Method for releasing Newcastle disease virus by cracking cells

Abstract

The invention discloses a method for releasing Newcastle disease virus by cracking cells, which comprises the following steps: (1) freezing and unfreezing NDV cell virus liquid, repeating for multiple times, and then carrying out ultrasonication treatment to obtain cell suspension; (2) centrifuging the cell suspension to remove cell debris and cell nucleuses, and collecting supernate; by adopting the method disclosed by the invention, the cell disruption rate is high, and the released Newcastle disease virus is high in hemagglutination titer.

Description

Technical Field

[0001] The invention belongs to the technical field of virus preparation, and in particular relates to a method for lysing cells to release Newcastle disease virus. Background Art

[0002] At present, the production of Newcastle disease vaccine in my country is mainly through the inoculation of 9-10 day old chicken embryos to cultivate and prepare vaccines. Direct use of chicken embryos for virus amplification has the following potential difficulties: (1) It requires a lot of manpower and material resources, which is a labor-intensive process; (2) This process is very dependent on the sufficient supply of SPF chicken embryos. Once there is a disease in the chicken flock or the SPF status of the chicken flock is interrupted, there may be a shortage of SPF chicken embryos; (3) Chicken embryo allantoic fluid contains complex proteins and yolk, which brings certain difficulties to the downstream purification process; (4) Chickens are the natural hosts of many bacteria and are easily infected. Studies have shown that the potential contamination inherited from the mother to the chicken embryo is much greater than the contamination caused by human operation. Therefore, it is urgent to select a new Newcastle disease virus vaccine production medium for the production of Newcastle disease virus.

[0003] In recent years, multiple cells have been used for the proliferation of Newcastle disease virus (NDV), such as MDBK cells, bovine kidney embryonic cell lines (BS / BEK), DF-1 cells (chicken embryo fibroblasts), suspended Vero cells, etc. Since NDV belongs to intracellular viruses and has a strong cell binding activity, it is necessary to collect and effectively break cells during virus harvesting to obtain a large amount of free viral particles. At present, in the large-scale production process for viruses, freeze-thaw methods are mostly used to lyse cells and harvest intracellular viruses. However, freeze-thaw methods have the disadvantages of being difficult to amplify and unstable in yield, which limits the production scale of viral biological products. There are also methods for harvesting intracellular viruses by hypotonic or chemical lysis, but a large number of viruses combined on the cell membrane are not fully released, and many chemical lysis reagents can also destroy the envelope of enveloped viruses, making the viruses lose their biological activity. Summary of the invention

[0004] In view of this, an object of the present invention is to provide a method for lysing cells to release Newcastle disease virus. The method of the present invention can effectively lyse cells to release Newcastle disease virus.

[0005] The specific technical solution adopted by the present invention is: A method for lysing cells to release Newcastle disease virus comprises the following steps: (1) freezing and thawing the NDV cell virus solution repeatedly for several times, and then performing ultrasonic disruption to obtain a cell suspension; (2) Centrifuge the cell suspension to remove cell debris and cell nuclei, and collect the supernatant.

[0006] Furthermore, the freezing and thawing process in step (1) is to freeze the NDV cell virus liquid in liquid nitrogen for 4-6 minutes and then slowly thaw it at 23-27°C.

[0007] Furthermore, the freezing and thawing treatment in step (1) is repeated three times.

[0008] Furthermore, the ultrasonic disruption treatment is carried out in an ice bath, and the cell suspension is quickly placed on ice after the ultrasonic treatment.

[0009] Furthermore, the ultrasonic disruption treatment is performed at 180 W, the cell virus solution is ultrasonically treated for 1 minute, a single disruption lasts for 2 seconds, an interval of 2 seconds, and the disruption is performed 15 times.

[0010] Furthermore, the cell suspension was centrifuged at 4000 rpm for 10 minutes at 4°C to remove cell debris and cell nuclei.

[0011] Furthermore, the supernatant was collected and stored in a -80°C refrigerator.

[0012] Furthermore, the NDV cell virus liquid is obtained by growing and culturing BSR T7 / 5 cells and then inoculating Newcastle disease virus for culturing.

[0013] The beneficial effects of the present invention are: by adopting the method of the present invention, the cell disruption rate is high, and the released Newcastle disease virus has a high hemagglutination titer, that is, the method of the present invention can effectively lyse cells and release Newcastle disease virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Shown is the cell morphology under a microscope of BSR cells at a cell density of 80%; Figure 2 Shown is a typical syncytium formed in BSR cells infected with the LaSota strain at 72 hours, indicated by the red arrow; Figure 3 Display as 10 -1 -10 -6 Hemagglutination titer of cell supernatant of BSR cells infected with LaSota strain at different dilutions for 72 h (hemagglutination titer not treated by the four methods) (control group); Figure 4 Shown are the damage of BSR cells by four different treatment methods; Figure 5 Shown are the hemagglutination titers of the virus amplified after 72 h of collection using four different treatment methods; Figure 6 Shown is a summary of the hemagglutination titers of virus amplified 72 h after collection using four different treatment methods. DETAILED DESCRIPTION

[0016] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments and drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0017] Unless otherwise specified, the materials in the following examples are commonly used materials in the art and can be obtained from commercial sources.

[0018] Example 1 BSR T7 / 5 cells (a clone of BHK-21 cells adapted to the rabies virus CVS strain) were kindly donated by the State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute and placed in a 5% CO 2 , and cultured in a 37°C cell culture incubator.

[0019] The attenuated Newcastle disease virus strain LaSota was donated by the State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute and stored in an ultra-low temperature freezer at -80°C for future use.

[0020] (I) BSR cell culture Place the experimental consumables in the ultra-clean bench for ultraviolet irradiation disinfection for 30 minutes, take out the DMEM medium containing 10% fetal bovine serum, 100U / mL penicillin and 100μg / mL streptomycin, trypsin containing 0.25% EDTA, and 1×PBS from the 4℃ refrigerator and put them in room temperature for rewarming, wipe the water droplets on the outer surface with a paper towel, disinfect with 75% alcohol and put them in the ultra-clean bench. Observe under a microscope. Generally, BSR cells are passaged when they grow to 80-90% cell density. Open the culture bottle cap in the ultra-clean bench and discard the original culture medium. Taking the T75 culture bottle as an example, rinse the cells with 5mL of PBS and discard it, add 2mL of trypsin, and tilt the culture bottle to ensure that the trypsin covers the entire cell surface. Place it in the incubator and let it digest for 1 minute, then take out the culture bottle and observe it under a microscope. When the cells become round or partially detach from the bottle wall, immediately add 5mL of DMEM culture medium to terminate the trypsin digestion reaction. Repeatedly pipette and beat several times to disperse the cells into single cells. Pipette the cell suspension into a 50mL centrifuge tube and centrifuge at 1000rpm for 3min. Carefully use a Pasteur pipette to aspirate and discard the supernatant. Resuspend the cells in 10mL PBS, take 10μL of the cell suspension and mix with 10μL of table blue to count the cells. Centrifuge the cell suspension at 1000rpm for 3min. Carefully use a Pasteur pipette to aspirate and discard the supernatant. According to the cell counting results, 2×10 5Resuspend the cells in a 12-well cell culture plate with an appropriate volume of DMEM containing 10% fetal bovine serum, 100U / mL penicillin and 100μg / mL streptomycin, and gently shake up and down to mix the cell suspension evenly, and observe the cell state under a microscope. Place the 12-well plate in a cell culture incubator and continue to culture. When the cells grow to a single layer with a density of about 80%, the next step of the virus attack experiment can be carried out. Figure 1 This is the cell morphology of BSR cells under a microscope.

[0021] (II) Amplification of Newcastle Disease Virus by BSR Cells The LaSota strain of Newcastle disease virus was amplified with cells in the following manner: BSR cells were inoculated into a 12-well plate. When the cells grew to a monolayer with a density of about 80%, the 10% FBS DMEM medium was discarded, and the cells were gently washed once with 1× PBS buffer. 5 mL of the LaSota strain of Newcastle disease virus amplified with chicken embryos was taken and sterilized by filtration through a 0.45 μm microporous filter membrane. The virus was diluted with a 2% FBS DMEM medium, and a 10-fold series of gradient dilutions was made. 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 A total of 6 dilutions of 2% FBS DMEM medium were inoculated into 12-well plates, shaken to make them evenly distributed, and placed in a 5% CO 2 , and cultured in a 37°C cell culture incubator, and BSR cells without virus were set as negative control.

[0022] (III) Collecting virus fluid Cell morphological changes and viral cytopathic effects (CPE) were observed under an inverted microscope at 24 hours, 48 ​​hours, 72 hours, and 96 hours. The virus solution was collected at these four time points, and the cells were fully scraped and the cell supernatant was aspirated and transferred together to a 2mL EP tube, marked and frozen in a -80°C refrigerator. After the cell virus solution was thawed, it was vortexed and mixed and then treated with four different methods to measure the hemagglutination (HA) titer of the LaSota strain. Figure 2 This is a typical syncytium formed when Newcastle disease virus infects BSR cells for 72 hours. Figure 3 It is the hemagglutination titer of the cell supernatant of BSR cells infected with Newcastle disease virus for 72 hours (the hemagglutination titer before being treated with the four methods) (control group).

[0023] (IV) Lysis of cells releases Newcastle disease virus Collect NDV 10 -1 -10-6 The diluted cell virus solution was treated with four different methods to release Newcastle disease virus. The four methods were repeated freezing and thawing, ultrasonic disruption, supernatant followed by freezing, and freeze followed by supernatant. The BSR cells in the 12-well plate were scraped separately, mixed with the virus supernatant and collected together, and then the BSR cells were disrupted by four methods to release NDV. The detailed steps of the four methods are as follows: 1. Freeze-thaw cycle Freeze them in liquid nitrogen for 5 minutes, then slowly thaw them at room temperature (about 25°C), repeating this process three times. Freeze in liquid nitrogen for at least 5 minutes to ensure thorough freezing and complete thawing (about 30 minutes at room temperature). Usually, it takes about 2 hours to complete three freeze-thaw cycles to complete virus isolation (5 minutes of freezing in liquid nitrogen and 30 minutes of thawing at room temperature for each cycle).

[0024] 1. Ultrasonic fragmentation The experiment used an ultrasonic cell crusher (SCIENTZ-IID) at a power intensity of 35% (180W) to crush the cell virus solution for 1 minute, with a single crushing of 2 seconds and an interval of 2 seconds, and 15 crushings. The crushing operation was carried out under ice bath conditions. After the sample was ultrasonicated, it was quickly placed on ice.

[0025] 2. Super-freeze first and then freeze The experiment used an ultrasonic cell crusher (SCIENTZ-IID) at a power intensity of 35% for crushing. The cell virus solution was ultrasonically treated for 1 minute, with a single crushing of 2 seconds and an interval of 2 seconds, and the crushing was performed 15 times. The crushing operation was carried out under ice bath conditions. After the samples were ultrasonicated, they were quickly placed on ice. After freezing them in liquid nitrogen for 5 minutes, they were slowly thawed at room temperature (about 25°C), and repeated three times.

[0026] 3. Freeze first and then super Freeze the cell virus solution in liquid nitrogen for 5 minutes, then slowly thaw at room temperature (about 25°C), repeat three times. Then use an ultrasonic cell crusher (SCIENTZ-IID) at a power intensity of 35% to crush the cell virus solution for 1 minute, with a single crushing of 2 seconds, an interval of 2 seconds, and 15 crushings. The crushing operation is carried out under ice bath conditions. After the sample is ultrasonicated, it is quickly placed on ice.

[0027] All cell suspensions treated by the methods were centrifuged at 4000 rpm for 10 minutes at 4°C to remove cell debris and nuclei. 50uL of the supernatant was taken for hemagglutination titer, and the collected supernatants were stored in a -80°C refrigerator.

[0028] Observation of BSR cell lysis morphology after treatment with four methods After treating BSR cells with the four methods, the mixed cell suspension was aspirated and filled onto a counting plate before centrifugation, and the degree of cell fragmentation after treatment was observed using a Nikon ordinary microscope. The number of intact cells was observed under a microscope before and after treatment with all methods to calculate the cell fragmentation rate and compare the fragmentation effect. Figure 4 Table 1 shows the cell disruption rates of the four methods.

[0029] Table 1 Cell disruption rates of the four methods Different approaches <![CDATA[Number of cells before fragmentation / 10 4 > <![CDATA[Number of cells after fragmentation / 10 4 > Broken rate / % Freeze-thaw cycle 349 109 68.77 Ultrasonication 349 24 93.12 Super freeze method 349 18 94.84 Freeze first then super 349 5 98.57 Hemagglutination (HA) assay Use an 8-channel pipette to add 50 μL of 1× PBS buffer to a 96-well V-shaped plate, add 50 μL of virus solution to the first well on the left, mix thoroughly, then draw 50 μL and add it to the second well, and so on, dilute to the 12th well in multiple proportions, and discard 50 μL after mixing the 12th well. Add 50 μL of 1% chicken blood red blood cells to each well, mix thoroughly, and let stand at room temperature for 30 minutes; use the parent strain LaSota as a positive control and PBS as a negative control. Incubate at room temperature for about 30 minutes until RBC agglutination is observed in the positive control well and RBC clumping is observed in the negative control well. The dilution of the last well showing agglutination gives the titer of the virus. Read the HA titer by tilting the plate and observing whether there is a teardrop-shaped red blood cell flow. Figure 5 Serves 10 -1 -10 -6 Hemagglutination titer of virus fluid collected from BSR cells infected with Newcastle disease virus at different dilutions for 72 hours and treated with four different methods. Figure 6 Serves 10 -1 -10 -6 Summary of the hemagglutination titers of the cell virus fluid collected from BSR cells at 72 hours after incubation of LaSota strain with different dilutions and treated with four different methods and the control group.

[0030] In conclusion, the freeze-before-ultrasound method can effectively lyse cells and release Newcastle disease virus.

[0031] Although the above embodiments have been described, once those skilled in the art know the basic creative concepts, they can make additional changes and modifications to these embodiments. Therefore, the above description is only an embodiment of the present invention and does not limit the patent protection scope of the present invention. Any equivalent structure or equivalent process transformation made by using the contents of the specification and drawings of the present invention, or directly or indirectly used in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for lysing cells to release Newcastle disease virus, characterized in that: The method comprises the following steps: (1) freezing and thawing the NDV cell virus solution repeatedly, and then performing ultrasonic disruption to obtain a cell suspension; (2) centrifuging the cell suspension to remove cell debris and cell nuclei, and collecting the supernatant.

2. The method for releasing Newcastle disease virus by lysing cells according to claim 1, characterized in that: The freezing and thawing process in step (1) is to freeze the NDV cell virus liquid in liquid nitrogen for 4-6 minutes and then slowly thaw it at 23-27°C.

3. The method for releasing Newcastle disease virus by lysing cells according to claim 1, characterized in that: The freezing and thawing process in step (1) is repeated three times.

4. The method for releasing Newcastle disease virus by lysing cells according to claim 1, characterized in that: The ultrasonic disruption treatment is carried out under ice bath conditions, and the cell suspension is quickly placed on ice after the ultrasonic treatment.

5. The method for releasing Newcastle disease virus by lysing cells according to claim 1, characterized in that: The ultrasonic disruption treatment was carried out at a power intensity of 180 W. The cell virus solution was ultrasonically treated for 1 minute, with a single disruption of 2 seconds and an interval of 2 seconds, and the disruption was performed 15 times.

6. The method for releasing Newcastle disease virus by lysing cells according to claim 1, characterized in that: The cell suspension was centrifuged at 4000 rpm for 10 minutes at 4°C to remove cell debris and cell nuclei.

7. The method for releasing Newcastle disease virus by lysing cells according to claim 1, characterized in that: The supernatant was collected and stored in a -80°C refrigerator.

8. The method for releasing Newcastle disease virus by lysing cells according to claim 1, characterized in that: The NDV cell virus liquid is obtained by growing and culturing BSRT7 / 5 cells and then inoculating Newcastle disease virus for culturing.

Images