A Sheldrake embryo fibroblast cell line, its construction method and application

Abstract

The present invention belongs to the field of biomedicine, and particularly relates to a fibroblast cell line of mallard duck embryos, a construction method thereof, and an application thereof. Through a large amount of creative work by the inventors, the present invention for the first time isolated and obtained a fibroblast cell line from mallard duck embryos, and successfully domesticated it in serum-free suspension. The name of the cell line is DEF-Sfs, fibroblast suspension cells of mallard duck embryos, which was deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China on June 28, 2024, with the deposit number of CCTCC NO: C2024191. This cell line can be used for culturing, isolating, and detecting avian viruses, preparing avian vaccines, and screening drugs for preventing or treating diseases caused by avian virus infections.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a fibroblast cell line of mallard duck embryos, a construction method thereof, and an application thereof. Background Art

[0002] The poultry industry occupies an important position in the breeding production of our country and has become the main pillar industry of the rural economy in our country. The national poultry inventory fluctuated around 6 billion from 2014 to 2019; in 2020, the national poultry production capacity scale increased significantly, with the poultry inventory reaching 6.78 billion, an increase of 260 million compared with the end of the previous year, a year-on-year increase of 3.99%. With the continuous advancement of modern specialized, intensive, and large-scale poultry breeding technologies, our country has become a major global poultry breeding country. However, with the expansion of the scale, the problem of poultry diseases has become increasingly prominent. Some major poultry diseases are still one of the key factors affecting survival rate and breeding efficiency and hindering the healthy development of the domestic poultry industry. Therefore, the research and development of new vaccines for major poultry diseases is of great significance.

[0003] The selection of cell substrates is an important part of vaccine research and development. In production, cell substrates provide nutrients for viruses so that the viruses can multiply in large numbers, and then provide raw materials for vaccine preparation. Currently, the types of cell substrates commonly used in vaccine production are divided into primary cells, diploid cells, passaged cells, etc. The advantage of primary cells is that they are easy to obtain, but they are easily contaminated by exogenous substances. Moreover, because primary cells cannot be continuously passaged, cells need to be prepared multiple times, and the differences between batches are large, and it is difficult to control the cell homogeneity. Compared with primary cells, diploid cells have the ability to be continuously passaged. And passaged cells are derived from animals, such as BHK cells and MDCK cells, which have the characteristics of infinite passaging, easy culture, and easy amplification, and the production cost is relatively low. However, since some passaged cells are derived from tumor cells, there are certain potential biological safety risks; on the other hand, most of the currently used passaged cells are derived from mammals, and the problem of cell substrate heterology will affect the immunogenicity of avian vaccines.

[0004] At the present stage, the research on avian cell lines only stays in the preliminary stage, that is, after taking out animal tissues, the tissue blocks are repeatedly digested with liquids such as trypsin and erythrocyte lysate to separate and prepare primary cells, and avian cell lines are obtained by continuous passaging and other methods. This method is complex and cumbersome, time-consuming, causes greater damage to cells, and the isolated primary cells cannot be separated from the dependence on serum. At the same time, most primary cells cannot be continuously passaged to more than 15 generations. In addition, the adherent culture mode limits the expansion of subsequent industrial production, and there are many shortcomings. Summary of the Invention

[0005] The purpose of the first aspect of the present invention is to provide a cell line.

[0006] 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.

[0007] The object of the third aspect of the present invention is to provide the application of the fibroblast suspension cell line of mallard duck embryos in the research and development of veterinary vaccines, including but not limited to avian influenza H9 virus, Newcastle disease virus, and avian adenovirus type 4.

[0008] The object of the fourth aspect of the present invention is to provide a method for culturing viruses.

[0009] The object of the fifth aspect of the present invention is to provide a method for a virus vaccine.

[0010] The object of the sixth aspect of the present invention is to provide a virus vaccine.

[0011] In order to achieve the above objects of the present invention, the technical solutions adopted by the present invention are as follows:

[0012] The first aspect of the present invention provides a fibroblast cell line of mallard duck embryos, named fibroblast suspension cells of mallard duck embryos (DEF-Sfs), which was deposited on June 28, 2024, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the deposit number of CCTCC NO: C2024191.

[0013] The second aspect of the present invention provides a method for constructing the fibroblast cell line of mallard duck embryos of the first aspect of the present invention, including the following steps:

[0014] 1) Separate the trunk part of the duck embryo and cut it into tissue pieces;

[0015] 2) Add the first medium, mix well, culture, and passage for 10 - 20 generations;

[0016] 3) Culture with serum-free medium and passage for 5 - 15 generations to obtain the fibroblast cell line of mallard duck embryos.

[0017] Preferably, the first medium includes 4 - 8 v / v% fetal bovine serum and a basal medium.

[0018] Preferably, the basal medium includes at least one of MEM medium, DMEM medium, and DMEM / F12 medium.

[0019] In some embodiments of the present invention, the construction method includes:

[0020] 1) Isolation of DEF adherent cells: Take out 10-day-old duck embryos in a laminar flow hood, remove parts such as the head, cut the trunk into pieces with surgical scissors, then add a small amount of MEM with 5 v / v% fetal bovine serum, blow to break up the tissue blocks, use a 300-mesh cell sieve to remove large clumps, transfer to a culture flask for culture, and the culture conditions are 37 °C, 5% CO 2 ;

[0021] 2) Subculture of DEF adherent cells: After the primary DEF cells are separated and statically cultured for 6 h, based on the principle that different cells have different adherent speeds, discard the non-adherent cells and the supernatant containing some clumps, only leave the adherent cells, supplement with fresh MEM containing 5% fetal bovine serum, and continue culturing. After the cells grow into a monolayer, use trypsin to digest and passage the DEF cells, and continuously passage them more than 15 generations to obtain adherent cells;

[0022] 3) Serum-free suspension domestication of DEF: After trypsin digestion of the adherent cells cultured with MEM containing 5 v / v% fetal bovine serum, collect the cell suspension, centrifuge at 800 rpm for 5 min, discard the supernatant, leave the cell pellet, resuspend with serum-free medium (BSL-01 medium, Guangzhou Baisai Biomedical Technology Co., Ltd.), adjust the cell density to 1.0×10 6 cells / mL, and the culture conditions are 37 °C, 5% CO 2 , and culture on a shaker at 130 r / min. After 48 h of cell culture, precipitate the cells at 800 rpm for 5 min, resuspend with fresh serum-free BSL-01 medium, and continuously passage by adjusting the cell density. If large cell clumps appear during the serum-free domestication of the cells, they can be left standing in the laminar flow hood for 5 min, take the supernatant for cell centrifugation and passage, and continuously culture and passage more than 25 generations to obtain the serum-free fully suspended culture type of Muscovy duck embryo fibroblast cell line DEF-Sfs.

[0023] Preferably, the size of the tissue fragments described in step 1) is 1 - 2 cm 3 / piece.

[0024] Preferably, the culture conditions are 31 - 39 °C, 4 - 6% CO 2 ; further preferably 33 - 37 °C, 5% CO 2 .

[0025] The third aspect of the present invention lies in providing the application of the Muscovy duck embryo fibroblast cell line of the first aspect of the present invention in any one of 1) - 8):

[0026] 1) Culturing viruses;

[0027] 2) Preparing products for culturing viruses;

[0028] 3) Isolating viruses;

[0029] 4) Preparation of products for virus isolation;

[0030] 5) Detection of virus for non-diagnostic and non-therapeutic purposes;

[0031] 6) Preparation of products for virus detection;

[0032] 7) Preparation of virus vaccines;

[0033] 8) Drug screening;

[0034] The drug is a drug for preventing or treating diseases caused by virus infection.

[0035] Preferably, the virus includes at least one of avian influenza virus, Newcastle disease virus, and avian adenovirus.

[0036] Preferably, the avian influenza virus includes H9 subtype avian influenza virus.

[0037] Preferably, the avian adenovirus is avian adenovirus type 4.

[0038] In some embodiments of the present invention, there is provided the use of the fibroblast cell line from Sheldrake duck embryo of the first aspect of the present invention in the preparation of products for culturing avian viruses;

[0039] The use of the fibroblast cell line from Sheldrake duck embryo of the first aspect of the present invention in the isolation of avian viruses;

[0040] The use of the fibroblast cell line from Sheldrake duck embryo of the first aspect of the present invention in the preparation of products for isolating avian viruses;

[0041] The use of the fibroblast cell line from Sheldrake duck embryo of the first aspect of the present invention in the detection of avian viruses for non-diagnostic and non-therapeutic purposes;

[0042] The use of the fibroblast cell line from Sheldrake duck embryo of the first aspect of the present invention in the preparation of products for detecting avian viruses;

[0043] The use of the fibroblast cell line from Sheldrake duck embryo of the first aspect of the present invention in the preparation of avian vaccines.

[0044] In the fourth aspect of the present invention, there is provided a method for culturing a virus, which comprises inoculating the virus into the fibroblast cell line from Sheldrake duck embryo of the first aspect of the present invention for culturing.

[0045] Preferably, the virus includes at least one of avian influenza virus, Newcastle disease virus, and avian adenovirus.

[0046] Preferably, the avian influenza virus includes H9 subtype avian influenza virus.

[0047] Preferably, the avian adenovirus is avian adenovirus type 4.

[0048] Preferably, the culture conditions are 31 - 39°C and 4 - 6% CO 2 ; more preferably 33 - 37°C and 5% CO 2 .

[0049] In the fifth aspect of the present invention, a method for preparing a viral vaccine is provided. A virus is inoculated into the fibroblast cell line of Shelduck embryos of the first aspect of the present invention, cultured, and inactivated to obtain a viral vaccine.

[0050] Preferably, the virus includes at least one of avian influenza virus, Newcastle disease virus, and avian adenovirus.

[0051] Preferably, the avian influenza virus includes H9 subtype avian influenza virus.

[0052] Preferably, the avian adenovirus is avian adenovirus type 4.

[0053] Preferably, the culture conditions are 31 - 39°C and 4 - 6% CO 2 ; more preferably 33 - 37°C and 5% CO 2 .

[0054] In the sixth aspect of the present invention, a viral vaccine is provided, which is prepared by the method of the fifth aspect of the present invention.

[0055] The beneficial effects of the present invention are as follows:

[0056] Through a large amount of creative work by the inventors, the present invention for the first time isolated and obtained a fibroblast cell line from Shelduck embryos, and successfully domesticated it in serum-free suspension, named Shelduck embryo fibroblast suspension cells DEF-Sfs. It was deposited on June 28, 2024, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the deposit number CCTCC NO: C2024191. This cell line can be used for culturing, isolating, and detecting avian viruses, preparing avian vaccines, and screening drugs for preventing or treating diseases caused by avian virus infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 It shows the growth status results of the 20th generation of Shelduck embryo fibroblast suspension cells DEF-Sfs of the present invention.

[0058] Figure 2 It shows the growth curve results of the Shelduck embryo fibroblast suspension cells DEF-Sfs of the present invention.

[0059] Figure 3 It shows the cytopathic effect results of the Shelduck embryo fibroblast suspension cells DEF-Sfs inoculated with avian influenza H9 virus for 72 hours.

[0060] Figure 4 Cytopathic effect results of Muscovy duck embryo fibroblast suspension cells DEF-Sfs inoculated with Newcastle disease virus for 58 h.

[0061] Figure 5 Cytopathic effect results of Muscovy duck embryo fibroblast suspension cells DEF-Sfs inoculated with avian adenovirus type 4 for 72 h. Specific implementation manners

[0062] The following will clearly and completely describe the concept and technical effects generated by the present invention in combination with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0063] Example 1 Establishment of Muscovy duck embryo fibroblast suspension cells DEF-Sfs

[0064] 1. Rapid isolation and culture of primary Muscovy duck embryo fibroblasts

[0065] (1) Place the fertilized Muscovy duck embryos in an incubator and incubate them until they reach 10 days old. Screen them under a strong light, select relatively healthy embryo eggs, and mark the outline of the air chamber at the same time;

[0066] (2) After disinfecting the embryo eggs, break the eggshell along the outline of the air chamber on a sterile operating table, tear open the shell membrane, expose the embryo body, gently pick up the embryo head with a curved forceps, take out the embryo body, and place it in a sterile petri dish;

[0067] (3) Remove the head, limbs and other parts of the embryo body, tear off the obvious blood vessels, cut the trunk part into pieces with surgical scissors, then add a small amount of MEM with 5 v / v% fetal bovine serum, blow and break the tissue blocks, use a 300-mesh cell sieve to remove larger lumps, and transfer them to a culture flask for culture. The culture conditions are 37 °C, 5% CO 2 ;

[0068] (4) 6 h after cell isolation and culture, gently shake the cell flask, discard the supernatant containing non-adherent cells and debris such as red blood cells, and only leave the adherent cells. Add fresh MEM with 5 v / v% fetal bovine serum and continue to culture. After the cells grow into a monolayer, digest and passage the Muscovy duck embryo fibroblasts (DEF) with trypsin, and continuously passage them to the 15th generation to obtain DEF adherent cells.

[0069] 2. Suspension domestication of Muscovy duck embryo fibroblasts

[0070] After trypsinizing the adherent DEF cells cultured in MEM supplemented with 5 v / v% fetal bovine serum, the cell suspension was collected, centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the cell pellet was left. The cells were resuspended in serum-free medium (BSL-01 medium, Biosai Biotechnology Co., Ltd., Guangzhou), and the cell density was adjusted to 1.0×10 6 cells / mL. The culture conditions were 37°C, 5% CO 2 , and cultured on a shaker at 130 r / min. If large cell aggregates appeared during the serum-free suspension acclimation of the cells, the cells could be left standing in the laminar flow hood for 5 min, and the supernatant was taken for cell centrifugation and subculture. The cells were continuously cultured and subcultured up to 25 passages to obtain fibroblast suspension cells of mallard duck embryos.

[0071] During the construction process of the above fibroblast suspension cells of mallard duck embryos, the cells were cryopreserved every 5 passages to establish a cell bank.

[0072] When the above fibroblast suspension cells of mallard duck embryos were subcultured to the 20th passage, they were named fibroblast suspension cells of mallard duck embryos DEF-Sfs, and were deposited on June 28, 2024 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the deposit number CCTCC NO: C2024191.

[0073] Figure 1 They are the fibroblast suspension cells of mallard duck embryos DEF-Sfs in Example 1. Figure 2 It is the growth curve of the fibroblast suspension cells of mallard duck embryos DEF-Sfs in Example 1.

[0074] Example 2 Testing the inoculation of avian influenza H9 virus on fibroblast suspension cells of mallard duck embryos DEF-Sfs

[0075] Take the 20th passage cells of fibroblast suspension cells of mallard duck embryos DEF-Sfs, place them at 37°C, 5% CO 2 , and culture at 130 r / min for 48 h. Add fresh medium to dilute the cells to 4×10 6 cells / mL. At the same time, inoculate avian influenza H9 virus at 5 v / v‰ (virus titer HA = 9log2, EID50 = 10 -8.0 / 0.1 mL), and add TPCK trypsin with a final concentration of 3 μg / mL. Adjust the temperature to 35°C, and harvest the virus solution after culturing for 72 h to detect HA. The results showed that the fibroblast suspension cell line of mallard duck embryos was sensitive to avian influenza H9 virus, and HA≥8Log 2 . Figure 3 It is the cytopathic effect diagram of the fibroblast suspension cells of mallard duck embryos DEF-Sfs inoculated with avian influenza H9 virus for 72 h in Example 2. The cells began to die in large numbers, and the cell debris increased.

[0076] The above-mentioned strain has been disclosed in the literature of Qi, Xuefeng et al. “Down-regulation of cellular protein heme oxygenase-1 inhibits 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.

[0077] Example 3: Test of inoculating duck embryo fibroblast suspension cells DEF-Sfs with Newcastle disease virus

[0078] Take the 20th passage cells of duck embryo fibroblast suspension cells DEF-Sfs, place them at 37 °C, 5% CO 2 , and culture at 130 r / min for 48 h. Add fresh medium to dilute the cells to 4×10 6 cells / mL. At the same time, inoculate Newcastle disease virus at 3 v / v‰ (virus titer HA = 9log2, EID50 = 10 -8.0 / 0.1 mL), and add TPCK trypsin with a final concentration of 3 μg / mL. Adjust the temperature to 35 °C. Harvest the virus solution after culturing for 58 h and detect HA. The results show that the duck embryo fibroblast suspension cell line is sensitive to Newcastle disease virus, and HA ≥ 9Log 2 . Figure 4 Figure showing the cytopathic effect of duck embryo fibroblast suspension cells DEF-Sfs inoculated with Newcastle disease virus for 58 h in Example 3. A large number of cells died, there were fewer surviving cells, it was not transparent, and there were more cell debris.

[0079] The above-mentioned strain has been disclosed in the literature of Wang, Jing-Yu et al. “Characterization of emerging Newcastle disease virus isolates in China.” Virology journal vol. 12 119. 7 Aug. 2015, doi:10.1186 / s12985-015-0351-z.

[0080] Example 4: Test of inoculating duck embryo fibroblast suspension cells DEF-Sfs with avian adenovirus type 4

[0081] Take the 20th passage cells of duck embryo fibroblast suspension cells DEF-Sfs, place them at 37 °C, 5% CO2 , cultured at 130 r / min for 72 h, and directly inoculated with fowl adenovirus type 4 at 1 v / v‰ (virus titer TCID50 = 10 -7.5 / 0.1 mL), the virus solution was harvested after 50 h of culture, and TCID 50 was detected. The results showed that the suspended cell line of mallard duck embryo fibroblasts was sensitive to fowl adenovirus type 4, and TCID 50 ≥10 -8.17 / 0.1 mL. Figure 5 It is the cytopathic effect map of the suspended cell DEF-Sfs of mallard duck embryo fibroblasts inoculated with fowl adenovirus type 4 for 72 h in Example 4.

[0082] The above-mentioned strain has been published in the literature Wen, Bo et al. “Transcriptome Analysis Reveals thePotential Role of Long Noncoding RNAs in Regulating Fowl Adenovirus Serotype4-Induced Apoptosis in Leghorn Male Hepatocellular Cells.” Viruses vol. 13,81623. 17 Aug. 2021, doi:10.3390 / v13081623.

Claims

1. A strain of duck embryonic fibroblast suspension cells DEF-Sfs was deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China on June 28, 2024, with the deposit number CCTCC NO: C2024191.

2. Use of the duck embryonic fibroblast suspension cells DEF-Sfs according to claim 1 in any one of 1) to 8): 1) Cultivate the virus; 2) Preparation of products for cultured viruses; 3) Isolation of viruses; 4) Preparation of products for virus isolation; 5) Virus testing for non-diagnostic treatment destinations; 6) Prepare products for detecting viruses; 7) Preparation of viral vaccines; 8) Drug screening; The medicine is a medicine for preventing or treating a disease caused by a viral infection; The virus comprises at least one of avian influenza virus, Newcastle disease virus and avian adenovirus.

3. A method for culturing a virus, comprising inoculating the virus into the DEF-Sfs suspension cells of duck embryonic fibroblasts according to claim 1, and culturing; The virus comprises at least one of avian influenza virus, Newcastle disease virus and avian adenovirus.

4. A method for preparing a virus vaccine, comprising inoculating the virus into the DEF-Sfs of the duck embryonic fibroblast suspension cells of claim 1, culturing, inactivating, and obtaining the virus vaccine; The virus comprises at least one of avian influenza virus, Newcastle disease virus and avian adenovirus.

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