A duck myocardial cell line and its construction method and application

Abstract

The present invention belongs to the field of biological medicine, and particularly relates to a duck cardiomyocyte line and its construction method and application. The present invention provides a duck cardiomyocyte line named Sheldrake Duck Myocardial Cells SDMC, which was deposited on July 5, 2024 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the deposit number of CCTCC NO: C2024190. It has been passaged to 80 generations at present, and the morphology is mainly spindle-shaped cells; the virus sensitivity test shows that this duck cardiomyocyte line is sensitive to infectious bursal disease virus, avian adenovirus and Duck Hepatitis A Virus Type I; 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 invention belongs to the field of biomedicine, and specifically relates to a duck myocardial cell system and a construction method and application thereof. Background Art

[0002] my country has a large population and a large demand for poultry products, which has promoted the rapid development of the domestic poultry farming industry, with the scale of farming increasing and the number of varieties increasing. Poultry production has played an important role in promoting my country's economic development and improving the living standards of urban and rural residents. With the development of global economic integration and the demand for international trade, my country, as a major poultry country, has broad development space. However, in recent years, the complexity of poultry diseases has become a major factor that troubles and restricts the development of the poultry industry. According to relevant statistics, there are more than 80 diseases that pose a threat and harm to my country's poultry industry, involving infectious diseases, parasitic diseases, nutritional metabolic diseases and toxic diseases. Among them, infectious diseases are the most common, accounting for more than 75% of the total number of diseases, and the losses caused are also the greatest. Among infectious diseases, viral infectious diseases occur the most, accounting for about 70%, and there is a trend of increasing year by year. The occurrence of poultry diseases is still mainly viral infectious diseases. For example, the dominant diseases in chickens are Newcastle disease, avian influenza, Marek's disease, infectious bursal disease, infectious bronchitis and infectious laryngotracheitis, etc. The main diseases in ducks are duck plague, duck parvovirus disease, duck reovirus disease, duck viral hepatitis, etc. The main diseases in geese are gout, gosling plague, goose paramyxovirus disease, etc.

[0003] Vaccine immunization is undoubtedly the most effective way to prevent poultry diseases caused by infectious viruses. Cell culture is increasingly favored by vaccine producers in vaccine production. Compared with the traditional chicken embryo culture method, cell culture has many advantages in separating and culturing viruses: first, it can adapt to the large-scale culture required for production, with high output and relatively lower cost; second, the quality is controllable and the difference between batches is small; third, it can effectively exclude latent infection of viruses and is conducive to virus growth; fourth, it is easy to obtain and it is easy to select susceptible cells.

[0004] However, there are currently few immortalized cell lines suitable for the growth of avian viruses at home and abroad, which makes it difficult to isolate avian virus antigens. There are many types of viruses and relatively few commercial vaccines. In addition, there is a lack of application of avian cells from different target organs in the development of related vaccines, and there is currently no strain of duck cardiomyocytes for vaccine development. Summary of the invention

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

[0006] The purpose 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 third aspect of the present invention aims to provide an application.

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

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

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

[0011] To achieve the above objectives of the present invention, the technical solutions adopted by the present invention are as follows:

[0012] In the first aspect of the present invention, a duck cardiomyocyte line named Sheldrake Duck Myocardial Cells SDMC is provided. It was deposited on June 28, 2024, at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, with the deposit number CCTCC NO: C2024190.

[0013] In the second aspect of the present invention, a method for constructing the duck cardiomyocyte line of the first aspect of the present invention is provided, including the following steps:

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

[0015] 2) Digest the tissue pieces with protease, separate the solid and liquid, and culture the cell precipitate in a first culture medium;

[0016] 3) After the cells adhere to the wall, change the culture medium to a second culture medium, culture, and passage 5 - 15 generations;

[0017] 4) Culture with a third culture medium and passage 5 - 15 generations;

[0018] 5) Culture with a fourth culture medium and passage 5 - 15 generations to obtain.

[0019] Preferably, the first culture medium includes 9 - 11 v / v% fetal bovine serum and a basal medium.

[0020] Preferably, the second culture medium includes 7 - 9 v / v% fetal bovine serum, 0.05 - 0.15 mmol / L BrdU, and a basal medium.

[0021] Preferably, the third culture medium includes 4 - 6 v / v% fetal bovine serum and a basal medium.

[0022] Preferably, the fourth culture medium includes 2 - 3 v / v% fetal bovine serum and a basal medium.

[0023] Preferably, the basal medium includes at least one of MEM medium, DMEM medium, and DMEM / F12 medium.

[0024] In one embodiment of the present invention, the construction method includes: 1) Take 3 duck embryos at 7-9 days old, isolate the heart under sterile conditions, cut the heart tissue into pieces, perform the first digestion with trypsin, discard the digestive fluid, and then perform the second digestion with trypsin to obtain tissue fragments and cell suspension; 2) Add the suspension to a centrifuge tube containing the first culture medium, mix well, filter through a stainless steel sieve to remove tissue fragments, obtain a cell suspension, add the first culture medium to re-cell, and use the differential attachment method. After static culture for 1 h, aspirate the cell suspension (without shaking) and continue static culture; 3) After the cells adhere and grow, replace them with the second culture medium for culture. After the cells grow into a monolayer, digest them with trypsin, and then inoculate them into a culture plate at a density of 0.1-1 cell / well. Select the cells with good growth and obvious pulsation in the single-cell wells to grow into clone clusters to obtain a duck myocardial single-cell strain; 4) Digest the duck myocardial single-cell strain with trypsin, subculture, and after the cells grow into a monolayer, digest and centrifuge to obtain a cell pellet, and culture it with the third culture medium to obtain duck myocardial cells with reduced serum acclimation; 4) Culture the duck myocardial cells with reduced serum acclimation with the fourth culture medium to obtain a low-serum adherent culture type duck myocardial cell line.

[0025] Preferably, the size of the tissue fragments in step 1) is 1-2 cm 3 / piece.

[0026] Preferably, the culture conditions are 31-39 °C, 4-6% CO2; further preferably, 33-37 °C, 5% CO2.

[0027] The third aspect of the present invention lies in providing the application of the duck myocardial cell line of the first aspect of the present invention in any one of 1)-8):

[0028] 1) Culturing viruses;

[0029] 2) Preparing products for culturing viruses;

[0030] 3) Isolating viruses;

[0031] 4) Preparing products for isolating viruses;

[0032] 5) Detecting viruses for non-diagnostic and therapeutic purposes;

[0033] 6) Preparing products for detecting viruses;

[0034] 7) Preparing virus vaccines;

[0035] 8) Drug screening;

[0036] The drug is a drug for preventing or treating diseases caused by viral infections.

[0037] Preferably, the virus includes at least one of infectious bursal disease virus, avian adenovirus, and type I duck viral hepatitis virus.

[0038] Preferably, the bursal disease virus is IBDV-X1.

[0039] Preferably, the avian adenovirus is FAdV-4.

[0040] Preferably, the type I duck viral hepatitis virus is DHV-1C80.

[0041] In the fourth aspect of the present invention, a method for culturing a virus is provided. The virus is inoculated into the duck cardiomyocyte line of the first aspect of the present invention and cultured.

[0042] Preferably, the virus includes at least one of bursal disease virus, avian adenovirus and type I duck viral hepatitis virus.

[0043] Preferably, the bursal disease virus is IBDV-X1.

[0044] Preferably, the avian adenovirus is FAdV-4.

[0045] Preferably, the type I duck viral hepatitis virus is DHV-1C80.

[0046] Preferably, the culture conditions are 31-39°C and 4-6% CO2; more preferably 33-37°C and 5% CO2.

[0047] In the fifth aspect of the present invention, a method for preparing a virus vaccine is provided. The virus is inoculated into the duck cardiomyocyte line of the first aspect of the present invention, cultured and inactivated to obtain the virus vaccine.

[0048] Preferably, the virus includes at least one of bursal disease virus, avian adenovirus and type I duck viral hepatitis virus.

[0049] Preferably, the bursal disease virus is IBDV-X1.

[0050] Preferably, the avian adenovirus is FAdV-4.

[0051] Preferably, the type I duck viral hepatitis virus is DHV-1C80.

[0052] Preferably, the culture conditions are 31-39°C and 4-6% CO2; more preferably 33-37°C and 5% CO2.

[0053] In the sixth aspect of the present invention, a virus vaccine is provided, which is prepared by the method of the fifth aspect of the present invention.

[0054] The beneficial effects of the present invention are:

[0055] The present invention provides a Sheldrake duck myocardial cell line SDMC, which has been passaged to the 80th generation, and the cells are mainly spindle-shaped. The virus sensitivity test shows that this duck myocardial cell line is sensitive to infectious bursal disease virus, avian adenovirus, and duck hepatitis virus type I. 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 of the Drawings

[0056] Figure 1 It is a result diagram of the 30th generation of normal culture of Sheldrake duck myocardial cell line SDMC.

[0057] Figure 2 It is a result diagram of the Sheldrake duck myocardial cell line SDMC inoculated with IBDV-X1 strain at a virus inoculation amount of 2.5‰ for 47 hours in Example 3.

[0058] Figure 3 It is a result diagram of the Sheldrake duck myocardial cell line SDMC inoculated with FAdV-4 strain at a virus inoculation amount of 1‰ for 40 hours in Example 4.

[0059] Figure 4 It is a result diagram of the Sheldrake duck myocardial cell line SDMC inoculated with FAdV-4 strain at a virus inoculation amount of 2.5‰ for 36 hours in Example 4.

[0060] Figure 5 It is a result diagram of the Sheldrake duck myocardial cell line SDMC inoculated with DHV-1 C80 strain at a virus inoculation amount of 1‰ for 65 hours in Example 5.

[0061] Figure 6 It is a result diagram of the Sheldrake duck myocardial cell line SDMC inoculated with DHV-1 C80 strain at a virus inoculation amount of 5‰ for 44 hours in Example 5. Detailed Embodiments

[0062] The following will clearly and completely describe the concept and technical effects of 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. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0063] Example 1

[0064] 1. Isolation and culture of primary duck myocardial cells:

[0065] (1) Take 5 duck embryos hatched for 8 days and mark the positions of the air chamber and the embryo body;

[0066] (2) Place the embryonated eggs on an egg rack, first wash the eggshell with warm water, and then dry it with a 75% alcohol cotton ball;

[0067] (3) Under sterile conditions, use forceps to open the air chamber, carefully remove the eggshell with forceps to expose the embryo body, take out the embryo body with angled forceps, and place it in a sterile petri dish;

[0068] (4) Gently lift the skin of the chest and abdomen of the embryo body with forceps, and then use ophthalmic scissors to cut open the skin of the chest and abdomen of the embryo body to expose the heart;

[0069] (5) Use ophthalmic scissors to separate the heart, place it in a clean sterile petri dish, remove blood vessels and connective tissues, wash it 3 times with pre-cooled D-Hank's solution, and cut the heart tissue into small pieces of 1-2 mm 3 in size;

[0070] (6) Wash the tissue fragments obtained in step (5) 3 times with D-Hank's solution, add 3 mL of 2.5 g / L trypsin and let it act for 10 min, then discard the digestive solution;

[0071] (7) Add another 3 mL of 2.5 g / L trypsin to the tissue fragments obtained in step (6) and let it act for 20 min, disperse the cells with a pipette, and quickly add the cell suspension to a centrifuge tube containing 7 mL of growth medium (MEM medium containing 100 mL / L serum), and pipette and mix well repeatedly;

[0072] (8) Filter the suspension obtained in step (7) through a 200-mesh stainless steel filter screen to remove tissue debris, then centrifuge the filtered cell solution at 800 r / min for 10 min, discard the supernatant, add 10 mL of growth medium (MEM medium containing 100 mL / L serum), resuspend the cells, add them to a 25-mL cell culture flask, and statically culture them in an incubator at 37°C and 5% CO2;

[0073] (9) Static culture the cell solution in step (8) for 1 h, then aspirate the cell suspension (do not shake), add the cell suspension to another cell culture flask, and statically culture it in an incubator at 37°C and 5% CO2.

[0074] 2. Obtaining duck cardiomyocytes:

[0075] (1) After the cells in step 1 adhere and grow for 12 h, discard the original culture medium, wash it 3 times with growth medium, then add 10 mL of growth medium (MEM medium containing 0.1 mmol / L BrdU and 100 mL / L serum), statically culture it in an incubator at 37°C and 5% CO2, discard half of the culture supernatant every 24 h, and add an equal amount of culture medium, and observe the cell growth situation under an inverted microscope;

[0076] (2) When the cells grow into a monolayer, discard the culture medium, wash once with 2.5 g / L trypsin, then add trypsin to digest into single cells, and inoculate into a 96-well plate at a density of 0.5 cells / well on average. Mark the wells inoculated with single cells, and select the cells with good growth and obvious pulsation to grow into clone clusters, thus obtaining the duck myocardial cell line.

[0077] 3. Subculture and domestication of duck myocardial cells: Digest the single cell strain in clusters with 5 g / L trypsin solution for 30 s, and then subculture for 10 generations to obtain a duck myocardial cell line with uniform morphology.

[0078] 4. Subculture and domestication of duck myocardial cells with reduced serum: When the duck myocardial adherent cells in step 3 grow into a monolayer, digest and disperse the duck myocardial adherent cells with 0.25 wt% EDTA-trypsin, centrifuge at 1000 rpm for 5 min to obtain cell precipitate, and culture with MEM medium containing 5% fetal bovine serum. The culture conditions are 37 °C and 5% CO2; after continuous culture for 5 generations, duck myocardial cells with reduced serum domestication are obtained;

[0079] 5. Low serum domestication of duck myocardial cells: Directly change the medium and subculture the duck myocardial cells in the last generation of culture in step 4 with MEM medium containing 2.5% fetal bovine serum, and continue to culture for 10 generations. The culture conditions are 37 °C and 5% CO2, and a low serum adherent culture type duck myocardial cell line is obtained through domestication.

[0080] During the construction process of the above low serum adherent culture type duck myocardial cell line, cells are cryopreserved every 5 subcultures to establish a cell bank.

[0081] The above low serum adherent culture type duck myocardial cell line is named Sheldrake Duck Myocardial Cells SDMC. When subcultured to the 35th generation, 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: C2024190.

[0082] Example 2 Characteristics of Sheldrake Duck Myocardial Cells SDMC

[0083] Morphological observation:

[0084] Observe the cell morphology of the 30th generation of the above duck myocardial cell line through an inverted microscope, and the results are as Figure 1 shown: The cells are spindle-shaped, the cytoplasm is transparent, the morphology is full, and the cell nucleus is oval and located in the center. They can converge into a monolayer 3 - 4 days after subculture.

[0085] Example 3 Sensitivity of Infectious Bursal Disease Virus to Duck Myocardial Cell Line

[0086] Take the 30th generation of SDMC of Muscovy duck myocardial cells. After culturing for 48 h at 37 °C and 5% CO2 until growth is stable, discard most of the culture medium in the laminar flow hood, leaving only 1 mL. Add 10 μL, 25 μL, and 50 μL of IBDV-X1 (TCID 50 = 10 -8.5 / 0.1 mL) virus strain (which has been disclosed in the literature: Xiang Rui. Study on the Immunoprotective Effects of Recombinant Proteins against Newcastle Disease, Avian Influenza, and Infectious Bursal Disease Viruses in Chickens [D]. Northwest A&F University, 2021.) and incubate for 30 min. After discarding the liquid in the flask, add 10 mL of MEM medium containing 2.5% fetal bovine serum, using the 30th generation of duck myocardial cells in Example 2 as a control. 24 h after virus inoculation (the culture conditions after virus inoculation are 37 °C and 5% CO2), observe the cytopathic effect every 2 - 3 h, and collect the cell venom for TCID 50 detection. The results are as shown in Figure 1 , Figure 2 and Table 1: In the control group, the surface of SDMC of Muscovy duck myocardial cells was smooth and plump, with a fusiform shape, and could grow confluent in about 48 h under normal passage; after inoculating with infectious bursal disease virus, cytopathic phenomena occurred: the cell edges began to shrink, the cells still attached to the bottom of the flask were in a filamentous shape, and most cells detached and adhered to the surface; 51 h after inoculating duck myocardial cells with a virus inoculation amount of 1‰, TCID 50 reached 10 -7.32 / 0.1 mL, 47 h after inoculating with a virus inoculation amount of 2.5‰, TCID 50 reached 10 -7.83 / 0.1 mL, 35 h after inoculating with a virus inoculation amount of 5‰, TCID 50 reached 10 -7.0 / 0.1 mL; it can be seen that duck myocardial cells are sensitive to infectious bursal disease virus.

[0087] Table 1 Results of Duck Myocardial Cells Inoculated with IBDV-X1 Virus Strain

[0088] Inoculum volume of IBDV-X1 strain Virus harvest time <![CDATA[TCID 50 > 10 μL (1‰) 51h <![CDATA[10 -7.32 / 0.1mL]]> 25 μL (2.5‰) 47h <![CDATA[10 -7.83 / 0.1mL]]> 50 μL (5‰) 35h <![CDATA[10 -7.0 / 0.1mL]]>

[0089] Example 4 Sensitivity of Avian Adenovirus to Duck Myocardial Cell Line

[0090] Take the 30th generation of SDMC of Muscovy duck myocardial cells. After culturing for 48 h at 37 °C and 5% CO2 until growth is stable, discard most of the culture medium in the laminar flow hood, leaving only 1 mL. Add 10 μL, 25 μL, and 50 μL of FAdV-4 (TCID 50 = 10 -8.0 / 0.1 ml) strain (already disclosed in the literature: Wuning. Research on the expression changes of early miRNAs and mRNAs in LMH cells infected with avian adenovirus serotype 4 [D]. Northwest A&F University, 2020.) was incubated for 30 min. After discarding the liquid in the flask, 10 mL of MEM medium containing 2.5% fetal bovine serum was added, using the control group in Example 3 as a control. After 24 h of virus inoculation (the culture conditions after virus inoculation were 37 °C and 5% CO2), the cytopathic effect was observed every 2 - 3 h, and the cell venom was collected for TCID 50 detection. The results are as Figure 3 、 Figure 4 and Table 2 show that: in the control group, the surface of duck myocardial cells was smooth and plump, with a spindle shape, and could grow confluent in about 48 h under normal passage; after inoculation with avian adenovirus, cytopathic phenomena occurred: the cells shrank, most cells became round and detached, and adhered to the surface; after 48 h of inoculating duck myocardial cells with a virus inoculation amount of 1‰, the TCID 50 reached 10 -7.0 / 0.1 mL. After 42 h of inoculating with a virus inoculation amount of 2.5‰, the TCID 50 reached 10 -7.5 / 0.1 mL. After 24 h of inoculating with a virus inoculation amount of 5‰, the TCID 50 reached 10 -7.16 / 0.1 mL; it can be seen that duck myocardial cells are sensitive to avian adenovirus.

[0091] Table 2 Results of duck myocardial cells inoculated with FAdV-4 strain

[0092] Inoculum volume of FAdV-4 strain Virus harvest time <![CDATA[TCID 50 > 10 μL (1‰) 48h <![CDATA[10 -7.0 / 0.1mL]]> 25 μL (2.5‰) 42h <![CDATA[10 -7.5 / 0.1mL <!-- 5 -->]]> 50 μL (5‰) 24h <![CDATA[10 -7.16 / 0.1mL]]>

[0093] Example 5 Sensitivity of duck hepatitis A virus type I to duck myocardial cell line

[0094] Take the 30th passage cells of the duck myocardial cell line. After culturing at 37 °C and 5% CO2 for 48 h until growth was stable, most of the culture medium was discarded in the laminar flow hood, leaving only 1 mL. 25 μL and 50 μL of DHV-1 C80 strain (TCID 50 = 10 -3.6 / 0.1 ml) strain (already disclosed in the literature: Wang Wenxiu, Zhang Qian, Qu Guanggang, etc. Establishment of a SYBR-Green real-time fluorescence quantitative PCR detection method for duck hepatitis A virus type I [J]. Chinese Journal of Veterinary Science, 2014, 34(08): 1248 - 1252.) was incubated for 30 min. After discarding the liquid in the flask, 10 mL of MEM medium containing 2.5% fetal bovine serum was added, using the control group in Example 3 as a control. After 24 h of virus inoculation (the culture conditions after virus inoculation were 37 °C and 5% CO2), the cytopathic effect was observed every 2 - 3 h, and the cell venom was collected for TCID 50 detection. The results are asFigure 5 , Figure 6 and as shown in Table 3: The surface of duck myocardial cells in the control group was smooth and plump, spindle-shaped, and could grow confluent in about 48 h under normal passage; after inoculation with avian adenovirus, pathological changes occurred: cell shrinkage, most cells became round and detached, and the detached cells showed agglomeration; after inoculating duck myocardial cells with a virus inoculation amount of 1‰ for 65 h, the TCID 50 reached 10 -3.2 / 0.1 mL, after inoculating with a virus inoculation amount of 2.5‰ for 60 h, the TCID 50 reached 10 -3.5 / 0.1 mL, after inoculating with a virus inoculation amount of 5‰ for 48 h, the TCID 50 reached 10 -4.16 / 0.1 mL; it can be seen that duck myocardial cells are sensitive to avian adenovirus.

[0095] Table 3 Results of duck myocardial cells inoculated with DHV-1 C80 strain

[0096] Inoculum volume of FAdV-4 strain Virus harvest time <![CDATA[TCID 50 > 10 μL (1‰) 65h <![CDATA[10 -3.2 / 0.1mL]]> 25 μL (2.5‰) 60h <![CDATA[10 -3.5 / 0.1mL]]> 50 μL (5‰) 48h <![CDATA[10 -4.16 / 0.1mL]]>

Claims

1. A duck myocardial cell line named Sheldrake Duck Myocardial Cells SDMC was deposited on June 28, 2024, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the deposit number CCTCC NO: C2024190.

2. Use of the duck myocardial cell line according to claim 1 in any one of 1) to 8): 1) Culturing a virus; 2) Preparing a product for culturing a virus; 3) Isolating a virus; 4) Preparing a product for isolating a virus; 5) Detecting a virus for non-diagnostic and therapeutic purposes; 6) Preparing a product for detecting a virus; 7) Preparing a virus vaccine; 8) Drug screening; The drug is a drug for preventing or treating diseases caused by viral infections; The virus includes at least one of infectious bursal disease virus, fowl adenovirus, and duck hepatitis A virus type 1.

3. A method for culturing a virus, comprising inoculating the virus into the duck myocardial cell line according to claim 1 and culturing; The virus includes at least one of infectious bursal disease virus, fowl adenovirus, and duck hepatitis A virus type 1.

4. A method for preparing a virus vaccine, comprising inoculating the virus into the duck myocardial cell line according to claim 1, culturing, and inactivating to obtain a virus vaccine; The virus includes at least one of infectious bursal disease virus, fowl adenovirus, and duck hepatitis A virus type 1.

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