CN114350601B - Cherry valley duck fibroblast line and construction method and application thereof - Google Patents
Cherry valley duck fibroblast line and construction method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and discloses a cherry valley duck fibroblast line and a construction method and application thereof. The cherry valley duck fibroblast line is named as cherry valley duck fibroblast yDEF, and is preserved in China center for type culture collection located in the university of Wuhan, wuhan in 2021, 11 months and 4 days, wherein the preservation number is CCTCC NO: c2021288, which has been transmitted to 85 generations at present, has smooth and full cell surface, no spindle-shaped fibroblast and uniform shape; the cells are polygonal in shape and are closely arranged; the virus susceptibility test shows that the cherry valley duck fibroblast cell line is susceptible to avian influenza virus, bursal disease virus, duck reovirus, avian adenovirus, newcastle disease virus and duck hepatitis virus; the cell line can be used for culturing, separating and detecting poultry viruses, preparing poultry vaccines and screening medicaments for preventing or treating diseases caused by poultry virus infection.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a cherry valley duck fibroblast line and a construction method and application thereof.
Background
Among the various avian diseases, infectious diseases are still the most serious diseases harming the poultry breeding industry, particularly viral infectious diseases. Highly pathogenic avian influenza represented by subtype avian influenza such as H5, H7 and H9 has serious influence on poultry breeding, and H5N6 avian influenza virus is a new subtype of avian influenza virus, is influenza virus generated after reassortment of avian influenza virus H5N1 and H6N6, and has high pathogenicity to poultry. In addition, in the poultry industry, the development of the livestock and poultry industry is also severely restricted by novel epidemic diseases such as newcastle disease, infectious bronchitis, infectious bursal disease, frequent infectious arthritis of chicken, relatively popular duck parvovirus disease, duck viral hepatitis, duck reovirus disease, duck paramyxovirus disease and the like in the duck industry.
Vaccine immunization is a means for effectively preventing the poultry disease pandemic at present, and the domestic production of the poultry vaccine is mainly realized by the traditional process of a chicken embryo method. The process has the advantages of long culture period, complex operation steps and high production cost, the problems of unstable quality of the chick embryos and potential exogenous virus pollution exist in large-scale production, the harvested waste embryos are difficult to harmlessly treat, and the problems of virus diffusion risk and environmental pollution exist. Therefore, the production of vaccines by continuous cell lines instead of chicken embryos is a great trend. With the continuous development of cell culture technology, cell matrix becomes an important material for the development of virus vaccines. Mature cell lines used for vaccine production are reported and researched at present, and comprise mammalian cells such as Vero, MDCK, marc145 and the like, however, the existing cell source vaccine mainly adopts heterologous cells as a cell matrix for production, and the immunogenicity is poor.
At present, the research and development and results of the avian cells are less, and the duck source cell lines are less. However, the separation of the viruses of ducks is mostly carried out by using Muscovy duck embryos, but because no commercial Specific Pathogen Free (SPF) Muscovy duck embryos exist at home and abroad at present, the separation of the viruses is influenced by the conditions of endogenous virus interference and the like when the ducks are separated by using the Muscovy duck embryos. In order to effectively isolate duck viruses, detect the viruses as soon as possible and prevent disease outbreaks, many scholars at home and abroad research on avian cell lines. Therefore, the isolation of primary avian cells and the establishment of immortalizable cell lines are the focus of breakthrough for the production of cell-derived vaccines. At present, a cherry valley duck fibroblast line capable of being stably and quickly passaged needs to be established urgently and applied to various poultry viruses.
Disclosure of Invention
The first aspect of the invention aims to provide a cherry valley duck fibroblast line.
The second aspect of the present invention is to provide the method for constructing the fibroblast cell line of the cherry valley duck of the first aspect.
The third aspect of the invention aims to provide the application of the cherry valley duck fibroblast line of the first aspect.
In a fourth aspect, the present invention provides a method for culturing a virus.
The fifth aspect of the present invention is to provide a method for preparing a viral vaccine.
In a sixth aspect, the present invention provides a viral vaccine.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides a cherry valley duck fibroblast line, which is named as cherry valley duck fibroblast yDEF and is preserved in the China center for type culture collection at the university of Wuhan, in 2021, 11 months and 4 days, wherein the preservation number is CCTCC NO: C2021288.
in a second aspect of the present invention, there is provided a method for constructing the fibroblast cell line of the cherry valley duck of the first aspect of the present invention, comprising the steps of: 1) Mixing trunk tissues of cherry valley duck embryos with collagenase, carrying out primary digestion to obtain primarily digested tissue fragments, mixing the primarily digested tissue fragments with pancreatin, and carrying out secondary digestion to obtain second tissue fragments; 2) Grinding the second tissue fragments, performing solid-liquid separation to obtain cells, adding erythrocyte lysate, performing solid-liquid separation to obtain cell precipitates, resuspending the cell precipitates by using an MEM (minimum essential medium) culture medium containing 9.5-10.5% fetal calf serum, culturing, replacing the MEM culture medium containing 7.5-8.5% fetal calf serum for culture after the cells grow adherently, digesting the cells by using pancreatin after the cells grow into a monolayer, then inoculating the cells into a culture plate according to the density of 0.1-1 cell/hole, taking the cells in a single cell hole to grow into a clone mass, and obtaining a cherry valley duck fibroblast single cell strain; 3) Digesting the cherry valley duck fibroblast single-cell strain by using pancreatin, subculturing, after the cells grow into a single layer, digesting by using pancreatin, carrying out solid-liquid separation to obtain cell precipitate, and carrying out subculturing by using an MEM (minimum medium) culture medium containing 4-6% fetal calf serum to obtain the cherry valley duck fibroblast domesticated by reducing serum; 4) And (3) subculturing the cherry valley duck fibroblasts domesticated by the serum-reducing medium by using MEM (minimum essential medium) containing 2-3% fetal calf serum to obtain the cherry valley duck fibroblasts.
Preferably, the preparation method of the trunk tissue of the cherry valley duck embryo in the step 1) is as follows: removing eggshell of embryo egg of cherry valley duck, taking out embryo body, and removing head, tail, viscera and blood vessel of embryo to obtain body tissue of cherry valley duck embryo.
Preferably, the collagenase used in step 1) includes collagenase of the tetrasaccharide type, bovine serum albumin of the fetus, penicillin, streptomycin and HANKS liquid.
Preferably, the first digestion in the step 1) is carried out at the temperature of 36-38 ℃ and the rpm of 12-18 for 10-20 min.
Preferably, the second digestion in step 1) is carried out at 20-30 ℃ for 15-25 min.
Preferably, the size of the second tissue fragment in step 2) is 1-2 cm 3 A/one.
Preferably, the generation number of the subculture in the step 3) is 8 to 12.
Preferably, the conditions of the subculture in step 3) are 36 to 38 ℃,4 to 6% 2 。
Preferably, the subculture in step 3) has 4 to 6 generations.
Preferably, the subculture in step 4) has 8 to 12 generations.
Preferably, the conditions of the subculture are 36 to 38 ℃,4 to 6% 2 。
In a third aspect of the invention, there is provided the use of the cherry valley duck fibroblast cell line of the first aspect of the invention:
the application of the cherry valley duck fibroblast line in the first aspect of the invention in culturing avian viruses;
the application of the cherry valley duck fibroblast line in the first aspect of the invention in preparing products for culturing avian viruses;
the application of the cherry valley duck fibroblast line in the first aspect of the invention in the separation of avian viruses;
the application of the cherry valley duck fibroblast line in the first aspect of the invention in preparing products for separating avian viruses;
the application of the fibroblast cell line of the cherry valley duck in the first aspect of the invention in detecting avian viruses at a non-diagnostic treatment destination;
the application of the cherry valley duck fibroblast line in the first aspect of the invention in preparing products for detecting avian viruses;
the application of the cherry valley duck fibroblast line in the first aspect of the invention in preparing poultry vaccines;
the application of the cherry valley duck fibroblast line in the first aspect of the invention in drug screening is a drug for preventing or treating diseases caused by poultry virus infection.
Preferably, the avian virus is at least one of avian influenza virus, bursal disease virus, duck reovirus, avian adenovirus, newcastle disease virus and duck hepatitis virus.
Preferably, the avian influenza virus is at least one of an avian influenza H5 subtype virus and an avian influenza H7 subtype virus; further at least one of H5-rFJ56 and H7-rGD 76.
Preferably, the bursal disease virus is IBDV-X1.
Preferably, the duck reovirus is NDRV-DH13.
Preferably, the avian adenovirus is FAdV-4.
Preferably, the newcastle disease virus is LaSota.
Preferably, the duck hepatitis virus is duck hepatitis CH60 attenuated vaccine.
In a fourth aspect of the present invention, a method for culturing avian viruses is provided, wherein avian viruses are inoculated into the cherry valley duck fibroblast cell line of the first aspect of the present invention and cultured.
Preferably, the avian virus is at least one of avian influenza virus, bursal disease virus, duck reovirus, avian adenovirus, newcastle disease virus and duck hepatitis virus.
Preferably, the avian influenza virus is at least one of an avian influenza H5 subtype virus and an avian influenza H7 subtype virus; further at least one of H5-rFJ56 and H7-rGD 76.
Preferably, the bursal disease virus is IBDV-X1.
Preferably, the duck reovirus is NDRV-DH13.
Preferably, the avian adenovirus is FAdV-4.
Preferably, the newcastle disease virus is LaSota.
Preferably, the duck hepatitis virus is duck hepatitis CH60 attenuated vaccine.
Preferably, the culture conditions are 31 to 39 ℃ and 4 to 6% CO 2 (ii) a Further 33 to 37 ℃ and 5% of CO 2 。
In a fifth aspect of the present invention, a method for preparing a poultry vaccine is provided, wherein a poultry virus is inoculated to the first aspect of the present invention of the cherry valley duck fibroblast cell line, cultured and inactivated to obtain the poultry vaccine.
Preferably, the avian virus is at least one of avian influenza virus, bursal disease virus, duck reovirus, avian adenovirus, newcastle disease virus and duck hepatitis virus.
Preferably, the avian influenza virus is at least one of an avian influenza H5 subtype virus and an avian influenza H7 subtype virus; further at least one of H5-rFJ56 and H7-rGD 76.
Preferably, the bursal disease virus is IBDV-X1.
Preferably, the duck reovirus is NDRV-DH13.
Preferably, the avian adenovirus is FAdV-4.
Preferably, the newcastle disease virus is LaSota.
Preferably, the duck hepatitis virus is duck hepatitis CH60 attenuated vaccine.
Preferably, the culture conditions are 31 to 39 ℃ and 4 to 6% CO 2 (ii) a Further 33 to 37 ℃ and 5% 2 。
In a sixth aspect of the present invention, there is provided an avian vaccine obtained by the process of the fifth aspect of the present invention.
The beneficial effects of the invention are:
the invention obtains a cherry valley duck fibroblast line which is named as cherry valley duck fibroblast yDEF through a large amount of creative labor of an inventor, and is preserved in China center for type culture preservation located in China, wuhan university at 11 months and 4 days 2021, wherein the preservation number is CCTCC NO: c2021288, which has been transmitted to 85 generations at present, has smooth and full cell surface, no spindle-shaped fibroblast and uniform shape; the cells are polygonal and are closely arranged; the virus susceptibility test shows that the cherry valley duck fibroblast cell line is susceptible to avian influenza virus, bursal disease virus, duck reovirus, avian adenovirus, newcastle disease virus and duck hepatitis virus; the cell line can be used for culturing, separating and detecting avian viruses, preparing avian vaccines and screening medicaments for preventing or treating diseases caused by avian virus infection.
Drawings
FIG. 1 is a cell morphology map of primary 5 th generation cells in example 2.
FIG. 2 is a cell morphology of 15 th generation cells of low serum adherent culture type cherry valley duck fibroblast cell line in example 2.
FIG. 3 is a karyotype analysis chart of the 10 th, 35 th and 45 th generation cells (DEF 10, DEF35 and DEF 45) of the low serum adherent culture type cherry valley duck fibroblast line in example 2.
FIG. 4 is a graph of a normal control group of fibroblasts of cherry valley duck cultured adherent to low serum in example 3.
FIG. 5 is a graph showing the results of inoculating 2.5% o of fibroblast cells of cherry valley duck cultured adherent to low serum in example 3 to H5-rFJ56 strain for 48H.
FIG. 6 is a graph of the results of the low serum adherent culture of cherry valley duck fibroblasts in example 3 inoculated with H7-rGD76 strain at a inoculation dose of 5% o for 48H.
FIG. 7 is a graph showing the results of 48 hours after the low serum adherent culture type cherry valley duck fibroblasts in example 4 were inoculated with IBDV-X1 strain at a inoculation amount of 2.5 ‰.
FIG. 8 is a graph showing the results of example 5 in which the low serum adherent culture type cherry valley duck fibroblasts were inoculated with NDRV-DH13 strain at a inoculation amount of 5 ‰ for 40 hours.
FIG. 9 is a graph showing the results of the inoculation of 5% o of low serum adherent culture type cherry valley duck fibroblasts into FAdV-4 strain for 48 hours in example 6.
FIG. 10 is a graph showing the results of inoculating low serum adherent culture type cherry valley duck fibroblasts to Newcastle Disease Virus (NDV) LaSota strain at a inoculation amount of 5 ‰ for 48 hours in example 7.
FIG. 11 is a graph showing the results of the administration of 10% of low serum adherent culture type cherry valley duck fibroblasts to duck hepatitis CH60 attenuated vaccine for 48 hours in example 8.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Experimental procedures without specific conditions noted in the following examples, generally according to conventional conditions, or according to conditions recommended by the manufacturer. The materials, reagents and the like used in the present examples are commercially available reagents and materials unless otherwise specified.
The reagents used in this example were analytical grade reagents and were commercially available from regular channels.
Example 1 construction of Low serum adherent culture type cherry valley duck fibroblast cell line
1. Separating and culturing fibroblast of cherry valley duck:
(1) Selecting embryo eggs of cherry valley duck hatched to proper age of day (5 d), selecting embryo bodies with abundant blood vessels and motion, and marking the positions of an air outlet chamber and the embryo bodies according to the eggs;
(2) Placing the embryo eggs on an egg rack, cleaning the eggshells with warm water, and wiping the eggshells with 75% alcohol cotton balls;
(3) After being sterilized by 75 percent alcohol, the air chamber is opened by a middle-sized tweezers in an aseptic method under the aseptic condition, and the eggshells are removed along the edge of the air chamber;
(4) Tearing off the eggshell by using ophthalmological forceps to expose the duck embryo, lightly picking up the embryo head by using elbow forceps, taking out the embryo body, placing the embryo body into a sterile plate, dissecting and taking materials;
(5) Carefully scratching tissues such as the head, the tail, the internal organs, the blood vessels and the like of the embryo by using scissors, adding sterile PBS solution to the trunk part of the duck embryo for cleaning twice to obtain a duck embryo trunk tissue sample, and cutting the duck embryo trunk tissue sample to 0.5-2 mm 3 Obtaining tissue fragments;
(6) Placing the tissue fragments obtained in the step (5) into a collagenase solution (the collagenase solution is prepared by four-type collagenase, fetal bovine serum albumin, penicillin, streptomycin and HANKS solution, wherein the concentration of the four-type collagenase is 0.5mg/mL, the concentration of the fetal bovine serum albumin is 0.5mg/mL, the concentration of the penicillin is 100U/mL, and the concentration of the streptomycin is 0.1 mg/mL) for digestion (the digestion condition is that the tissue fragments are vibrated for 15min at the vibration frequency of 15rpm at 37 ℃), and filtering to obtain preliminarily digested tissue fragments; digesting the primarily digested tissue fragment in 0.25% (v/v) EDTA-pancreatin at room temperature (25 deg.C) for 20min, and filtering to obtain a second tissue fragment;
(7) Adjusting the second tissue fragment to 1-2 cm 3 After the last one of the two or more,grinding, filtering, centrifuging (1000 rpm for 10 min), and collecting cells; adding erythrocyte lysate, blowing, standing at room temperature, centrifuging (1000 rpm for 10 min) to obtain cell precipitate, and suspending the cell precipitate in MEM medium containing 10% fetal calf serum (cell density of 2 × 10) 4 one/mL) of the cells, placing the obtained cell suspension into a T25 cell culture flask, and then transferring the cell suspension into a water jacket type constant-temperature carbon dioxide incubator for culture (the culture condition is that the temperature is 37 ℃, and the concentration of carbon dioxide is 5%).
2. Obtaining fibroblast of cherry valley duck: after the cells in the step 1 grow adherently, replacing a fresh culture medium (MEM culture medium containing 8% fetal calf serum) every day until the adherently cultured cells grow into a monolayer, digesting the cells by using 0.25% EDTA-pancreatin until the cells grow into single cells, then inoculating the cells into a 96-well plate according to the average density of 0.5 cell/hole, marking the holes for inoculating the single cells, and obtaining the cherry valley duck fibroblast single cell strain after the cells in the single cell holes grow into a clone cluster.
3. Carrying out passage domestication on cherry valley duck fibroblasts: digesting the clustered single-cell strain with 5g/L trypsin solution for 30s, and then subculturing for 10 generations (the culture condition is 37 ℃ and the carbon dioxide concentration is 5%), thereby obtaining the cherry valley duck fibroblast strain with uniform morphology.
4. Carrying out passage domestication on fibroblast blood serum-reducing cells of cherry valley ducks: after the fibroblasts of the cherry valley duck in step 3 grew into a monolayer, digesting and dispersing the fibroblasts of the cherry valley duck with 0.25% EDTA-trypsin, centrifuging at 1000rpm 5min to obtain cell precipitates, culturing in MEM medium containing 5% fetal bovine serum under the conditions of 37 ℃ and 5% CO 2 (ii) a Continuously culturing for 5 generations to obtain the serum-reducing domesticated cherry valley duck fibroblast.
5. Low serum acclimation of cherry valley duck fibroblast: directly subjecting the fibroblast of the cherry valley duck cultured in the last generation in step 4 to liquid replacement and subculture in MEM medium containing 2.5% fetal calf serum under 37 deg.C and 5% CO for 10 generations 2 And domesticating to obtain a low serum adherent culture type cherry valley duck fibroblast line.
And (3) freezing and storing the cells every 5 times of passage in the process of constructing the low serum adherent culture type cherry valley duck fibroblast line, and establishing a cell bank.
The low serum adherent culture type cherry valley duck fibroblast line is named as cherry valley duck fibroblast yDEF, and is preserved in China type culture collection of Wuhan, wuhan university in 11 months and 4 days 2021, wherein the preservation number is CCTCC NO: C2021288.
example 2 characterization of cherry valley duck fibroblast cell lines
1. Morphological observation
The cell morphology of the 30 th generation cells of the low serum adherent culture type cherry valley duck fibroblast line and the 5 th generation cells of the primary cells obtained in step 1 (7) of example 1 was observed by an inverted microscope, and the results are shown in fig. 1 and 2: the primary isolated cells are mostly arranged in a polygonal compact way, and part of spindle-shaped fibroblasts are arranged in the primary isolated cells; the low serum adherent culture type cherry valley duck fibroblast obtained in the embodiment 1 has a smooth and full surface, does not have spindle-shaped fibroblast, and has uniform shape; and the cells are polygonal in shape and are closely arranged.
2. Karyotyping analysis
The 10 th, 35 th and 45 th generation cells (DEF 10, DEF35 and DEF 45) of the low serum adherent culture type cherry valley duck fibroblast line are respectively taken for karyotype analysis (completed by entrusted Tianjin Sell biotechnology Co., ltd.), and the details are as follows:
(1) Cells were treated with colchicine at 37 ℃ for 2h (colchicine concentration 20. Mu.g/mL) during the logarithmic growth phase of mitosis.
(2) Hypotonic treatment: discarding colchicine, transferring cell digestion to a 50mL centrifuge tube, adding 0.5% KCl hypotonic solution, treating at 37 ℃ for 20min, and centrifuging at 800-1000 rmp for 5min.
(3) Fixing: removing supernatant, adding appropriate amount of freshly prepared Carnot solution (methanol: glacial acetic acid =1, glacial acetic acid swelling, methanol fixation) for pre-fixing for 20min, centrifuging for 5min at 800-1000 rmp, replacing fixing solution, and continuously fixing for 10min; centrifuging at 800-1000 rmp for 5min, and then preparing cell suspension by using stationary liquid.
(4) Tabletting: taking a frozen slide, dripping 1 drop of cell suspension to the slide at a height of about 50cm from the slide, and naturally drying in the air; the drop is quickly passed over the alcohol burner flame, back and forth 2-3 times, and the sample is fixed on the slide.
(5) Dyeing: dripping 3-5 drops of reagent of nuclear type analysis dye liquor (purchased from Nanjing institute of bioengineering) on the glass slide, and dyeing for about 1 minute; keeping the first reagent, continuously dripping 6-10 drops of a second reagent of nuclear type analysis dye solution (purchased from Nanjing to build a bioengineering institute), and slightly shaking the glass slide to fully and uniformly mix the dye solution and dye for 5-8 min; washing with water for 30s, and performing microscopic examination after drying.
(6) Karyotyping analysis: cells were counted under a microscope and analyzed for 50 metaphase.
The results are shown in table 1 and fig. 3: at the 10 th generation 92% of the cells contained 78 chromosomes, at the 35 th and 45 th generations 90% of the cells contained 78 chromosomes.
TABLE 1 karyotype analysis results for different generations of yDEF
Example 3 susceptibility of avian influenza H5 subtype virus and avian influenza H7 subtype virus to infection with low serum adherent culture type cherry valley duck fibroblast cell line
Taking 35 th generation cell of low serum adherent culture type fibroblast cell line of cherry valley duck, at 37 deg.C and 5% CO 2 After 48H of incubation, most of the culture medium was discarded in a clean bench leaving only 1-2 mL, and 25. Mu.L and 50. Mu.L of H5-rFJ56 (HA =9 Log) were added, respectively 2 ) Strains (already in the literature: research on proliferation conditions and amplification process of H5 subtype avian influenza virus by different MDCK monoclonal cell strains, namely, zhuhui, a source of yellow and bright bacteria, pengxien, xihaikang and Jingzheng [ J]Animal medicine development, 2020,41 (04): 38-41 published) incubate for 30min, discard the liquid in the flask and add 10mL of MEM medium containing 1% fetal bovine serum;control groups were added with 10mL MEM medium containing 1% fetal bovine serum. 24h after inoculation (33 ℃ C., 5% CO in the culture conditions after inoculation) 2 ) And observing the cytopathic condition every 4h, and collecting the cell venom for HA detection. The results are shown in fig. 4, 5 and table 2: the surfaces of the fibroblasts of the low-serum adherent culture type cherry valley ducks in the control group are smooth and full, the cells are polygonal and are closely arranged, and the cells can overgrow about 48 hours under normal passage; after the avian influenza H5 subtype virus is inoculated, a pathological change phenomenon appears: gaps begin to appear between cells, vacuoles begin to appear inside the cells, and a part of the cells directly die and float above the liquid; after the fibroblasts of the cherry valley duck cultured by the low serum adherent culture are inoculated for 48 hours according to the inoculation amount of 2.5 per mill, HA reaches 8Log 2 After inoculation for 32 hours according to 5 per mill of inoculation amount, HA reaches 9Log 2 (ii) a Therefore, the low serum adherent culture type cherry valley duck fibroblast is sensitive to the avian influenza H5 subtype virus.
TABLE 2 result of H5-rFJ56 strain inoculation of fibroblast of cherry valley duck in low serum adherent culture
| H5-rFJ56 strain inoculation dose | Time of taking poison | HA |
| 25μL(2.5‰) | 48h | 9Log 2 |
| 50μL(5‰) | 32h | 9Log 2 |
Collecting 35 th generation cells of low serum adherent culture type fibroblast cell line of Prunus Pseudocerasifera and Valley duck, and removing CO at 37 deg.C and 5% 2 After 48H of incubation, most of the culture medium was discarded in the clean bench leaving only 1-2 mL, and 25. Mu.L of H7-rGD76 (HA =9 Log) was added separately 2 ) Strains (already in the literature: development of inactivated vaccine of rGD76 strain of leaf hajia, vengesafranin, qi-Qi wenbao, wanhong, limin, schlenna, huxiumei, lioming, lizhao H7N9 subtype recombinant avian influenza virus [ J]The animal medicine was advanced, 2019,40 (08): 44-48, published) and incubated for 30min, the liquid in the flask was discarded and 10mL of MEM medium containing 1% fetal bovine serum was added; the control group was used as a control, and the cell mass was inoculated for 24 hours (after inoculation, the culture conditions were 33 ℃ and 5% CO 2 ) And observing the cytopathic condition every 4h, and collecting the cell venom for HA detection. The results are shown in FIGS. 4 and 6: the surfaces of the fibroblasts of the low-serum adherent culture type cherry valley ducks in the control group are smooth and full, the cells are polygonal and are closely arranged, and the cells can overgrow about 48 hours under normal passage; after the avian influenza H7 subtype virus is inoculated, a pathological change phenomenon appears: gaps begin to appear between cells, vacuoles begin to appear inside the cells, and a part of the cells directly die and float above the liquid; after inoculation for 48 hours according to 5 per mill of inoculation amount, HA reaches 9Log 2 (ii) a Therefore, the low serum adherent culture type cherry valley duck fibroblast is sensitive to the avian influenza H7 subtype virus.
Example 4 sensitivity of Fa's bursa Virus infection on Low serum adherent culture type cherry valley Duck fibroblast cell line
Collecting 35 th generation cells of low serum adherent culture type fibroblast cell line of Prunus Pseudocerasifera and Valley duck, and removing CO at 37 deg.C and 5% 2 After culturing for 48h and stable growth, most of the culture solution is discarded in a clean bench, only 1-2 mL of the culture solution is left, and 10 muL, 25 muL and 50 muL of IBDV-X1 (TCID) are added 50 ＝10 -8.5 0.1 mL) strain (already in literature: [3]Immune protection effect study of Newcastle disease, avian influenza, infectious bursal disease Virus recombinant protein [ D]University of agriculture and forestry, 2021), 10mL of MEM medium containing 1% fetal bovine serum was added after discarding the liquid in the flask, and the control group of example 3 was used as a control. After 24h of inoculation (culture conditions after inoculation are 37)℃、5％CO 2 ) Observing cytopathic condition every 4h, and collecting cell venom as TCID 50 And (6) detecting. The results are shown in fig. 4, 7 and table 3: the surfaces of the fibroblasts of the low-serum adherent culture type cherry valley ducks in the control group are smooth and full, the cells are polygonal and are closely arranged, and the cells can overgrow about 48 hours under normal passage; after inoculation of bursal disease virus, pathological changes appear: most cells become round and shrink, fall off and float on the liquid level of the culture medium; a small part of cells still at the bottom of the bottle become slender in shape, and bubbles are generated in the cells; after the fibroblasts of the cherry valley duck cultured by adherence with low serum are inoculated for 56 hours according to the inoculation amount of 1 per mill, TCID 50 Up to 10 -7.67 0.1mL, after inoculation for 48 hours according to the inoculation amount of 2.5 thousandth, TCID 50 Up to 10 -8.16 0.1mL, after being inoculated for 28h according to 5 per thousand inoculation amount, TCID 50 Up to 10 -7.5 0.1mL; therefore, the low serum adherent culture type cherry valley duck fibroblasts are sensitive to the bursal disease virus.
TABLE 3 result of IBDV-X1 strain inoculation of fibroblast of low-serum adherent culture type cherry valley duck
| IBDV-X1 strain inoculation amount | Time of taking poison | TCID 50 |
| 10μL(1‰) | 56h | 10 -7.67 /0.1mL |
| 25μL(2.5‰) | 48h | 10 -8.16 /0.1mL |
| 50μL(5‰) | 28h | 10 -7.5 /0.1mL |
Example 5 sensitivity of Duck reovirus infection to Low serum adherent culture type cherry valley Duck fibroblast cell line
Collecting 35 th generation cells of low serum adherent culture type fibroblast cell line of Prunus Pseudocerasifera and Valley duck, and removing CO at 37 deg.C and 5% 2 After culturing for 48h and stable growth, most of the culture solution is discarded in a clean bench, only 1-2 mL of the culture solution is left, and 25 μ L and 50 μ L of NDRV-DH13 (TCID) are added respectively 50 ＝10 -4.0 0.1 mL) strain (already in literature: prokaryotic expression and immunogenicity analysis of structural protein sigma B of novel duck reovirus strain DH13 of Yanghua lake, huanghuilan, yuan Sheng, liwenjun, yaoxin Yan, zhangyuqian, lizhao, huangshujian, zhang saussurea involucrata [ J/OL ] are provided]Chinese veterinary drug 1-9[2021-10-25 ]]Published.)) was incubated for 30min, the liquid in the flask was discarded and 10mL of MEM medium containing 1% fetal bovine serum was added; the control group of example 3 was used as a control. 24h after inoculation (37 ℃ C., 5% CO after inoculation) 2 ) Observing cytopathic condition every 4h, and collecting cell venom as TCID 50 And (6) detecting. The results are shown in fig. 4, fig. 8 and table 4: the fibroblast cells of the low-serum adherent culture type cherry valley duck in the control group have smooth and full surfaces, are polygonal and are closely arranged, and can grow fully after about 48 hours of normal passage; after the duck reovirus is inoculated, a pathological change phenomenon appears: the cells begin to become round and shrink, part of the cells fall off and float in the culture medium liquid, and syncytial lesion phenomena mainly occur; after the fibroblasts of the cherry valley duck cultured by adherence with low serum are inoculated for 52 hours according to 2.5 per mill of inoculation amount, TCID 50 Up to 10 -3.83 0.1mL, after inoculation for 40h according to 5 per mill of inoculation amount, TCID 50 Up to 10 -4.25 0.1mL; it can be seen that the low serum adherent culture type cherry valley duck fibroblasts are sensitive to duck reovirus.
TABLE 4 result of NDRV-DH13 strain inoculation of fibroblast of low serum adherent culture type cherry valley duck
| Inoculation dose of NDRV-DH13 strain | Time of taking poison | TCID 50 |
| 25μL(2.5‰) | 52h | 10 -3.83 /0.1mL |
| 50μL(5‰) | 40h | 10 -4.25 /0.1mL |
Example 6 sensitivity of avian adenovirus infection on Low serum adherent culture type cherry valley Duck fibroblast cell line
Collecting 35 th generation cells of low serum adherent culture type fibroblast cell line of Prunus Pseudocerasifera and Valley duck, and removing CO at 37 deg.C and 5% 2 After culturing for 48h, most of the culture medium is discarded in a clean bench, leaving only 1-2 mL, and 50. Mu.L of FAdV-4 (TCID) is added 50 ＝10 -8.0 0.1 ml) strain (already in literature: study on early miRNAs and mRNAs expression changes in avian adenovirus serum type 4 infection by Wenning LMH cells [ D]Published in 2020), the flask is discarded and 10mL of MEM medium containing 1% fetal bovine serum is added, using the control group of example 3 as a control. 24h after inoculation (37 deg.C, 5% CO after inoculation) 2 ) Observing cytopathic condition every 4h, and collecting cell venom as TCID 50 And (6) detecting. The results are shown in FIGS. 4 and 9: low-serum adherent culture cherry in control groupThe peach valley duck fibroblast has smooth and full surface, is polygonal and is closely arranged, and can grow fully after about 48 hours of normal passage; after inoculation of the avian adenovirus, a pathological change phenomenon occurs: cell shrinkage, partial cell rounding and shedding; after the fibroblast of the cherry valley duck cultured by adherence with low serum is inoculated for 48 hours according to 5 per mill of inoculation amount, TCID 50 Up to 10 -7.5 0.1mL; it can be seen that the low serum adherent culture type cherry valley duck fibroblasts are sensitive to avian adenovirus.
Example 7 susceptibility of Newcastle disease Virus to infection by Low serum adherent culture of the fibroblast cell line cherry valley duck
Taking 35 th generation cell of low serum adherent culture type fibroblast cell line of cherry valley duck, at 37 deg.C and 5% CO 2 After 48h of incubation, most of the culture medium was discarded in a clean bench, leaving only 1-2 mL, and 50. Mu.L of Newcastle Disease Virus (NDV) LaSota (HA =9 Log) was added 2 ) Strains (already in the literature: process study on preparation of bivalent inactivated vaccine by culturing equine huitong, vengea scarlet, and Egyaha, newcastle disease and avian influenza virus in same embryo]Poultry and poultry disease control, disclosed in 2021 (05): 18-22)), for 30min, the liquid in the flask was discarded, and 10mL of MEM medium containing 1% fetal bovine serum was added, using the control group in example 3 as a control. 24h after inoculation (37 ℃ C., 5% CO after inoculation) 2 ) And observing the cytopathic condition every 4h, and collecting the cell venom for HA detection. The results are shown in FIGS. 4 and 10: the surfaces of the fibroblasts of the low-serum adherent culture type cherry valley ducks in the control group are smooth and full, the cells are polygonal and are closely arranged, and the cells can overgrow about 48 hours under normal passage; after inoculation of newcastle disease virus, a lesion phenomenon occurs: the cells are shriveled, vacuoles appear in the cells, and partial cells become round and fall off; after the fibroblasts of the cherry valley duck cultured by the low serum adherent culture are inoculated for 48 hours according to 5 per mill of inoculation amount, HA reaches 8Log 2 (ii) a Therefore, the low serum adherent culture type cherry valley duck fibroblast is sensitive to the Newcastle disease virus.
Example 8 sensitivity of Duck hepatitis Virus infection Low serum adherent culture type cherry valley Duck fibroblast cell line
Taking 35 th generation cell of low serum adherent culture type fibroblast cell line of cherry valley duck at 37 deg.C、5％CO 2 After culturing for 48h, most of the culture medium was discarded in an ultra-clean bench, leaving only 1-2 mL, 1mL of duck hepatitis CH60 attenuated vaccine (purchased from Hayao group biological vaccine Co., ltd., product name of Ganbui, http:// www.moa.gov.cn/govpublic/SYJ/201307/t 20130702-3509497. Htm, published as new veterinary registration) was added for incubation for 30min, and 10mL of MEM medium containing 1% fetal calf serum was added after removing the liquid in the bottle, and the control group in example 3 was used as a control. 24h after inoculation (37 ℃ C., 5% CO after inoculation) 2 ) Observing cytopathic condition every 4h, and collecting cell venom as TCID 50 And (6) detecting. The results are shown in FIGS. 4 and 11: the surfaces of the fibroblasts of the low-serum adherent culture type cherry valley ducks in the control group are smooth and full, the cells are polygonal and are closely arranged, and the cells can overgrow about 48 hours under normal passage; after the duck hepatitis new virus is inoculated, a pathological change phenomenon appears: the cells are shriveled, vacuoles appear in the cells, and partial cells become round and fall off; after the fibroblasts of the cherry valley duck cultured by low serum adherent culture are inoculated for 48 hours according to 10 percent of inoculation amount, TCID 50 Up to 10 -3.5 0.1mL; therefore, the low serum adherent culture type cherry valley duck fibroblast is sensitive to the Newcastle disease virus.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (6)
1. A cherry valley duck fibroblast line is named as cherry valley duck fibroblast yDEF, and is preserved in the China center for type culture collection (CCTCC NO) at 11 months and 4 days in 2021, wuhan university, with the preservation number of CCTCC NO: C2021288.
2. use of the cherry valley duck fibroblast cell line of claim 1 in any one of (1) to (4);
(1) Culturing the virus;
(2) Preparing a product of the cultured virus;
(3) Isolating the virus;
(4) Preparing a product of the isolated virus;
the virus is at least one of avian influenza virus, bursal disease virus, duck reovirus, avian adenovirus, newcastle disease virus and duck hepatitis virus.
3. Use of the cherry valley duck fibroblast cell line of claim 1 in any one of (5) to (6);
(5) Detecting the virus at a non-diagnostic treatment destination;
(6) Preparing a product for detecting viruses;
the virus is at least one of avian influenza virus, bursal disease virus, duck reovirus, avian adenovirus, newcastle disease virus and duck hepatitis virus.
4. Use of the cherry valley duck fibroblast cell line of claim 1 in any one of (7) to (8);
(7) Preparing a virus vaccine;
(8) Screening drugs;
the medicament is a medicament for preventing or treating diseases caused by viral infection;
the virus is at least one of avian influenza virus, bursal disease virus, duck reovirus, avian adenovirus, newcastle disease virus and duck hepatitis virus.
5. A method for culturing virus, inoculating virus to the cherry valley duck fibroblast cell line of claim 1, culturing;
the virus is at least one of avian influenza virus, bursal disease virus, duck reovirus, avian adenovirus, newcastle disease virus and duck hepatitis virus.
6. A method for preparing virus vaccine, inoculating virus to the cherry valley duck fibroblast line of claim 1, culturing, inactivating to obtain virus vaccine;
the virus is at least one of avian influenza virus, bursal disease virus, duck reovirus, avian adenovirus, newcastle disease virus and duck hepatitis virus.
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