 | 上海海洋大学 | A kind of construction method of hybridized prussian carp caudal fin cell system and its application |
Non-Patent Citations (3)
| Title |
|---|
| Comparative Genomics of Carp Herpesviruses;Davison, AJ;《JOURNAL OF VIROLOGY》;20130331;第87卷(第5期);第2908-2922页 * |
| Cyprinid herpesvirus 2 infection emerged in cultured gibel carp, Carassius auratus gibelio in China;JinXu;《Veterinary Microbiology》;20130927;第166卷(第1-2期);第138-144页 * |
| Establishment of a novel and highly permissive cell line for the efficient replication of cyprinid herpesvirus 2 (CyHV-2);Jie Ma;《Veterinary Microbiology》;20150630;第177卷(第3–4期);第315-325页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109294974A (en) | 2019-02-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109294974B (en) | Carassius auratus gibelio spinal cord tissue cell line as well as construction method and application thereof | |
| CN103667176B (en) | Carassius auratus gibelio brain tissue cell line sensitive to cyprinid herpesvirus II, and establishing method and application thereof | |
| CN112410290B (en) | Lateolabrax japonicus myocardial fibroblast line and application thereof | |
| CN110551689B (en) | Channa argus brain cell line and construction method and application thereof | |
| CN114854676B (en) | Construction method and application of grass carp skeletal muscle myoblast cell line | |
| CN109971710B (en) | Jian carp brain cell line and establishing method and application thereof | |
| CN111718899A (en) | Culture method for in vitro induced NK (natural killer) cells after resuscitation of cryopreserved human PBMCs (peripheral blood mononuclear cells) | |
| CN113249308A (en) | Perch arterial ball cell line and application and culture method thereof | |
| CN113717939B (en) | Red porgy brain cell line and its construction method and use | |
| CN105483075B (en) | A kind of culture medium and method for cultivating Urechis uniconctus trochophore cell line | |
| CN104974977B (en) | A kind of epinephelus lanceolatus fish nephridial tissue cell line and its construction method | |
| CN109207422B (en) | European eel kidney cell line EK and application thereof | |
| CN113755438B (en) | Mandarin fish spinal cord tissue cell line and construction method and application thereof | |
| CN112126619B (en) | Rhabdoviral sensitive finless eel kidney tissue cell line and application | |
| CN110951694B (en) | Preparation method of autologous trophoblast and culture method of SNK cells | |
| CN111718898B (en) | Method and reagent for improving stress tolerance of synovial membrane mesenchymal stem cells | |
| CN110283793B (en) | Method for separating and culturing porcine pseudorabies virus | |
| CN113512559A (en) | Mycoplasma bovis Mbov _0701 mutant gene and mutant strain and application thereof | |
| CN112280740A (en) | Gobiocypris rarus brain cell line and application thereof | |
| CN107304412A (en) | The culture medium of retinal pigment epithelium and its application | |
| CN111411087A (en) | Cyprinus carpioides herpesvirus II type low virulent strain and application thereof | |
| CN105543162B (en) | Crucian brain tissue cell line and application thereof | |
| CN115094028B (en) | Pomfret liver cell line and application thereof | |
| CN115572707B (en) | Fancy carp muscle cell line and in-vitro culture method and application thereof | |
| CN116836912B (en) | Lateolabrax japonicus kistrodon cell line and construction method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Shen Jinyu Inventor after: Cao Zheng Inventor after: Pan Xiaoyi Inventor after: Yao Jiabin Inventor after: Lin Lingyun Inventor after: Yin Wenlin Inventor after: Liu Yihan Inventor before: Shen Jinyu Inventor before: Xing Gang Inventor before: Yue Fengxiong Inventor before: Zhou Tao Inventor before: Huang Jie Inventor before: Cao Zheng Inventor before: Pan Xiaoyi Inventor before: Xia Yanchun Inventor before: Yao Jiabin Inventor before: Lin Lingyun Inventor before: Yin Wenlin Inventor before: Liu Yihan Inventor before: Lu Yuxiao |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20221207 Address after: No. 999, Hangchang Bridge South Road, Huzhou City, Zhejiang Province 313098 Patentee after: ZHEJIANG INSTITUTE OF FRESH WATER FISHERIES Address before: 313001 No. 999, hangchangqiao South Road, Wuxing District, Huzhou City, Zhejiang Province Patentee before: ZHEJIANG INSTITUTE OF FRESH WATER FISHERIES Patentee before: CHENGDU TECBOND BIOLOGICAL PRODUCTS CO.,LTD. |