CN112126619A - Rhabdoviral sensitive finless eel kidney tissue cell line and application - Google Patents
Rhabdoviral sensitive finless eel kidney tissue cell line and application Download PDFInfo
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Abstract
The invention belongs to the field of aquatic organism cells and the technical field of disease control in aquaculture, and discloses a rhabdovirus-sensitive finless eel kidney tissue cell line and application thereof, wherein the cell line is preserved in China center for type culture Collection with the preservation number as follows: CCTCC NO: C2019286. the finless eel kidney tissue cell line (CrE-K) has good growth state, and is sensitive to CrERV newly discovered and separated and identified at present; the CrERV can still detect the virus nucleic acid and stabilize the cytopathy after being continuously passaged on CrE-K cells to the 16 th generation; cells with pathological effect are made into ultrathin electron microscope slices, and a large amount of CrERV mature virus particles and the replication process thereof can be observed in CrE-K cells. The construction method of the finless eel kidney tissue cell line constructed by the invention has strong repeatability and scientific and reasonable conditions, is suitable for in vitro culture of rhabdovirus, and provides a technical platform for separation, detection and culture of the rhabdovirus and complete biological characteristic research thereof.
Description
Technical Field
The invention belongs to the field of aquatic organism cells and the technical field of aquaculture disease prevention and control, and particularly relates to a rhabdovirus-sensitive finless eel kidney tissue cell line and application thereof.
Background
Rhabdoviridae (Rhabdoviridae) are a class of negative strand RNA viruses with a broad host range, the particles of which are bullet-shaped or rod-shaped. The Rabies virus (Rabies virus) is the best known and can cause Rabies of human beings or other warm-blooded animals, the fatality rate is almost 100 percent, the wide attention in the world is caused, and the panic of the society on the safety of cultured pets is caused. Therefore, the intensive study of rhabdoviruses is of great significance. In addition, rhabdoviruses can cause a variety of acute infectious diseases in livestock (e.g., bovine epizootic fever), as well as infectious diseases in fish and various crops. The International Committee for virus Taxonomy (International Committee on Taxonomy of Viruses, ICTV) published the tenth report of the latest virus Taxonomy in 2017, dividing the Rhabdoviridae (Rhabdoviridae) into 18 genera, among which the rhabdoviruses capable of infecting fish are three genera, perhabdivirus, Sprivivirus and novirhabdivirus. At present, the three genera can infect various cultured fishes, have strong pathogenicity, the death rate can reach 100 percent, and cause great economic loss to the global sea fresh water culture industry, particularly, according to the inspection and quarantine protocol of live aquatic animals at the entrance and exit, the cultured fishes entering and exiting the border, such as culture and fingerling of weever, carp, rainbow trout, crucian, paralichthys olivaceus and the like, are subjected to rhabdovirus (IHNV, SVC V, HIRRV) epidemic disease detection, the entrance and exit safety significance is great, and the economic trade of the entrance and exit is influenced.
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). It has been found that finless eel rhabdovirus (CrERV) is serially passaged in cell lines commonly used for fish virus Isolation, and that the virus propagation titer is not high (Liu et al, Isolation, identification and classification of a novel rhabdovirus from purified chicken rice-field eel, Monopterus albus. archives of Virology (2019)164: 105-116.), including cell lines sensitive to other fish rhabdoviruses: carp epithelial tumor cells (EPC), salmon embryonic cells (CHINOOK salmony, CHSE-214), rainbow trout gonad cells (RAINBOW trout gonad, RTG-2) and grass carp gonad cells (Cteno pharyngodon idellus, GCO) have low proliferation titer to CrERV, only allogynogenetic crucian brain cells (Gibel carp, GiCB) can produce obvious cytopathic effect (CPE), but after the virus is transmitted to the 10 th generation on the CiCB cells, the proliferation titer of the virus is gradually reduced until the CPE phenomenon disappears. Because the research on CrERV is limited due to the lack of CrERV continuous passage sensitive cell lines, the establishment of CrERV sensitive cell lines and the research on the biological characteristics of the cell lines have important significance in the deep research on the characteristics of the virus by carrying out continuous passage expanded culture on CrERV.
The present invention establishes a sensitive cell line CrE-K for isolated culture of rhabdoviruses, applications of which also include, but are not limited to: the physicochemical characteristics, morphogenesis, virus infection ways and infection mechanisms of rhabdovirus and other fish viruses are developed on the molecular cell level; preparing, screening or evaluating the antiviral drugs for fish.
Disclosure of Invention
The invention aims to provide a rhabdovirus sensitive finless eel kidney tissue cell line. The cell line can be used for separating, detecting, separating and culturing rhabdovirus. The cell line is sent to China center for type culture Collection for preservation in 2019, 11/1, and is classified and named: finless eel kidney cell line CrE-K, accession number: CCTCC NO: c2019286, address: wuhan university in Wuhan, China.
Another object of the present invention is to provide an application of a rhabdovirus sensitive finless eel kidney tissue cell line. The cell line can be used for isolating, detecting and culturing rhabdoviruses; the method is used for in vitro continuous passage and expansion culture of rhabdovirus so as to deeply research the characteristics of the virus.
In order to achieve the purpose, the invention adopts the following technical measures:
a method for establishing a rhabdovirus-sensitive finless eel kidney tissue cell line comprises the following steps:
(1) treatment of renal tissue: taking out the kidney tissue of the finless eel under the aseptic condition, and carrying out aseptic treatment to 50-100 mm3The tissue mass of (a);
(2) primary culture: digesting the tissue block obtained in the step (1) in a trypsin digestive fluid special for tissue separation for 10-15 min, shaking for 3-4 times, adding a culture fluid special for finless eel kidney histiocyte (hereinafter referred to as culture fluid) with the same volume, uniformly blowing, centrifugally collecting digested cells, sucking and removing supernatant, adding the culture fluid, blowing cell precipitates, adding the prepared cell suspension into a cell culture bottle for culture, and replacing the culture fluid half a day;
(3) subculturing: after primary culture finless eel kidney tissue cells grow into a monolayer, 0.25% W/V trypsin digestive juice is added for standing and digesting for 2 minutes, the cells are suspended by the culture solution, and subculture is carried out in a mode of transferring 1 bottle to 2 bottles. And after the cells form a monolayer again, carrying out next subculture according to the subculture method of the cells to obtain a stably inherited rhabdovirus sensitive cell line CrE-K derived from the kidney tissue of the finless eel, wherein the cell line is sent to a China Center for Type Culture Collection (CCTCC) for collection on 11/1 of 2019, and is classified and named: finless eel kidney cell line CrE-K, accession number: CCTCC NO: c2019286, address: wuhan university in Wuhan, China.
The finless eel kidney tissue cell line CrE-K is fibroblast-like cell, the optimal culture medium is M199, the optimal serum volume fraction is 20% (V/V), and the optimal culture temperature is 28 ℃. CrE-K cells were thawed after freezing with liquid nitrogen, and by staining, about 80% of the cells had cell viability and remained in the original growth state, and the cells had been stably passaged 58 times.
The trypsin digestive juice special for tissue separation is 0.5-0.7% W/V trypsin digestive juice, the temperature of cell culture and passage is 25-28 ℃, and the pH value is 7.0-7.4.
The special culture solution for the finless eel kidney histiocyte is an M199 culture solution containing 10-20% V/V fetal calf serum, 10-20 ng/ml human basic fibroblast growth factor, 10-20 ng/ml human epidermal growth factor, 100U/ml penicillin, 100 mu g/ml streptomycin, 0.25 mu g/ml amphotericin B and having a pH value of 7.0-7.4.
An application of a rhabdovirus sensitive finless eel kidney tissue cell line comprises the application of the cell line in rhabdovirus culture; the use of the cell line for the detection of rhabdoviruses; the use of such cell lines for the isolation of rhabdoviruses; the application of the cell line in preparing rhabdovirus vaccine; the cell line is applied to the separation and detection of other fish viruses; the application of the cell line as an anti-rhabdovirus drug screening biological model; the cell line is applied to gene transfection of fish cells and research on gene functions.
The rhabdovirus is fish rhabdovirus, including Perhabdivirus, Sprivivirus or/and Novirrabivirus.
Compared with the prior art, the invention has the following advantages:
(1) the invention detects and verifies the finless eel rhabdovirus cDNA continuously passaged on CrE-K cells, and the result proves that virus nucleic acid can be still detected at the 16 th generation of CrE-K cells continuously passaged; cytopathic effect (CPE) was stable and significant; the cells with pathological effect are observed by ultrathin electron microscope slices, and the result of a transmission electron microscope shows that CrERV mature virus particles and the replication process thereof exist in CrE-K cells, so that the CrERV has good biological activity in CrE-K cells.
(2) The titer of the CrERV virus of generation 10 propagated by the CrE-K cell line of the invention was determined. The virus titer is measured in 96-well micro-culture plate, the virus liquid is diluted by 10 times gradient of serum-free M199 culture medium, and is respectively inoculated in 96-well micro-culture plate which is full of CrE-K cells, each dilution is provided with 8 multiple wells, each well is 100 muL, the control group is provided with 8 wells, and each well is added with 100 muL of serum-free M199 culture medium to simulate infection. After completion of the adsorption, the virus solution was discarded, and 100. mu.L of a cell maintenance solution (M199 medium containing 2% (V/V) serum) was added to all wells of the microplate, and the microplate was cultured at 28 ℃ to observe the results day by day and calculate the virus titer (TCID50/0.1mL) by the Reed-Muench method (Reed et al, 1938). The results show that CrERV can be stably proliferated in CrE-K cells, and the titer TCI D50 is 107.67/0.1ml。
(3) For the CrERV suspected clinical disease material to be detected, on the basis of positive RT-PCR detection, cell pathological changes such as cell vacuolation, cell mutual fusion, cell monolayer shedding and the like can be observed after the CrE-K cell line established by the invention is used for inoculating the suspected disease material for 48 hours; the CrERV suspected clinical disease material to be detected is continuously passaged for more than 15 times, and the existence of the virus can still be detected by RT-PCR.
(4) The CrE-K cell line established by the invention is stable in heredity, provides a necessary technical platform for the separation and identification of rhabdovirus and the research of the complete biological characteristics of the rhabdovirus, and lays an important foundation for the prevention and control of the rhabdovirus.
Drawings
FIG. 1 is a schematic diagram of different generations of finless eel kidney tissue cells CrE-K.
In FIG. 1, A is primary finless eel kidney tissue cells; in FIG. 1, B is the 39 th generation xenogenic finless eel kidney tissue cell line.
FIG. 2 is a chromosome representation of 18 th generation finless eel kidney tissue cell line.
FIG. 3 is a schematic diagram of a finless eel kidney tissue cell line infected with CrERV.
FIG. 3A is CrE-K normal control cells; b in FIG. 3 is CrE-K cells infected with CrERV passage 1, 4 d; c in FIG. 3 is CrE-K cells infected with CrERV 3 rd generation 3 d; in FIG. 3D is CrE-K cells infected with CrERV passage 5 2D.
FIG. 4 is a diagram showing RT-PCR detection results of CrERV of different generations of culture.
M: DL2000 Marker; line 1: CrERV tissue toxicity control; line 2: culturing CrERV cytotoxicity on the 1 st generation CrE-K cells; line 3: culturing CrERV cell virus in CrE-K generation 2; line 4: culturing CrERV cell virus in CrE-K3 rd generation cell; line 5: artificially infecting tissue homogenate, culturing CrERV cell virus by CrE-K cells at the 1 st generation; line 6: artificially infecting tissue homogenate, culturing CrERV cell virus by CrE-K cells at the 2 nd generation; line 7: artificially infecting tissue homogenate, culturing CrERV cell virus by CrE-K cells at the 3 rd generation; line 8: culturing CrERV cytotoxicity on 5 th generation CrE-K cells; line 9: culturing CrERV cell virus in CrE-K generation 10; line 10: culturing CrERV cytotoxicity on 15 th generation CrE-K cells; line 11: negative control
FIG. 5 plaques formed by CrE-K cytopathic effect in Virus titer experiments
FIG. 6 is a schematic view of an ultrathin section of a 5 th generation CrERV cell infected with CrE-K by a cell electron microscope.
Detailed Description
The present invention is further described below with reference to specific examples, which are only exemplary and do not limit the scope of the present invention in any way. Modifications and substitutions in detail and form can be made to the technical solution of the present invention without departing from the spirit and scope of the present invention, but the modifications and substitutions are intended to be covered by the scope of the present invention, and reagents used in the examples of the present invention, if not specifically described, are 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 finless eel is about 67.5g in weight and about 35cm in length, and is sourced from the Xiantao experimental base of the Yangjiang aquatic product research institute of Chinese aquatic science. The experiment was temporarily kept indoors for 1 week. The preservation number is CCTCC NO: the cell line of the invention was functionally verified by the ricefield eel rhabdovirus (CrERV) from V201819, which belongs to the genus Perhabdovirus, and the remaining fish-infectable rhabdoviruses, Sprivivirus and Novirrabovirus, are also suitable for use in the cell line of the invention.
2) Main reagent and consumable
M199 cell culture medium, amphotericin B, penicillin/streptomycin, Phosphate Buffered Saline (PBS), trypsin-EDTA, dimethyl sulfoxide (DMSO), colchicine were purchased from Sigma; the human basic fibroblast growth factor and the human epidermal growth factor are products of Peprotech company; fetal bovine serum was purchased from GIBICO; the Trizol nucleic acid extraction reagent is an Invitrogen product; rTaq enzyme and dNTPs for RT-PCR are products of TaKaRa company. 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
Class II biosafety cabinets (ESCO); inverted microscope (Nikon); a CCD camera (Nikon NIS Elements F530); low speed refrigerated centrifuge (3K15, Sigma); thermostated incubator (Sanyo, MIR-153); liquid nitrogen tanks (MVE); microtomes (UC7, Leica); transmission electron microscope (H-7650, Hitachi)
Example 1:
the establishment of the finless eel kidney tissue cell line comprises the following steps:
(1) treatment of renal tissue: taking out the finless eel kidney tissue under aseptic condition, placing the finless eel kidney tissue in a culture dish, rinsing with PBS for 3 times, and shearing the finless eel kidney tissue into 50-100 mm with sterilized ophthalmic scissors3The tissue mass of (a);
(2) primary culture: placing the cut tissue blocks into 0.5W/V% trypsin digestive juice, digesting for 15min at 28 ℃, shaking for 3 times, adding special culture solution (hereinafter referred to as culture solution for short, the formula of the culture solution is M199 culture medium containing 20V/V% fetal calf serum, 10ng/ml human basic fibroblast growth factor, 10ng/ml human epidermal growth factor, 100U/ml penicillin, 100 mu g/ml streptomycin and 0.25 mu g/ml amphotericin B) for the finless eel kidney histiocyte after digestion, uniformly blowing, filtering once by a 100-mesh nylon gauze, collecting filtrate to a centrifuge tube, centrifuging at 1500rpm for 5min, collecting digested and filtered cells, removing supernatant, adding culture solution, blowing, filtering with 300 mesh nylon gauze, collecting filtrate, and centrifuging at 1000rpm for 5min to obtain cell precipitate; addingThe culture solution is blown to beat the cell sediment, and the prepared cell suspension is added into the culture solution with the volume of 25cm2Culturing at 25 deg.C in a cell culture bottle, and replacing culture solution once every 2 days;
(3) subculturing: after primary culture of finless eel kidney histiocyte to grow into a monolayer, absorbing the original culture solution, adding 2ml of 0.25W/V% trypsin digestive juice, standing and digesting for 2 minutes at a constant temperature, absorbing the trypsin digestive juice, adding 10ml of special culture solution for finless eel kidney histiocyte, blowing and beating the bottom cells of the bottle to prepare cell suspension, and carrying out subculture in a mode of transferring 2 bottles from 1 bottle. And after the cells form a monolayer again, carrying out next subculture according to the subculture method of the cells until a cell line is established. After subculturing to 6 th generation, the special culture solution for the finless eel kidney histiocyte is not added with human basic fibroblast growth factor, human epidermal cell growth factor, penicillin, streptomycin and amphotericin B. After the GiCB cells are subcultured for about 2 days or 3 days, about 80% of the bottom of the cell culture bottle can be fully paved to form a confluent cell monolayer, and a compact cell monolayer can be formed after 5 days, so that the next subculture can be carried out. As shown in fig. 1, a is primary finless eel kidney tissue cells; b is 39 th generation finless eel kidney tissue cell line.
The fifth generation of the cell line is sent to China center for type culture Collection for preservation in 2019, 11/1, with the preservation number: CCTCC NO: c2019286, classification name: finless eel kidney cell line CrE-K, address: wuhan university in Wuhan, China.
Example 2:
biological characteristics of the finless eel kidney tissue cell line CrE-K:
(1) morphology: the cell type is a fibroblast-like cell.
(2) Growth characteristics: the CrE-K cells after passage begin to adhere to the wall after 30min, and the wall is completely adhered after 8 h; the population doubling time was 45.5 h.
(3) Stability: the finless eel kidney tissue cell line CrE-K is transmitted to 58 generations before application date, and the proliferation is stable.
(4) Freezing and recovering:
CrE-K cells are attached to the wall quickly after recovery, and the growth form and condition are basically similar to those of cells without freezing storage, and no obvious difference occurs. The resuscitated cells were stained with trypan blue, and about (80.38 ± 5.10)% of the cells were not stained and had cell activity by cell count.
(5) Chromosome analysis
The 18 th generation of the finless eel kidney tissue cell line CrE-K is in a 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 28 ℃, pre-cooled carnot stationary liquid is added after hypotonic treatment is carried out for 25min by using 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 phase cells, the chromosome mode of 18 th generation eel kidney tissue source cells is 24 (figure 2), and the chromosome mode is matched with the chromosome characteristics of cultured eels (Liyucheng et al, the chromosome group type research of eels, Wuhan university report, 1982, 01: 55-116; Liu Han et al, the chromosome group types of two different body color eels, aquatic science, 2006, 06: 287-290), namely, the whole chromosomes of the eels are 24 chromosomes.
Example 3:
the application of the rhabdovirus sensitive finless eel kidney tissue cell line comprises the following steps:
(1) collection and treatment of disease material infected with ricefield eel rhabdovirus
Collecting dead diseased fish kidney and spleen infected with Monopteri albi rhabdovirus (CCTCC NO: V201819), cutting, adding PBS with equal volume to homogenate, centrifuging at 5000rpm at 4 deg.C for 30min, filtering with 0.22 μm filter membrane to obtain sterile tissue homogenate, packaging, and storing at-80 deg.C;
(2) proliferation of Monopteri albi Rhabdoviruses at CrE-K
CrE-K, removing the culture medium, washing with PBS for 2 times, inoculating 1ml of supernatant of the homogenate of the pathological tissue to CrE-K cell monolayer, adding Polybrene (Polybrene) with the final concentration of 10 mug/microliter, adsorbing for 2h at 25 ℃, and slightly shaking the culture flask once every 15-20 min during the period so as to uniformly adsorb. After adsorption, the M199 maintenance solution with the serum concentration of 2% is changed to continue culturing at 28 ℃, cytopathic effect (CPE) is observed day by day, and the virus is harvested until the pathological effect reaches 80%.
The virus titer is measured in 96-well micro-culture plate, the virus liquid is diluted by 10 times gradient of serum-free M199 culture medium, and is respectively inoculated in 96-well micro-culture plate which is full of CrE-K cells, each dilution is provided with 8 multiple wells, each well is 100 muL, the control group is provided with 8 wells, and each well is added with 100 muL of serum-free M199 culture medium to simulate infection. After adsorption, the virus solution was discarded, 100. mu.L of cell maintenance medium (M199 medium containing 2% (V/V) serum) was added to all wells of the microplate, the plate was cultured at 28 ℃ and the results were observed day by day to calculate the virus Titer (TCID) by the Reed-Muench method50/0.1mL)(Reed et al.，1938)。
(3) Extraction and RT-PCR detection of ricefield eel rhabdovirus cytotoxic nucleic acid
CrE-K cells with obvious lesions are repeatedly frozen and thawed for 2 times, and then virus RNA is extracted by Trizol. The virus is detected by using a method for detecting CrERV by RT-PCR.
Primer:
CrERV-F：GTGGCAGCAATTGACATGTTCT；
CrERV-R：CATATCCCATCACCTTATTGACCCT；
the amplification conditions were: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30 sec, annealing at 55 ℃ for 30 sec, extension at 72 ℃ for 1 min, and 35 cycles followed by 5min at 72 ℃; taking a positive control DNA template as a positive control, and taking pure water as a negative control. The amplification product was identified by 2% (W/V) agarose gel electrophoresis.
(4) Electron microscope observation of ricefield eel rhabdovirus infection CrE-K
CrE-K cells infected with CrERV cell poison of the 5 th generation are fixed by 2% glutaraldehyde, and then are fixed by osmium tetroxide, and then are dehydrated and embedded to be ultrathin sliced, and the slice is observed by a transmission electron microscope after uranium acetate-lead citrate double staining.
(5) Results
After the homogenate inoculation of the eel rhabdovirus disease fish tissue and the CrE-K cell monolayer for 3d, the cell becomes round or vacuolated, retracts, forms a syncytium and enlarges the cell gap; CrE-K cells fused and formed multinucleated giant cells after 4d, and the cell monolayer was shed, generating a phenomenon of net-pulling, presenting typical cytopathic effect (CPE), and being stable after passage to the 15 th generation of CPE (FIG. 3), while no change was seen in normal CrE-K cells.
And (3) extracting CrERV cell culture RNA of different culture generations, carrying out RT-PCR detection, obtaining 833bp fragments through amplification, wherein the sizes of the 833bp fragments are consistent with those of amplification bands of positive control (figure 4), and judging the results to be positive.
After the virus was inoculated in 96-well microplate, the cytopathic condition was observed day by day, and the number of diseased wells was photographed and recorded. As shown in fig. 5: plaques formed by the virus at CrE-K monolayers (FIG. 5, infected with passage 5 virus). On day 5, observation was stopped, the number of CPE wells was counted, and the tissue culture half-infection TCID50 was calculated according to the Reed-Muench method. I.e., the reciprocal of the dilution of the virus when it infects half of the tissue cells.
Observing cytopathic condition on day 5, 100, 10-1、10-2、10-3、10-4、10-5、10-6、10-7Plaque in all 4 parallels, 10-8Unequal number of plaques appeared in 4 replicates. The control cell monolayer was intact and no plaque was present.
Statistics are as follows:
TABLE 1 Virus titer titration (inoculum dose 0.1ml)
The calculation formula of the Reed-Muench method is as follows: lgTCID50 ═ the highest dilution log of percentage of positivity greater than 50% + distance ratio × -the log of the dilution factor, where distance ratio ═ the (percentage of positivity greater than 50% -50)/(percentage of positivity greater than 50% — percentage of positivity less than 50%)
(1) The number of positive wells (1) and the number of negative wells (2) for each virus dilution were calculated.
(2) The cumulative number of positive and negative wells was calculated. Accumulating the positive hole accumulation number from bottom to top (3); the negative well accumulation counts were accumulated from top to bottom (4).
(3) Calculate the percentage of positive wells: ratio (5) ═ 3/[ (3) + (4) ]
(4) Calculate distance ratio ═ (greater than 50% percent positive-50%)/(greater than 50% percent positive-less than 50% percent positive)
(5) Logarithm of dilution factor, 10-series logarithm of dilution lg10 ═ 1
The calculation results are as follows:
10-7(100%) and 10-8(25%) distance ratio between ═ 100% to 50%/(100% to 25%): 0.67
lgTCID50/0.1ml＝7+0.67×1＝7.67
The virus titer TCID50 was therefore 10 ═ 107.67/0.1ml。
According to the above method, the virus titers of the 12 th and 14 th passages of the infection have been detected as follows: 107.62±0.69，107.69 ±0.53Moreover, the virus pathological phenomenon and time are consistent during the continuous proliferation of CrERV on CrE-K cells, and the virus titer is stable by combining with the measurement of virus titer of 14 continuous generations.
CrE-K cells infected with 5 th generation CrERV cytotoxin are observed through a transmission electron microscope, a large number of mature CrERV virions and the replication process thereof can be observed, the CrERV has good biological activity in CrE-K cells (figure 6), and the sensitivity of the established finless eel kidney tissue cell line to viruses is further proved, so that the finless eel kidney 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 fish rhabdovirus, and can be used for preparing vaccines of the fish rhabdovirus and screening antiviral drugs.
Claims (7)
1. A rhabdovirus-sensitive finless eel kidney tissue cell line, characterized in that: the eel kidney tissue cell line is an eel kidney cell line CrE-K with a preservation number of CCTCC NO: C2019286.
2. use of the cell line of claim 1 in the isolation of fish rhabdovirus.
3. Use of the cell line of claim 1 in fish rhabdovirus culture.
4. Use of the cell line of claim 1 in the detection of fish rhabdovirus.
5. Use of the cell line of claim 1 for the preparation of a vaccine for fish rhabdovirus disease.
6. Use of the cell line of claim 1 as a biological model for screening for anti-fish rhabdovirus drugs.
7. The fish rhabdovirus of any one of claims 2 to 6, beingPerhabdovirus、SprivivirusOr/andNovirhabdovirusbelongs to the field of medicine.
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| CN111254225A (en) * | 2020-03-30 | 2020-06-09 | 中国水产科学研究院珠江水产研究所 | Mandarin fish frog virus and rhabdovirus double PCR detection kit and detection method |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2348689C1 (en) * | 2007-08-28 | 2009-03-10 | ГНУ ВНИИ ветеринарной вирусологии и микробиологии | Constant sso-2 cell line of parenchymic organ pool of siberian sturgeon acipenser baeri |
| CN103525749A (en) * | 2013-11-04 | 2014-01-22 | 大连海洋大学 | Triploid loach fin cell line TRMF and establishing method thereof |
| CN103555657A (en) * | 2013-11-04 | 2014-02-05 | 大连海洋大学 | Tetraploid loach fin cell line TEMF and establishment method thereof |
| CN104974977A (en) * | 2015-04-30 | 2015-10-14 | 肇庆大华农生物药品有限公司 | Epinephelus lanceolatus kidney tissue cell line and construction method thereof |
| CN109182277A (en) * | 2018-09-07 | 2019-01-11 | 中国水产科学研究院长江水产研究所 | A kind of swamp eel rhabdovirus CrERV and RT-PCR detection primer and application |
| CN110551689A (en) * | 2019-08-13 | 2019-12-10 | 中国水产科学研究院珠江水产研究所 | Channa argus brain cell line and construction method and application thereof |
-
2020
- 2020-01-16 CN CN202010048530.9A patent/CN112126619B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2348689C1 (en) * | 2007-08-28 | 2009-03-10 | ГНУ ВНИИ ветеринарной вирусологии и микробиологии | Constant sso-2 cell line of parenchymic organ pool of siberian sturgeon acipenser baeri |
| CN103525749A (en) * | 2013-11-04 | 2014-01-22 | 大连海洋大学 | Triploid loach fin cell line TRMF and establishing method thereof |
| CN103555657A (en) * | 2013-11-04 | 2014-02-05 | 大连海洋大学 | Tetraploid loach fin cell line TEMF and establishment method thereof |
| CN104974977A (en) * | 2015-04-30 | 2015-10-14 | 肇庆大华农生物药品有限公司 | Epinephelus lanceolatus kidney tissue cell line and construction method thereof |
| CN109182277A (en) * | 2018-09-07 | 2019-01-11 | 中国水产科学研究院长江水产研究所 | A kind of swamp eel rhabdovirus CrERV and RT-PCR detection primer and application |
| CN110551689A (en) * | 2019-08-13 | 2019-12-10 | 中国水产科学研究院珠江水产研究所 | Channa argus brain cell line and construction method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| CHANGSHENG DU 等: "Induction of apoptosis in a carp leucocyte cell line infected with turbot (Scophthalmus maximus L.) rhabdovirus", 《VIRUS RESEARCH》 * |
| TONG OU 等: "Isolation and identification of a lethal rhabdovirus from farmed rice field eels Monopterus albus", 《DISEASES OF AQUATIC ORGANISMS》 * |
| WENZHI LIU 等: "Isolation, identifcation, and classifcation of a novel rhabdovirus from diseased Chinese rice-feld eels (Monopterus albus)", 《ARCHIVES OF VIROLOGY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111254225A (en) * | 2020-03-30 | 2020-06-09 | 中国水产科学研究院珠江水产研究所 | Mandarin fish frog virus and rhabdovirus double PCR detection kit and detection method |
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