CN118956732A - A duck corneal epithelial cell suspension cell line and its construction method and application - Google Patents
A duck corneal epithelial cell suspension cell line and its construction method and application Download PDFInfo
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Abstract
本发明属于生物技术领域，具体涉及一种鸭眼角膜上皮细胞悬浮细胞株及其构建方法与应用。本发明首次在鸭上分离获得成眼角膜上皮细胞，并成功悬浮驯化，名称为麻鸭眼角膜上皮悬浮细胞DCE‑S，于2024年6月28日保藏于位于中国.武汉.武汉大学的中国典型培养物保藏中心，保藏编号为CCTCC NO：C2024189。该细胞株易于培养，生长速度较快，能够稳定传代，细胞密度高，可达6～8×106cells/mL，呈单个分散生长，形态饱满，大小均一，细胞活率高、适用于生物反应器无血清悬浮培养的细胞株。该细胞株可用于培养、分离、检测禽类病毒，制备禽类疫苗，筛选用于预防或治疗禽类病毒感染所致的疾病的药物。The present invention belongs to the field of biotechnology, and specifically relates to a duck corneal epithelial cell suspension cell line and its construction method and application. The present invention isolates and obtains adult corneal epithelial cells from ducks for the first time, and successfully suspends and domesticates them. The name is duck corneal epithelial suspension cells DCE-S, which were deposited in the China Center for Type Culture Collection at Wuhan University, Wuhan, China on June 28, 2024, with a deposit number of CCTCC NO: C2024189. The cell line is easy to culture, grows fast, can be stably passaged, has a high cell density of 6 to 8×10 6 cells/mL, grows in a single dispersed manner, has a full morphology, is uniform in size, has a high cell viability, and is suitable for serum-free suspension culture in bioreactors. The cell line can be used to culture, isolate, and detect avian viruses, prepare avian vaccines, and screen drugs for the prevention or treatment of diseases caused by avian virus infections.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a duck eye cornea epithelial cell suspension cell strain, a construction method and application thereof.
Background
The production and consumption of meat ducks are large, 30 or more hundred million meat ducks are produced annually, the annual output is over 1000 hundred million yuan, the frequency of fowl diseases is increased along with the increase of the feeding amount, and the diseases are the most serious diseases endangering the aquatic fowl breeding industry, such as avian influenza (subtype H5, H7, H9, and the like), duck plague, duck viral hepatitis, young duck gosling plague, muscovy duck parvovirus disease, duck coronavirus enteritis, duck pox, muscovy duck reovirus disease, novel viral infectious diseases such as duck reovirus disease, duck paramyxovirus disease, duck torus, duck tembusu virus disease, and the like, and the development of the fowl breeding industry is severely restricted.
Vaccine immunization is the most effective means for preventing animal epidemic diseases, and domestic poultry vaccine production mainly depends on a chick embryo culture process, a poultry primary cell culture process and a passage cell culture process. The chick embryo culture process has the advantages of long culture period, complicated operation steps, high production cost, unstable chick embryo quality and potential exogenous virus pollution during large-scale production, difficult innocent treatment of waste embryo after harvesting, and virus diffusion risk and environmental pollution. The fowl primary cell culture process needs to prepare fresh primary cells each time, and can only produce virus vaccine by the adherence culture process, on one hand, serum maintenance culture is needed, and the cost is high; on the other hand, limited scale up, limited matrix surface area, results in reduced virus yields, susceptibility to contamination and batch-to-batch quality inconsistencies. The existing cell-derived vaccine mainly adopts heterologous cells (such as canine kidney cells (MDCK suspension cells) to culture avian influenza virus) as a culture medium for virus production, and the vaccine immune effect is slightly poorer than that of chicken embryo culture.
The research and development of poultry cells in China are less, and a commercial duck-origin cell line is not available. In order to better and more effectively separate, proliferate and produce avian viruses, develop a special vaccine for waterfowl and prevent disease outbreaks, develop a duck-origin cell suspension cell strain suitable for industrialized mass production, and have great significance.
The application number 202111338851.3 of goose retina epithelial cell line, a construction method and application thereof mainly obtains 1 strain of low-serum adherence culture type goose retina epithelial cell line which is not a duck source cell line and can not be subjected to serum-free suspension culture.
The application number 202210113778.8 of the pig retina epithelial cell line, the construction method and the application thereof mainly obtains 1 pig retina epithelial cell line which is not a duck source cell line and can not be subjected to serum-free suspension culture.
Disclosure of Invention
The object of the first aspect of the invention is to provide a cell line.
The object of the second aspect of the present invention is to provide a method for constructing the cell line of the first aspect of the present invention.
A third aspect of the invention is directed to a use.
The fourth aspect of the present invention is directed to a method for culturing viruses.
The object of the fifth aspect of the present invention is to provide a method for viral vaccine.
The sixth aspect of the present invention is directed to a viral vaccine.
In order to achieve the above purpose of the present invention, the present invention adopts the following technical scheme:
in a first aspect of the present invention, a duck eye cornea epithelial cell line, named sheldrake eye cornea epithelial suspension cell DCE-S (shelduck), is deposited at the China center for type culture collection, having a deposit number of cctccc NO: C2024189.
In a second aspect of the present invention, there is provided a method for constructing a duck eye cornea epithelial cell line according to the first aspect of the present invention, comprising the steps of:
1) Separating eyeballs of duck embryos, separating corneas of the eyeballs, and shearing into tissue blocks;
2) Adding a first culture medium, uniformly mixing, culturing, and carrying out passage for 5-15 generations;
3) Culturing by using a second culture medium, and carrying out passage for 3-5 generations;
4) Culturing by using a third culture medium, and carrying out passage for 3-5 generations;
5) Culturing with a fourth culture medium, carrying out passage for 3-5 generations,
6) Culturing by using a serum-free suspension culture medium, and carrying out passage for 20-30 generations to obtain the strain.
Preferably, the first medium comprises 8-10 v/v% fetal bovine serum, and a basal medium.
Preferably, the second medium comprises 6-8 v/v% fetal bovine serum, and a basal medium.
Preferably, the third medium comprises 4-6 v/v% fetal bovine serum, and a basal medium.
Preferably, the fourth medium comprises 2-3 v/v% fetal bovine serum, and a basal medium.
Preferably, the basal medium comprises at least one of MEM medium, DMEM/F12 medium.
In some embodiments of the invention, the construction method comprises: 1) Taking 3 duck embryos of 7-11 days old, separating eyeballs by using an ophthalmic scissors, placing the duck embryos into a clean sterile plate, washing 3 times by using precooled D-Hank's liquid, separating cornea of the eyes, shearing, adding a small amount of MEM (MEM) of 8-10 v/v% fetal bovine serum, blowing broken tissue blocks, and transferring the crushed tissue blocks into a culture flask for culture under the culture condition of 37 ℃ and 5% CO 2 culture box; 2) After 12-24 h of cell separation culture, gently shaking the cell bottle, discarding the supernatant, leaving only adhered cells, supplementing MEM with fresh 8-10 v/v% fetal bovine serum, and continuously culturing; after the cells grow into a single layer, digesting the single cells by 0.25wt% EDTA-pancreatin, then inoculating the single cells into a 96-well plate according to the average density of 0.5 cells/well, marking the wells inoculated with the single cells, and after the cells in the single cell wells grow into clone clusters, obtaining single cell strains of the duck eye cornea epithelium, and continuously carrying out passage for more than 10 generations; 3) Culturing with MEM culture medium containing 6-8 v/v% foetus calf serum at 37 deg.c in 5% CO 2 incubator. Culturing continuously for 3-5 generations, and culturing by adopting a MEM culture medium containing 4-6 v/v% of fetal calf serum; culturing continuously for 3-5 generations, and culturing by adopting a MEM culture medium containing 2-3 v/v% of fetal calf serum; continuously culturing for 3-5 generations to obtain low-serum adherence culture type duck eye cornea epithelial cells; 4) After low serum adherence culture type duck eye cornea epithelial cells grow into a single layer, the single layer is digested by 0.25wt% EDTA-pancreatin, cell suspension is collected, the cell suspension is centrifuged at 800-1000 rpm for 6min, the supernatant is discarded, cell sediment is left, the cell sediment is resuspended by a serum-free suspension culture medium, the cell density is adjusted to 1.0X10 6 cells/mL, and the culture condition is 37 ℃ and 5% CO 2, and the shake culture is carried out at 100-130 r/min. Cell morphology was observed by sampling every 24 hours and counted. After 2-3 days of culture, the cells are subjected to centrifugal liquid exchange and passage until the cell number of each generation of cells is more than or equal to 6.0X10 6 cells/mL. And (3) stably and continuously culturing the cells for more than 30 generations to obtain serum-free total suspension culture type duck eye cornea epithelial cells, namely duck eye cornea epithelial cell suspension cell strains.
Preferably, the size of the tissue fragments in step 1) is 1-2 cm 3/min.
Preferably, the culture conditions are 31-39 ℃ and 4-6% CO 2; further, the temperature is 33 to 37 ℃ and the concentration of CO 2 is 5 percent.
In a third aspect, the invention provides the use of a duck eye cornea epithelial cell line according to the first aspect of the invention in any one of 1) to 8):
1) Culturing the virus;
2) Preparing a product for culturing viruses;
3) Isolating the virus;
4) Preparing a virus-isolated product;
5) Detecting the virus at a non-diagnostic treatment destination;
6) Preparing a product for detecting viruses;
7) Preparing a virus vaccine;
8) Screening medicines;
The medicine is used for preventing or treating diseases caused by virus infection.
Preferably, the virus comprises at least one of an avian influenza virus, a duck circovirus, and a duck tembusu virus.
Preferably, the avian influenza virus comprises an H9 subtype avian influenza virus.
In a fourth aspect of the invention, there is provided a method of culturing a virus by inoculating the virus into the duck eye corneal epithelial cell line of the first aspect of the invention and culturing.
Preferably, the virus comprises at least one of an avian influenza virus, a duck circovirus, and a duck tembusu virus.
Preferably, the avian influenza virus comprises an H9 subtype avian influenza virus.
Preferably, the culture conditions are 31-39 ℃ and 4-6% CO 2; further, the temperature is 33 to 37 ℃ and the concentration of CO 2 is 5 percent.
In a fifth aspect of the invention, there is provided a method of preparing a viral vaccine by inoculating a virus into the duck eye cornea epithelial cell line of the first aspect of the invention, culturing, and inactivating to obtain a viral vaccine.
Preferably, the virus comprises at least one of an avian influenza virus, a duck circovirus, and a duck tembusu virus.
Preferably, the avian influenza virus comprises an H9 subtype avian influenza virus.
Preferably, the culture conditions are 31-39 ℃ and 4-6% CO 2; further, the temperature is 33 to 37 ℃ and the concentration of CO 2 is 5 percent.
In a sixth aspect of the invention there is provided a viral vaccine prepared by the method of the fifth aspect of the invention.
The beneficial effects of the invention are as follows:
The invention provides a duck eye cornea epithelial cell line, namely a shepherd' S eye cornea epithelial suspension cell DCE-S, which is preserved in China Center for Type Culture Collection (CCTCCNO) in the university of Wuhan and Wuhan in 2024 at 6 months and 28 days through a great deal of creative labor of the inventor, wherein the duck eye cornea epithelial cells are firstly separated from ducks and successfully suspended and domesticated, and the preservation number is CCTCCNO: C2024189. the cell strain is easy to culture, has a high growth speed, can be stably passaged, has a high cell density which can reach 6-8 multiplied by 10 6 cells/mL, is single and dispersive to grow, has full shape and uniform size, has a high cell activity rate, and is suitable for serum-free suspension culture of a bioreactor. The cell strain can be used for culturing, separating and detecting avian viruses, preparing avian vaccines and screening medicines for preventing or treating diseases caused by avian virus infection. The cell strain provides an important culture medium for large-scale suspension culture, cost reduction and synergy of poultry vaccines.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a low serum wall-mounted cultured duck eye corneal epithelial cells of example 1; wherein the left side is the result after 24 hours of culture, and the right side is the result after 48 hours of culture.
FIG. 2 shows the results of growth state of the DCE-S generation 25 of the sheldrake eye corneal epithelial suspension cells.
FIG. 3 shows stable passaging of the sheldrake eye corneal epithelium suspension cells DCE-S (F9-F30) for different generations.
FIG. 4 shows cytopathic results of the DCE-S inoculation of the sheldrake eye corneal epithelial suspension cells with H9 subtype avian influenza virus for 72H.
FIG. 5 shows cytopathic results of duck eye corneal epithelial suspension cells DCE-S inoculated with duck circovirus virus for 60 h.
FIG. 6 shows cytopathic results of duck eye corneal epithelial suspension cells DCE-S inoculated with duck Tembusu virus for 50 h.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
EXAMPLE 1 construction of Duck eye corneal epithelial cell suspension cell lines
1. Quick separation and culture of primary duck eye cornea epithelial cells
(1) Taking duck embryo hatched for 10 days old, and drawing air chamber boundary and fetal position after checking;
(2) Sterilizing embryo eggs with 75% alcohol, tapping and crushing the egg shells of the air chamber parts with sterilized forceps in a sterile operation table, removing the shell membranes of the air chamber parts with another sterile forceps, taking out embryo bodies with the other sterile forceps, and placing the embryo bodies in a sterile plate;
(3) Separating eyeball with ophthalmic scissors, placing into a clean sterile plate, washing with precooled D-Hank's liquid for 3 times, separating cornea, shearing, adding a small amount of MEM (MEM) of 10v/v% fetal bovine serum, blowing broken tissue blocks, transferring into a culture bottle, and culturing under the condition of 37 ℃ and 5% CO 2 incubator;
(4) After 24 hours of cell separation and culture, gently shaking the cell bottle, discarding the supernatant, leaving only adherent cells, supplementing MEM with fresh 10% fetal bovine serum, and continuously culturing;
(5) After the cells grow into a monolayer, digestion treatment is carried out on the single cells by 0.25wt% of EDTA-pancreatin, then the single cells are inoculated into a 96-well plate according to the average density of 0.5 cells/well, the wells inoculated with the single cells are marked, and after the cells in the single cell wells grow into clone clusters, the duck eye cornea epithelial single cell strain can be obtained.
(6) Continuously passaging the duck eye cornea epithelial single cell strain to 10 generations to obtain an adherence culture type duck eye cornea epithelial cell with uniform morphology.
2. Construction of duck eye cornea epithelial cell suspension cell strain
(1) Obtaining low serum adherence culture type duck eye cornea epithelial cells: after the duck eye cornea epithelial cells in the step (6) of the step 1 grow into a single layer, the single layer is digested by 0.25wt% EDTA-pancreatin, and the single layer is cultured by adopting MEM culture medium containing 8v/v% fetal bovine serum, wherein the culture condition is 37 ℃ and 5% CO 2 incubator. After 5 passages of continuous culture, adopting MEM culture medium containing 6v/v% of fetal bovine serum for culture; after 5 passages of continuous culture, adopting MEM culture medium containing 3v/v% of fetal bovine serum for culture; after 5 passages of continuous culture, low serum adherence culture type duck eye cornea epithelial cells are obtained. The results are shown in FIG. 1.
(2) Obtaining duck eye cornea epithelial cell suspension cell strain: when low serum adherence culture type duck eye cornea epithelial cells grow into a single layer, digestion is carried out by 0.25wt% EDTA-pancreatin, cell suspension is collected, centrifugation is carried out at 800rpm for 6min, supernatant is discarded, cell precipitation is left, cell precipitation is resuspended by serum-free suspension medium (BSL-01 medium, guangzhou Baisai biological medicine technology Co., ltd.) and cell density is adjusted to 1.0X10 6 cells/mL, and culture conditions are 37 ℃ and 5% CO 2 and 120r/min shaking culture is carried out. Cell morphology was observed by sampling every 24 hours and counted. After 3 days of culture, the cells are subjected to centrifugal liquid exchange and passage until the cell number of each cell is more than or equal to 6.0X10 6 cells/mL, and the cells are stably and continuously cultured and passage is carried out for 30 generations, so that serum-free full-suspension culture type duck eye cornea epithelial cells, namely duck eye cornea epithelial cell suspension cell strains, are obtained.
And freezing and storing cells and establishing a cell bank every 5 passages in the construction process of the duck eye cornea epithelial cell suspension cell strain.
The serum-free total suspension culture type duck eye cornea epithelial cells are named as DCE-S of the duck eye cornea epithelial cells, and are preserved in China center for type culture collection (CCTCC NO) with the preservation number of CCTCC NO: C2024189. FIG. 2 shows the results of growth state of the DCE-S generation 25 of the sheldrake eye corneal epithelial suspension cells. FIG. 3 shows the stable passaging of different generations of DCE-S (F9-F30) cells, wherein the cell density of DCE-SD cells is stabilized above 6.0X106 cells/mL when the generation number of DCE-S cells is F1, F9-F30.
Example 2 test of DCE-S inoculation of sheldrake eye corneal epithelial suspension cells with H9 subtype avian influenza Virus
Taking DCE-S generation 25 cells of the sheldrake eye cornea epithelial suspension cells, placing the cells at 37 ℃, carrying out shake culture for 72 hours at 5% CO 2 and 120r/min, adding fresh culture medium to dilute the cells to 4X 10 6 cells/mL when the cell density reaches more than 6X 10 6 cells/mL, inoculating H9 subtype avian influenza virus (virus titer HA=9 log 2,EID50＝10-8.0/0.1 mL) at the same time according to 1 millv/v, adding TPCK pancreatin with a final concentration of 5ug/mL, adjusting the temperature to 35 ℃, culturing for 72 hours to harvest virus liquid, and detecting HA. The result shows that DCE-S of the sheldrake eye cornea epithelial suspension cells is sensitive to H9 subtype avian influenza virus, and HA is more than or equal to 9log 2. FIG. 4 is a cytopathic chart of the DCE-S inoculation of the sheldrake eye corneal epithelial suspension cells with H9 subtype avian influenza virus for 72H, massive cell death and cell lysis, more cell debris and obvious reduction of living cells.
Such strains are disclosed in Qi,Xuefeng et al."Down-regulation of cellular protein heme oxygenase-1inhibits proliferation of avian influenza virus H9N2 in chicken oviduct epithelial cells."The Journal of general virology vol.99,1(2018):36-43.doi:10.1099/jgv.0.000986.
Example 3 Duck eye corneal epithelial suspension cell DCE-S inoculated Duck circovirus (DuCV) test
Taking DCE-S generation 25 cells of the sheldrake eye cornea epithelial suspension cells, placing the cells at 37 ℃, carrying out shake culture for 72 hours at 5% CO 2 and 120r/min, adding fresh culture medium to dilute the cells to 4X 10 6 cells/mL when the cell density reaches more than 6X 10 6 cells/mL, inoculating duck circovirus (virus titer TCID 50＝10-6.0/0.1 mL) at 5%v/v, adjusting the temperature to 37 ℃, culturing for 60 hours, harvesting virus liquid, and detecting TCID 50. The results show that DCE-S of the sheldrake eye corneal epithelium suspension cells is sensitive to duck circovirus, and TCID 50≥10-7.0/0.1 mL. FIG. 5 is a cytopathic chart of DCE-S of the sheldrake eye corneal epithelium suspension cells inoculated with duck circovirus for 60h, with a large number of cells dying and lysing, more cell debris and significantly reduced living cells.
The strain is disclosed in New Yong and the like, duck hepatitis A virus type 3 and duck circovirus double fluorescence quantitative PCR detection methods [ J ]. Chinese fowl literature.
Example 4 Duck eye corneal epithelial suspension cell DCE-S inoculated Duck tembusu Virus (DTMUV) test
Taking DCE-S generation 25 cells of the sheldrake eye cornea epithelial suspension cells, placing the cells at 37 ℃, carrying out shake culture for 72 hours at 5% CO 2 and 120r/min, adding fresh culture medium to dilute the cells to 4X 10 6 cells/mL when the cell density reaches more than 6X 10 6 cells/mL, inoculating duck tembusu virus (virus titer TCID 50＝10-7.0/0.1 mL) at 0.5%v/v, adjusting the temperature to 37 ℃, and harvesting virus liquid after culturing for 50 hours, and detecting TCID 50. The results show that the duck eye cornea epithelial cell suspension cell strain is sensitive to duck tembusu virus, and TCID 50≥10-7.5/0.1 mL. FIG. 6 is a cytopathic chart of duck eye cornea epithelial suspension cells DCE-S inoculated with duck Tembusu virus for 50h, cells dying and lysing in large quantity, cell fragments are more, and living cells are obviously reduced.
The establishment and preliminary application of one-step RT-PCR detection method of duck tembusu virus are disclosed in 2015 (volume 40), 5 th stage and 37-40 literature.
Claims (10)
1. A duck eye cornea epithelial cell line, named as a sheldrake eye cornea epithelial suspension cell DCE-S, is preserved in China center for type culture collection (CCTCC NO) of university of Wuhan, 2024, 6 months and 28 days: C2024189.
2. The method for constructing a duck eye cornea epithelial cell line according to claim 1, comprising the following steps:
1) Separating eyeballs of duck embryos, separating corneas of the eyeballs, and shearing into tissue blocks;
2) Adding a first culture medium, uniformly mixing, culturing, and carrying out passage for 5-15 generations;
3) Culturing by using a second culture medium, and carrying out passage for 3-5 generations;
4) Culturing by using a third culture medium, and carrying out passage for 3-5 generations;
5) Culturing with a fourth culture medium, carrying out passage for 3-5 generations,
6) Culturing by using a serum-free suspension culture medium, and carrying out passage for 20-30 generations to obtain the strain.
3. The construction method according to claim 2, wherein:
the first culture medium comprises 8-10 v/v% of fetal bovine serum and a basic culture medium; and/or
The second culture medium comprises 6-8 v/v% of fetal bovine serum and a basic culture medium; and/or
The third culture medium comprises 4-6 v/v% of fetal bovine serum and a basic culture medium; and/or
The fourth culture medium comprises 2-3 v/v% of fetal bovine serum and a basal culture medium.
4. A method of construction according to claim 3, wherein:
The basal medium comprises at least one of MEM medium, DMEM medium and DMEM/F12 medium.
5. Use of the duck eye cornea epithelial cell line of claim 1 in any one of 1) to 8):
1) Culturing the virus;
2) Preparing a product for culturing viruses;
3) Isolating the virus;
4) Preparing a virus-isolated product;
5) Detecting the virus at a non-diagnostic treatment destination;
6) Preparing a product for detecting viruses;
7) Preparing a virus vaccine;
8) Screening medicines;
The medicine is used for preventing or treating diseases caused by virus infection.
6. The use according to claim 5, characterized in that:
The virus includes at least one of avian influenza virus, duck circovirus, and duck tembusu virus.
7. A method of culturing a virus by inoculating the virus into the duck eye cornea epithelial cell line of claim 1 and culturing.
8. A method for preparing a viral vaccine, comprising inoculating a virus into the duck eye cornea epithelial cell line of claim 1, culturing, and inactivating to obtain the viral vaccine.
9. The method according to claim 7 or 8, characterized in that:
The virus includes at least one of avian influenza virus, duck circovirus, and duck tembusu virus.
10. A viral vaccine prepared by the method of claim 8 or 9.
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