Culture method of human metapneumovirus

Abstract

The invention relates to the technical field of biomedicine, and discloses a human metapneumovirus culture method which comprises the following steps: a culture medium is prepared and put into a culture bottle, and the culture medium comprises a DMEM culture medium, glucose, sodium bicarbonate, an HEPES buffer solution, chondroitin sulfate and hyaluronic acid; host cells are selected; preparing a culture solution, wherein the culture solution comprises L-glutamine, bovine serum albumin, antibiotics and N-acetylcysteine; and inoculating the human metapneumovirus into the culture medium for adsorption and infection. The chondroitin sulfate and the hyaluronic acid are added into the culture medium, the chondroitin sulfate and the hyaluronic acid can be combined with glycosaminoglycans on the surfaces of the host cells to form a compound, interaction sites of viruses and the host cells are increased, then virus receptor expression on the surfaces of the host cells is enhanced, the cell membrane environment is optimized, and therefore the anti-tumor effect is achieved. Therefore, the attachment and entry efficiency of the hMPV is improved, and the rapid proliferation of the virus is obviously promoted.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a method for culturing human metapneumovirus. Background Art

[0002] Human-Metapneumovirus (hMPV) is an important respiratory pathogen that is widely distributed around the world, especially causing acute respiratory infections in children, and has great public health significance. The laboratory isolation and identification of hMPV mainly rely on cell culture technology, and efficient virus amplification is a key prerequisite for related research and vaccine development.

[0003] Existing hMPV culture optimization strategies mostly focus on the selection of cell lines, optimization of the virus infection multiplicity, and adjustment of culture conditions. Although these methods have improved the virus yield to a certain extent, their applicability is relatively narrow and it is difficult to significantly increase the virus yield, which directly restricts the efficiency of virus isolation and identification and the progress of subsequent scientific research. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a method for culturing human metapneumovirus, which solves the problem that, although the prior art improves the virus yield to a certain extent, its applicability is narrow and it is difficult to significantly increase the virus yield, which restricts the efficiency of virus isolation and identification and the progress of subsequent scientific research work.

[0005] To achieve the above objectives, the present invention is implemented by the following technical scheme: A method for culturing human metapneumovirus, comprising the following steps: Prepare a culture medium and put it into a culture bottle: the culture medium includes the following components in mass fractions: DMEM culture medium: 800-950 parts, glucose: 1000-1200 parts, sodium bicarbonate: 800-1000 parts, HEPES buffer: 2000-2300 parts, chondroitin sulfate: 30-50 parts, hyaluronic acid: 10-40 parts; Among them, DMEM medium provides cells with basic nutrients (such as amino acids and vitamins) to support the normal growth of host cells; Glucose provides energy substrates for cells and can significantly enhance cellular metabolic activity during viral replication; Sodium bicarbonate and HEPES buffer together maintain the pH stability of the culture environment. The buffering capacity of HEPES is particularly suitable for high-density cell culture; Chondroitin sulfate and hyaluronic acid can serve as components of the extracellular matrix, forming a glycosaminoglycan-like network structure on the cell surface, simulating the microenvironment of natural virus receptors, and promoting human metapneumovirus (hMPV) to attach to and enter host cells more efficiently, thereby significantly improving the infection efficiency.

[0006] Selecting host cells and inoculating them into culture medium for primary cultivation; Among them, HEp-2 cells are an epithelial cell line with a clear origin and good sensitivity to hMPV, making them suitable as hosts for viral infection; controlling the appropriate cell density can maintain a balance between cell spacing and nutrient supply, prevent excessive fusion or malnutrition, and provide sufficient cell surface receptor sites for virus adsorption.

[0007] Prepare a culture solution and add it into a culture bottle, wherein the culture solution comprises the following components in parts by weight: 150-200 parts of L-glutamine, 30-70 parts of bovine serum albumin, 100-120 parts of antibiotics and 170-200 parts of N-acetylcysteine; Among them, L-glutamine is an important nitrogen source and energy supplier, which can enhance cell metabolic activity and support efficient viral replication; Bovine serum albumin (BSA) can stabilize the culture environment, reduce toxin adsorption, and promote the stable existence of virus particles in the culture system; Antibiotics (penicillin and streptomycin) inhibit bacterial contamination and ensure pure virus growth; N-acetylcysteine ​​(NAC) has antioxidant function, can remove ROS (reactive oxygen species), improve the stability of cell membrane structure, and cooperate with the complex formed by chondroitin sulfate and hyaluronic acid to further optimize cell membrane permeability and promote the fusion and entry of viruses into cell membranes.

[0008] Human metapneumovirus was inoculated into the culture medium for adsorption and infection.

[0009] Preferably, the culture medium is prepared and loaded into a culture bottle, and after the culture medium components are loaded into the same container, they are stirred for 10 to 15 minutes at a temperature of 25 to 37° C. and a rotation speed of 150 to 200 rpm, and then loaded into the culture bottle.

[0010] Preferably, the host cells include HEp-2 cells, and the host cell density is: 1×10 5 ~1×10 6 cells / mL, the volume ratio of the host cells to the culture medium is: 1-10:10-100, and the hyaluronic acid includes sodium hyaluronate.

[0011] Among them, HEp-2 cells are an epithelial cell line with a clear origin and good sensitivity to hMPV, making them suitable as hosts for viral infection; controlling the appropriate cell density can maintain a balance between cell spacing and nutrient supply, prevent excessive fusion or malnutrition, and provide sufficient cell surface receptor sites for virus adsorption.

[0012] Preferably, the initial culture is carried out at a temperature of 36-38°C and 5% CO 2Culture under the same conditions for 2 to 3 hours.

[0013] Among them, this condition simulates the physiological environment in the body, which is conducive to the cells entering a stable growth period and adjusting their state, helping to achieve the optimal physiological state before virus inoculation and enhancing the ability to respond to the virus.

[0014] Preferably, in the preparation of the culture medium, L-glutamine, bovine serum albumin, antibiotics and N-acetylcysteine ​​are mixed and stirred at a temperature of 36 to 38° C. and a rotation speed of 200 to 280 rpm for 10 to 16 minutes.

[0015] This process can promote the full and uniform mixing of the components, while enhancing the solubility and stability of NAC at a suitable temperature, ensuring that it continues to play an antioxidant and membrane stabilizing role during subsequent viral infection. Preferably, the antibiotics include penicillin and streptomycin, and the mass ratio of the two is 1-1.2:1-1.2.

[0016] Among them, this ratio can effectively prevent bacterial contamination and ensure the stability of the culture system without inhibiting the activity of host cells.

[0017] Preferably, the concentration of human metapneumovirus in the inoculation is 1×10 3 ~1×10 5 PFU / mL, and the volume ratio of human metapneumovirus to host cells is 1-10:10-100.

[0018] Among them, controlling the virus inoculation concentration helps to achieve synchronous infection and prevent experimental errors caused by too high or too low MOI (multiplicity of infection); a reasonable volume ratio ensures that the virus particles can fully contact the cell surface receptors and increase the infection initiation rate.

[0019] Preferably, the adsorption and infection are carried out at a temperature of 36-38°C and 5% CO 2 Carry out under the conditions for 1.5 to 2.5 hours.

[0020] This condition can simulate the physiological environment in the body, which is conducive to the cells entering a stable growth period and adjusting their state, helping to achieve the optimal physiological state before virus inoculation and enhancing the ability to respond to the virus.

[0021] The present invention provides a method for culturing human metapneumovirus, which has the following beneficial effects: 1. The present invention adds chondroitin sulfate and hyaluronic acid to the culture medium, and utilizes that chondroitin sulfate and hyaluronic acid can bind to glycosaminoglycans on the surface of host cells to form a complex, thereby increasing the interaction sites between the virus and the host cells, thereby enhancing the expression of viral receptors on the surface of the host cells, optimizing the cell membrane environment, thereby improving the attachment and entry efficiency of hMPV, and significantly promoting the rapid proliferation of the virus.

[0022] 2. The present invention adds N-acetylcysteine ​​to the culture medium and uses NAC as an antioxidant. It can provide sulfhydryl reducing free radicals to achieve the purpose of removing free radicals in cells, maintain the redox balance in cells, protect cells from oxidative damage, and enhance the fluidity of cell membranes, thereby reducing the damage of oxidative stress to cells, thereby improving the vitality and stability of cells and promoting the interaction between viruses and host cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic diagram of the method flow of the present invention. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] In order to better understand the present invention, the above contents are described in detail below in conjunction with specific embodiments.

[0026] Please refer to the attached Figure 1 : Example 1

[0027] Prepare DMEM medium: 900 parts, glucose: 1150 parts, sodium bicarbonate: 950 parts, HEPES buffer: 2100 parts, chondroitin sulfate: 40 parts, hyaluronic acid (sodium hyaluronate): 25 parts. Stir at 32°C and 180 rpm for 13 minutes, then put into culture bottle; The host cells (HEp-2, cell density was 5×10 5 cells / mL) and inoculated into the culture medium for primary culture (volume ratio of host cells to culture medium was 5:70). The initial culture conditions were as follows: transfer the culture flask to an incubator at 37°C and 5% CO 2 The conditions were cultured for 2.3 h; Prepare culture medium: 175 parts of L-glutamine, 55 parts of bovine serum albumin, 115 parts of antibiotics (penicillin:streptomycin=1:1.1) and 180 parts of N-acetylcysteine, stir at 37°C and 240 rpm for 14 minutes, and add to the culture bottle; Human metapneumovirus (concentration: 1×10 4 PFU / mL) was inoculated into the culture medium, and then the culture flask was transferred to an incubator at 37°C and 5% CO 2 Adsorption and infection were carried out for 2 h under the same conditions.

[0028] Example 2

[0029] Prepare DMEM culture medium: 800 parts, glucose: 1000 parts, sodium bicarbonate: 800 parts, HEPES buffer: 2000 parts, chondroitin sulfate: 30 parts, hyaluronic acid (sodium hyaluronate): 10 parts. Stir at 25°C and 150 rpm for 10 minutes, then put into culture bottle; The host cells (HEp-2, cell density was 1×10 5 cells / mL) and inoculated into the culture medium for primary culture (the volume ratio of host cells to culture medium was 1:100). The initial culture conditions were as follows: the culture flask was transferred to an incubator at 37°C and 5% CO 2 Cultured for 2 h under the same conditions; Prepare culture medium: 150 parts of L-glutamine, 30 parts of bovine serum albumin, 100 parts of antibiotics (penicillin:streptomycin=1:1.2) and 170 parts of N-acetylcysteine, stir at 37°C and 200 rpm for 10 min, and add to the culture bottle; Human metapneumovirus (concentration: 1×10 3 PFU / mL) was inoculated into the culture medium, and then the culture flask was transferred to an incubator at 37°C and 5% CO 2 Adsorption and infection were carried out for 1.5 h under the same conditions.

[0030] Example 3

[0031] Prepare DMEM culture medium: 950 parts, glucose: 1200 parts, sodium bicarbonate: 1000 parts, HEPES buffer: 2300 parts, chondroitin sulfate: 50 parts, hyaluronic acid (sodium hyaluronate): 40 parts. Stir at 37°C and 200 rpm for 15 minutes, then put into culture bottle; The host cells (HEp-2, cell density was 1×10 6cells / mL) and inoculated into the culture medium for primary culture (volume ratio of host cells to culture medium was 10:90). The initial culture conditions were as follows: transfer the culture flask to an incubator at 37°C and 5% CO 2 Cultured for 3 h under the same conditions; Prepare culture medium: 200 parts of L-glutamine, 70 parts of bovine serum albumin, 120 parts of antibiotics (penicillin:streptomycin=1.2:1) and 200 parts of N-acetylcysteine, stir at 37°C and 280 rpm for 16 min, and add to the culture bottle; Human metapneumovirus (concentration: 1×10 5 PFU / mL) was inoculated into the culture medium, and then the culture flask was transferred to an incubator at 37°C and 5% CO 2 Adsorption and infection were carried out for 2.5 h under the same conditions.

[0032] Comparative Example 1: Based on Example 1, without chondroitin sulfate, the rest remain the same.

[0033] Comparative Example 2: Based on Example 2, 20 parts of chondroitin sulfate, and the rest remain the same.

[0034] Comparative Example 3: Based on Example 3, 60 parts of chondroitin sulfate were used, and the rest remained the same.

[0035] Comparative Example 4: Based on Example 1, without hyaluronic acid (sodium hyaluronate), the rest remain the same.

[0036] Comparative Example 5: Based on Example 2, hyaluronic acid (sodium hyaluronate) is 9 parts, and the rest remain the same.

[0037] Comparative Example 6: Based on Example 3, hyaluronic acid (sodium hyaluronate) is 50 parts, and the rest remains the same.

[0038] Comparative Example 7: Based on Example 1, without hyaluronic acid (sodium hyaluronate) and chondroitin sulfate, the rest remain the same.

[0039] Comparative Example 8: Based on Example 1, without N-acetylcysteine, the rest remain the same.

[0040] Comparative Example 9: Based on Example 2, 160 parts of N-acetylcysteine ​​were used, and the rest remained the same.

[0041] Comparative Example 10: Based on Example 3, 210 parts of N-acetylcysteine ​​were used, and the rest remained the same.

[0042] Experiment 1: Purpose This experiment aims to verify the effects of chondroitin sulfate and hyaluronic acid on enhancing the sensitivity of host cells (HEp-2 cells) to human metapneumovirus (hMPV) and promoting the rapid proliferation of the virus. By comparing the virus titer, cell viability and cell number under different conditions (including changes in the amount of chondroitin sulfate and hyaluronic acid added), the key role of these two components in hMPV culture was clarified.

[0043] Sample setup: Examples 1-3, Comparative Examples 1-7.

[0044] Data Collection: 1. Virus titer (PFU / mL) determination Equipment: Cell culture incubator (37°C, 5% CO 2 ), microscope (for observing cytopathic effect, CPE), multichannel pipette, 6-well plate or 12-well plate (for virus titer determination), sterile PBS buffer, cell stain (neutral red).

[0045] Method: Plaque-Assay Steps: Cell seeding: HEp-2 cells were plated at 1×10 5 The cells were seeded at a density of cells / mL in 6-well plates and incubated at 37°C and 5% CO 2 The cells were cultured for 24 h to allow them to adhere to the wall and form a monolayer.

[0046] Virus dilution: The virus sample was diluted 10-fold in sterile PBS buffer (10 -1 , 10 -2 , 10 -3 ).

[0047] Virus inoculation: Remove the culture medium from the 6-well plate, add 100 µL of diluted virus solution to each well, shake gently to evenly distribute the virus, and incubate at 37°C and 5% CO 2 Adsorbed for 1 h under the conditions.

[0048] Agar overlay: Gently add agar medium (containing 2% low melting point agar and 1% fetal bovine serum) preheated to 42°C to each well to cover the cell layer. After the agar solidifies, transfer the 6-well plate to 37°C and 5% CO. 2 Culture in an incubator for 5 days.

[0049] Staining and counting: Aspirate the agar layer, stain the cell monolayer with neutral red, and observe and count the number of plaques.

[0050] Calculate the virus titer: Calculate the virus titer (PFU / mL) based on the number of plaques and the dilution factor.

[0051] 2. Cell viability (%) determination Equipment: Cell culture incubator (37°C, 5% CO 2 ), microplate reader (for absorbance measurement), 96-well plate, and cell counting kit.

[0052] Method:CCK-8 method.

[0053] Steps: Cell seeding: HEp-2 cells were plated at 1×10 4 The cells were seeded at a density of 10 cells / well in a 96-well plate and incubated at 37°C and 5% CO 2 The cells were cultured for 24 h to allow them to adhere.

[0054] Reagent addition: Add 10 µL CCK-8 reagent to each well and continue culturing for 4 h.

[0055] Stop the reaction: measure the absorbance directly.

[0056] Absorbance determination: The absorbance was measured at a wavelength of 450 nm using an enzyme reader.

[0057] Calculate cell viability: Calculate cell viability (%) based on the absorbance value. The formula is: Cell viability (%) = (absorbance of experimental group / absorbance of control group) × 100%.

[0058] 3. Cell Number Determination Equipment: cell counting chamber, microscope.

[0059] Method: Cell counting method.

[0060] Steps: Cell cultures were harvested on days 3 and 7 after virus adsorption infection.

[0061] After diluting the cell suspension, take 10 µL and add it to a cell counting plate to count the number of cells under a microscope.

[0062] Calculate the number of cells per unit volume (mL).

[0063] The test results are shown in Table 1: Table 1:

[0064] Experimental Summary It can be seen from the experimental data that the virus titers of Examples 1 to 3 are significantly higher than those of Comparative Examples 1 to 7, indicating that the addition of chondroitin sulfate and hyaluronic acid significantly enhances the sensitivity of host cells to hMPV and promotes the rapid proliferation of the virus. The specific analysis is as follows: Virus titers of Examples 1 to 3: The virus titer of Example 1 was 1.2×10 6 PFU / mL, Example 2 is 8.7×10 5 PFU / mL, Example 3 is 1.5×10 6 PFU / mL, which were significantly higher than the virus titers of Comparative Examples 1 to 7.

[0065] This indicates that adding chondroitin sulfate and hyaluronic acid to the culture medium can significantly improve the proliferation efficiency of hMPV.

[0066] Virus titers of Comparative Example 1 and Comparative Example 4: The virus titer of comparative example 1 (without chondroitin sulfate) was 6.3×10 4 PFU / mL, decreased by about 80% compared with Example 1.

[0067] The virus titer of Comparative Example 4 (without hyaluronic acid) was 5.9×10 4 PFU / mL decreased by about 85% compared with Example 1.

[0068] This shows that after completely removing chondroitin sulfate or hyaluronic acid, the virus titer was significantly reduced, indicating that these two components play a key role in the virus adsorption and infection process.

[0069] Virus titers of Comparative Example 2 and Comparative Example 5: The virus titer of Comparative Example 2 (20 parts of chondroitin sulfate) was 7.8×10 4 PFU / mL decreased by about 93% compared with Example 1.

[0070] The virus titer of Comparative Example 5 (9 parts of hyaluronic acid) was 6.7×10 4 PFU / mL, decreased by about 94% compared with Example 1.

[0071] This shows that after reducing the content of chondroitin sulfate or hyaluronic acid, the viral titer is also significantly reduced, further verifying the importance of these two components.

[0072] Virus titers of Comparative Examples 3 and 6: The virus titer of Comparative Example 3 (60 parts of chondroitin sulfate) was 1.1×10 5 PFU / mL, decreased by about 90% compared with Example 1.

[0073] The virus titer of Comparative Example 6 (50 parts of hyaluronic acid) was 9.2×10 4 PFU / mL decreased by about 93% compared with Example 1.

[0074] This shows that after increasing the content of chondroitin sulfate or hyaluronic acid, the virus titer increased, but the increase was limited, indicating that there is an optimal range for the addition of these two ingredients.

[0075] Virus titer of Comparative Example 7: The virus titer of Comparative Example 7 (without chondroitin sulfate and hyaluronic acid) was 4.8×10 4 PFU / mL, decreased by about 96% compared with Example 1.

[0076] At the same time, the cell viability of Comparative Example 7 also decreased significantly (81.5%), indicating that the synergistic effect of these two components is essential for viral proliferation and cell protection.

[0077] Moreover, the number of cells in Examples 1 to 3 reached 3.8×10 5 / mL and 7.2×10 5 / mL, which is 2 to 3 times higher than that of comparative examples 1 to 7, further verifying the promoting effect of chondroitin sulfate and hyaluronic acid on virus proliferation.

[0078] This shows that chondroitin sulfate and hyaluronic acid form complexes by binding to glycosaminoglycans on the surface of host cells, increasing the interaction sites between the virus and the host cells, thereby optimizing the cell membrane environment and improving the attachment and entry efficiency of hMPV. This effect significantly enhances the sensitivity of host cells to hMPV and promotes the rapid proliferation of the virus. The synergistic effect of chondroitin sulfate and hyaluronic acid further improves the efficiency and stability of virus culture, providing important technical support for the research and application of hMPV.

[0079] Experiment 2: Purpose This experiment aims to verify the sensitivity of N-acetylcysteine ​​to human metapneumovirus (hMPV) and its effect in promoting rapid viral proliferation. By comparing the virus titer and cell viability under different conditions (varying amounts of N-acetylcysteine ​​added), the key role of these two components in hMPV culture was clarified.

[0080] Sample setup: Examples 1-3, Comparative Examples 8-10.

[0081] Data Collection: 1. Virus titer (PFU / mL) determination Equipment: Cell culture incubator (37°C, 5% CO 2), microscope (for observing cytopathic effect, CPE), multichannel pipette, 6-well plate or 12-well plate (for virus titer determination), sterile PBS buffer, cell stain (neutral red).

[0082] Method: Plaque-Assay Steps: Cell seeding: HEp-2 cells were plated at 1×10 5 The cells were seeded at a density of cells / mL in 6-well plates and incubated at 37°C and 5% CO 2 The cells were cultured for 24 h to allow them to adhere to the wall and form a monolayer.

[0083] Virus dilution: The virus sample was diluted 10-fold in sterile PBS buffer (10 -1 , 10 -2 , 10 -3 ).

[0084] Virus inoculation: Remove the culture medium from the 6-well plate, add 100 µL of diluted virus solution to each well, shake gently to evenly distribute the virus, and incubate at 37°C and 5% CO 2 Adsorbed for 1 h under the conditions.

[0085] Agar overlay: Gently add agar medium (containing 2% low melting point agar and 1% fetal bovine serum) preheated to 42°C to each well to cover the cell layer. After the agar solidifies, transfer the 6-well plate to 37°C and 5% CO. 2 Culture in an incubator for 5 days.

[0086] Staining and counting: Aspirate the agar layer, stain the cell monolayer with neutral red, and observe and count the number of plaques.

[0087] Calculate the virus titer: Calculate the virus titer (PFU / mL) based on the number of plaques and the dilution factor.

[0088] 2. Cell viability (%) determination Equipment: Cell culture incubator (37°C, 5% CO 2 ), microplate reader (for absorbance measurement), 96-well plate, and cell counting kit.

[0089] Method:CCK-8 method.

[0090] Steps: Cell seeding: HEp-2 cells were plated at 1×10 4 The cells were seeded at a density of 10 cells / well in a 96-well plate and incubated at 37°C and 5% CO 2 The cells were cultured for 24 h to allow them to adhere.

[0091] Reagent addition: Add 10 µL CCK-8 reagent to each well and continue culturing for 4 h.

[0092] Stop the reaction: measure the absorbance directly.

[0093] Absorbance determination: The absorbance was measured at a wavelength of 450 nm using an enzyme reader.

[0094] Calculate cell viability: Calculate cell viability (%) based on the absorbance value. The formula is: Cell viability (%) = (absorbance of experimental group / absorbance of control group) × 100%.

[0095] 3. Cell Number Determination Equipment: cell counting chamber, microscope.

[0096] Method: Cell counting method.

[0097] Steps: Cell cultures were harvested on days 3 and 7 after virus adsorption infection.

[0098] After diluting the cell suspension, take 10 µL and add it to a cell counting plate to count the number of cells under a microscope.

[0099] Calculate the number of cells per unit volume (mL).

[0100] The test results are shown in Table 2: Table 2:

[0101] Experimental Summary It can be seen from the experimental data that the virus titer, cell viability and cell number of Examples 1 to 3 are significantly higher than those of Comparative Examples 8 to 10, indicating that the addition of N-acetylcysteine ​​significantly enhances the sensitivity of host cells to hMPV and promotes the rapid proliferation of the virus. The specific analysis is as follows: The virus titers of Examples 1 to 3 were 1.2×10 6 PFU / mL, 8.7×10 5 PFU / mL and 1.5×10 6 PFU / mL, which is significantly higher than the virus titer of Comparative Examples 8 to 10. At the same time, the number of cells in Examples 1 to 3 reached 3.4×10 5 / mL and 3.9×10 5 / mL, reaching 6.8×10 in 7 days 5 / mL and 7.4×10 5 / mL, which is 2 to 3 times higher than that of comparative examples 8 to 10, further verifying the promoting effect of N-acetylcysteine ​​on viral proliferation.

[0102] After completely removing N-acetylcysteine ​​in Comparative Example 8, the virus titer decreased by about 40%, and the cell number also decreased significantly, indicating that N-acetylcysteine ​​played a key role in virus adsorption and infection. After reducing the content of N-acetylcysteine ​​in Comparative Example 9, the virus titer and cell number also decreased significantly, further verifying the importance of this component. After increasing the content of N-acetylcysteine ​​in Comparative Example 10, the virus titer and cell number increased, but the increase was limited, indicating that there is an optimal range for the addition of N-acetylcysteine.

[0103] N-acetylcysteine ​​scavenges free radicals, maintains intracellular redox balance, protects cells from oxidative damage, and enhances the fluidity of cell membranes, further promoting the interaction between viruses and host cells. This effect significantly improves the proliferation efficiency and culture stability of hMPV, providing important technical support for the research and application of hMPV.

[0104] Therefore, in summary, chondroitin sulfate, hyaluronic acid and N-acetylcysteine ​​play a synergistic role in hMPV culture, significantly improving the virus proliferation efficiency and culture stability. Chondroitin sulfate and hyaluronic acid bind to glycosaminoglycans on the surface of host cells to form a complex, increasing the interaction sites between the virus and the host cells, thereby optimizing the cell membrane environment and improving the attachment and entry efficiency of hMPV. N-acetylcysteine ​​protects cells from oxidative damage by scavenging free radicals and maintaining intracellular redox balance, while enhancing the fluidity of the cell membrane, further promoting the interaction between the virus and host cells. The synergistic effect of these three components significantly enhances the sensitivity of host cells to hMPV and promotes the rapid proliferation of the virus.

[0105] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for culturing human metapneumovirus, characterized in that: The following steps are involved: Prepare a culture medium and put it into a culture bottle: the culture medium includes the following components in mass fractions: DMEM culture medium: 800-950 parts, glucose: 1000-1200 parts, sodium bicarbonate: 800-1000 parts, HEPES buffer: 2000-2300 parts, chondroitin sulfate: 30-50 parts, hyaluronic acid: 10-40 parts; Selecting host cells and inoculating them into culture medium for primary cultivation; Prepare a culture solution and add it into a culture bottle, wherein the culture solution comprises the following components in parts by weight: 150-200 parts of L-glutamine, 30-70 parts of bovine serum albumin, 100-120 parts of antibiotics and 170-200 parts of N-acetylcysteine; Human metapneumovirus was inoculated into the culture medium for adsorption and infection.

2. The method for culturing human metapneumovirus according to claim 1, characterized in that: The culture medium is prepared and loaded into a culture bottle. After the culture medium components are loaded into the same container, they are stirred for 10 to 15 minutes at a temperature of 25 to 37° C. and a rotation speed of 150 to 200 rpm, and then loaded into the culture bottle.

3. The method for culturing human metapneumovirus according to claim 1, characterized in that: The host cells include HEp-2 cells, and the host cell density is: 1×10 5 ~1×10 6 cells / mL, the volume ratio of the host cells to the culture medium is: 1-10:10-100, and the hyaluronic acid includes sodium hyaluronate.

4. The method for culturing human metapneumovirus according to claim 1, characterized in that: The initial culture is carried out at a temperature of 36-38° C. and 5% CO 2 for 2-3 hours.

5. The method for culturing human metapneumovirus according to claim 1, characterized in that: In the preparation of the culture solution, L-glutamine, bovine serum albumin, antibiotics and N-acetylcysteine ​​are mixed and stirred at a temperature of 36 to 38° C. and a rotation speed of 200 to 280 rpm for 10 to 16 minutes.

6. The method for culturing human metapneumovirus according to claim 1, characterized in that: The antibiotics include penicillin and streptomycin, and the mass ratio of the two is 1-1.2:1-1.

2.

7. The method for culturing human metapneumovirus according to claim 1, characterized in that: The concentration of human metapneumovirus in the inoculation was 1×10 3 ~1×10 5 PFU / mL, and the volume ratio of human metapneumovirus to host cells is 1-10:10-100.

8. The method for culturing human metapneumovirus according to claim 1, characterized in that: The adsorption and infection are carried out at a temperature of 36-38° C. and 5% CO 2 for 1.5-2.5 hours.

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