A preparation method for increasing the yield of live attenuated varicella vaccine virus

Abstract

The invention discloses a method for preparing a virus solution of a live attenuated varicella vaccine, comprising the following steps: preparing a mixed culture medium, passage human diploid cells, inoculating the cells with the varicella zoster virus Oka strain and performing primary and secondary culture, harvesting the virus multiple times, and finally freezing the virus harvest solution; the culture solution of the human diploid cell passage and the cells after virus inoculation are both composed of a mixed culture medium and bovine serum. The invention is based on a mixed culture medium combined with secondary culture and multiple virus harvesting methods, which can greatly improve the virus yield and cell utilization rate and save the cost of passaged cells.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine production, and more specifically, to a preparation method for improving the yield of live attenuated varicella vaccine virus. Background Art

[0002] The full name of the varicella virus is the varicella-zoster virus. The first infection of the human body with the virus can lead to primary infection and cause chickenpox, which is more common in children. After the disease is cured, the virus can continue to lurk in the sensory ganglion cells of the spinal cord and cranial nerves. When the cellular immunity is reduced, the virus will be activated again, and then reach the skin cells controlled along the sensory nerve axons and proliferate inside them, thus causing shingles. The varicella vaccine is prepared by the passage of varicella virus strains and is the only means to prevent varicella infection. Vaccination with the varicella vaccine can not only prevent chickenpox, but also prevent complications caused by varicella zoster.

[0003] At present, the varicella live attenuated vaccine uses human diploid cells as the cell matrix for culturing the virus, and the cell matrix has been used in vaccine production for more than 40 years. For example, Chinese patent CN104188923A proposes a novel method for producing a freeze-dried varicella live attenuated vaccine, which includes the following steps: 1) resuscitation and subculture of human diploid cell lines; 2) culturing human diploid cells in a bioreactor using microcarriers; 3) inoculating the varicella virus Oka strain to proliferate the virus, and when the cell pathological changes reach more than 75%, the varicella virus culture fluid is ready to be harvested to obtain a preliminary culture fluid; 4) adding a lyophilization protective agent to the preliminary culture fluid, harvesting the varicella virus culture fluid, and preparing it into a varicella vaccine stock solution after purification; 5) packaging the above-mentioned varicella vaccine stock solution and freeze-drying it to obtain the novel freeze-dried varicella live attenuated vaccine. When implementing the patented method, technicians in this field found that, on the one hand, human diploid cell cell matrix generally proliferates slowly after growing to a monolayer, and senescence occurs. The cell density, growth state and maintenance time of the cells on the culture dish all affect the virus yield, and the lesions are prone to uneven infection, some cells are not infected, thereby reducing the utilization rate of the cells, and some cells are easily shed due to excessive infection, resulting in loss of virus fluid; on the other hand, when the virus is harvested, since the one-time digestion harvesting method is basically adopted, the metabolites of the virus are not conducive to the growth and maintenance of the cells, and the cells are prone to aging and shedding, which not only loses the virus fluid, but also limits the continued amplification of the virus, so it must be harvested when the lesions are about 75%. Therefore, the utilization rate of the cell matrix in the existing varicella attenuated live vaccine preparation method is not high, and the virus harvest rate also has a lot of room for improvement. Summary of the invention

[0004] Based on this, it is necessary to provide a preparation method for increasing the yield of varicella live attenuated vaccine virus in response to the above technical problems.

[0005] In order to solve the above technical problems, the present invention provides a preparation method for improving the yield of varicella live attenuated vaccine virus, comprising the following steps: using a mixed culture medium to culture to obtain high-density human diploid cells with a long maintenance time, inoculating varicella-zoster virus Oka strain for the first culture, culturing to the early stage of lesions using high-concentration recombinant trypsin to re-digest the virus-carrying cells, making the lesion cells evenly distributed and then performing a secondary culture, using different concentrations of recombinant trypsin to culture and digest and harvest for multiple times according to the degree of cell lesions, and finally freezing the virus harvested liquid; the human diploid cell culture and the culture liquid after the virus inoculation are both composed of a mixed culture medium and bovine serum.

[0006] Furthermore, the mixed culture medium is composed of MEM culture medium, Biote D126C culture medium and Source Culture HDC culture medium in a volume percentage of 40% to 60%: 25 to 35%: 15% to 25%.

[0007] Furthermore, the human diploid cells are MRC-5 cells, and the passage ratio is 1:2 to 1:4.

[0008] Furthermore, the culture medium for subculturing the human diploid cell MRC-5 is composed of a mixed culture medium and bovine serum in a volume ratio of 88% to 92%: 8% to 12%.

[0009] Furthermore, in the first culture of cells after inoculation with the varicella-zoster virus Oka strain, the secondary culture after re-digestion in the early stage of lesions, and multiple digestion and culture steps according to the degree of cell pathology, the culture medium is composed of mixed culture medium and bovine serum in a volume ratio of 97% to 99%: 1% to 3%.

[0010] Furthermore, MRC-5 cells are inoculated with varicella-zoster virus Oka strain at an MOI of 0.01 to 0.1 and cultured for the first time. When the cell pathology reaches 5% to 10%, the cells are digested with high concentration of recombinant trypsin and then cultured for the second time. In this step, the cells are re-digested to redistribute the virus-carrying cells in the culture medium, making the diseased cells more uniform and increasing the final density of the diseased cells.

[0011] Further, when the secondary cultured cells have a lesion of 40% to 60%, a low concentration of recombinant trypsin is used for the first digestion, and when the cell surface peels off 30% to 40%, the first harvest is performed; the cells that have not shed off are continued to be cultured until the lesion reaches 40% to 60%, a low concentration of recombinant trypsin is used for the second digestion, and when the cell surface peels off 45% to 55%, the second harvest is performed; the cells that have not shed off are continued to be cultured until the lesion reaches more than 75%, and a high concentration of recombinant trypsin is used for the third digestion and harvest. The multiple harvests in this step use recombinant trypsin with mild digestion strength to achieve an ideal digestion and separation effect at a specific concentration, and realize the layer-by-layer peeling of cells.

[0012] Furthermore, the concentration of the high-concentration recombinant trypsin is 0.2-0.4 mg / ml.

[0013] Furthermore, the concentration of the low-concentration recombinant trypsin is 0.1-0.2 mg / ml.

[0014] Furthermore, the cell suspensions harvested multiple times were centrifuged at 2-8°C and 3000-4000 rpm for 8-10 minutes, the lower precipitate was removed, virus protection solution was added to resuspend the suspension and cryopreserved at below -60°C.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention creatively provides a mixed culture medium, which is based on the mixed culture medium and can greatly improve the density and maintenance time of cells, solve the problem of slow growth and easy aging and shedding of cells after monolayer, and lay a foundation for subsequent improvement of virus yield; combined with the present invention, the virus-inoculated cells are cultured twice, and when the cell pathology in the first culture reaches 5% to 10%, the cells are digested again and cultured twice, so that the virus-carrying cells are redistributed, the cell pathology is more uniform, and the infection rate and utilization rate of the cells are improved; finally, combined with the present invention, different concentrations of recombinant trypsin are used according to the degree of cell pathology for multiple digestion, multiple culture, and multiple harvesting, the loss of virus liquid shedding is reduced, and the space for virus amplification is increased, which greatly promotes the improvement of virus yield, and the cell utilization is basically saturated, saving the cost of raw materials in the production process. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the solutions in the present application or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1The cell pathological changes observed under a microscope during the three virus digestion harvests in Example 1;

[0019] Figure 2 The cell pathological changes observed under a microscope during the three virus digestion harvests in Example 2;

[0020] Figure 3 The cell pathological changes observed under a microscope during the three virus digestion harvests in Example 1;

[0021] Figure 4 The cell lesions were observed under a microscope during single virus digestion and harvest in Comparative Example 1;

[0022] Figure 5 The cell lesions were observed under a microscope during virus digestion and harvest in Comparative Example 2;

[0023] Figure 6 The cell lesions were observed under a microscope during virus digestion and harvest in Comparative Example 3;

[0024] Figure 7 The cell lesions were observed under a microscope during virus digestion and harvest in Comparative Example 4;

[0025] Figure 8 The cell lesions were observed under a microscope during virus digestion and harvest in Comparative Example 5;

[0026] Fig. 9 For Comparative Example 6, the cell pathological changes were observed under a microscope when the virus was harvested after three virus digestions. DETAILED DESCRIPTION

[0027] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings 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 should fall within the scope of protection of the present invention.

[0028] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0029] Description of raw materials for embodiments and comparative examples:

[0030] Human diploid MRC-5 cells: purchased from ATCC;

[0031] MEM medium: purchased from Gibco;

[0032] Bio-Tech D126C culture medium: purchased from Bio-Tech Biotech (Shanghai) Co., Ltd.;

[0033] Yuanpei HDC medium: purchased from Shanghai Yuanpei Biotechnology Co., Ltd.;

[0034] Bovine serum: purchased from Zhejiang Tianhang Biotechnology Co., Ltd.;

[0035] PBS (phosphate) buffer with a pH of 7.4: 10 mmol / L PBS buffer prepared in the applicant's laboratory;

[0036] Virus protection solution: purchased from Shanghai Yuanye Biotechnology Co., Ltd.;

[0037] Recombinant trypsin: purchased from Wuhan Hanhai New Enzyme Biotechnology Co., Ltd.

[0038] Example 1

[0039] This embodiment proposes a method for increasing the yield of live attenuated varicella vaccine virus, comprising the following steps:

[0040] S1. Prepare mixed culture medium: Mix MEM culture medium, Biot D126C culture medium and Source Culture HDC culture medium in a volume percentage of 40%:35%:25% and place on a shaker for 5 minutes;

[0041] S2. Cell passaging: The human diploid MRC-5 cells were passaged at a ratio of 1:2 using the mixed culture medium prepared in S1 and a culture medium with a volume ratio of 92%:8% bovine serum, and placed in a static constant temperature culture at 36.5°C;

[0042] S3. Virus inoculation and initial culture: MRC-5 cells grown to a dense monolayer were inoculated with the varicella zoster virus Oka strain at an MOI of 0.01, and then cultured with a mixed culture medium and bovine serum at a volume ratio of 99%:1%, and cultured at 35°C;

[0043] S4, secondary culture of the virus: When the cell lesion rate reaches 5%, the cells are digested with 0.2 mg / ml recombinant trypsin to remove the attached cells, and the culture medium in step S3 is added, and the cells are placed at 35°C for secondary culture;

[0044] S5. Digest, culture and harvest multiple times according to the degree of lesions: When the lesions reach 40%, use 0.1mg / ml recombinant trypsin for the first digestion, and the cells will peel off layer by layer. When the surface layer peels off 30%, the virus liquid is harvested, and the remaining cells that have not shed are added to the culture medium with a volume ratio of 99%:1% of mixed culture medium and bovine serum for further cultivation; When the cell lesions reach 40%, use 0.1mg / ml recombinant trypsin for the second digestion, and when the surface layer peels off 45%, the virus liquid is harvested, and the remaining cells that have not shed are added to the culture medium with a volume ratio of 99%:1% of mixed culture medium and bovine serum for further cultivation; When the cell lesions reach more than 75%, use 0.25mg / ml recombinant trypsin for the third digestion and harvest. Figure 1 The cell lesions observed under a microscope during the three digestion harvests are shown. The cell lesions were uniform during the three digestion harvests.

[0045] S6. Freezing of virus harvest solution: Centrifuge the cell suspension collected from three digestions at 2°C and 3000rpm for 8 minutes. After centrifugation, remove the lower layer of sediment and add virus protection solution to resuspend and disperse evenly by pipetting. This is the virus harvest solution, which is frozen below -60°C;

[0046] S7. Cell disruption and virus clarification: Take out the virus harvest liquid and place it in a room temperature running water bath to thaw naturally. When the ice begins to separate from the inner wall of the container, freeze-break the cells. After the ice surface is completely melted, use a stainless steel mesh to clarify and filter. Finally, obtain the virus filtrate and take samples for virus titer detection.

[0047] Example 2

[0048] This embodiment proposes a method for increasing the yield of live attenuated varicella vaccine virus, comprising the following steps: S1, preparing a mixed culture medium: mixing MEM culture medium, Biot D126C culture medium and Source Culture HDC culture medium in a volume percentage of 50%:30%:20% and placing on a shaker for 7.5 minutes;

[0049] S2. Cell passaging: The human diploid MRC-5 cells were passaged at a ratio of 1:3 using the mixed culture medium prepared in S1 and a culture medium with a volume ratio of 90%:10% of bovine serum, and placed in a constant temperature culture at 37°C;

[0050] S3. Virus inoculation and initial culture: MRC-5 cells grown to a dense monolayer were inoculated with the varicella zoster virus Oka strain at an MOI of 0.05, and then cultured with a mixed culture medium and bovine serum at a volume ratio of 98%:2%, and then cultured at 36°C;

[0051] S4. Secondary culture of the virus: When the cell lesion rate reaches 75%, the cells are re-digested with 0.3 mg / ml recombinant trypsin to remove the attached cells, and the culture medium in step S3 is continued to be added, and the cells are placed at 36°C for secondary culture;

[0052] S5. Digest, culture and harvest multiple times according to the degree of lesions: When the lesions reach 50%, use 0.15mg / ml recombinant trypsin for the first digestion, and the cells will peel off layer by layer. When the surface layer peels off 35%, the virus liquid is harvested, and the remaining cells that have not shed are added to the culture medium with a volume ratio of 98%:2% of mixed culture medium and bovine serum for further cultivation; When the cell lesions reach 50%, use 0.15mg / ml recombinant trypsin for the second digestion, and when the surface layer peels off 50%, the virus liquid is harvested, and the remaining cells that have not shed are added to the culture medium with a volume ratio of 98%:2% of mixed culture medium and bovine serum for further cultivation; When the cell lesions reach more than 75%, use 0.3mg / ml recombinant trypsin for the third harvest. Figure 2 The cell lesions observed under a microscope during the three digestion harvests are shown. The cell lesions were uniform during the three digestion harvests.

[0053] S6. Freezing of virus harvest solution: Centrifuge the cell suspension collected three times at 5°C and 3500rpm for 9 minutes. After centrifugation, take the precipitate and add virus protection solution to resuspend and disperse evenly by blowing, which is the virus harvest solution, and freeze it below -60°C;

[0054] S7. Cell disruption and virus clarification: Take out the virus harvest and place it in a room temperature running water bath to thaw naturally. When the ice begins to separate from the inner wall of the container, break the cells by freeze-breaking. After the ice surface is completely melted by freeze-breaking, use a stainless steel mesh to clarify and filter. Finally, obtain the virus filtrate and take samples for virus titer detection.

[0055] Example 3

[0056] This embodiment proposes a method for increasing the yield of varicella live attenuated vaccine virus liquid, comprising the following steps: preparing a mixed culture medium: mixing MEM culture medium, Biot D126C culture medium and Source Culture HDC culture medium in a volume percentage of 60%:25%:15% and placing on a shaker for 10 minutes;

[0057] S2. Cell passaging: The human diploid MRC-5 cells were passaged at a ratio of 1:4 using the mixed culture medium prepared in S1 and a culture medium with a volume ratio of 88%:12% of bovine serum, and placed in a constant temperature culture at 37.5°C;

[0058] S3. Virus inoculation and initial culture: MRC-5 cells grown to a dense monolayer were inoculated with the varicella zoster virus Oka strain at an MOI of 0.1, and then a culture medium containing a mixed culture medium and bovine serum in a volume ratio of 97%:3% was added, and then cultured at 37°C;

[0059] S4. Secondary culture of the virus: When the cell lesion rate reaches 10%, the cells are re-digested with 0.4 mg / ml recombinant trypsin to remove the attached cells, and the culture medium in step S3 is added, and the cells are placed at 37°C for secondary culture;

[0060] S5. Digest, culture and harvest multiple times according to the degree of lesions: When the lesions reach 60%, use 0.2mg / ml recombinant trypsin for the first digestion, and the cells peel off layer by layer. When the surface layer peels off 40%, the virus liquid is harvested, and the remaining cells that have not shed are added to the culture medium with a volume ratio of 97%:3% of the culture medium and bovine serum to continue culturing; when the cell lesions reach 60%, use 0.2mg / ml recombinant trypsin for the second digestion, and when the surface layer peels off 55%, the virus liquid is harvested, and the remaining cells that have not shed are added to the culture medium with a volume ratio of 97%:3% of the culture medium and bovine serum to continue culturing; when the cell lesions reach more than 75%, use 0.4mg / ml recombinant trypsin for the third harvest. Figure 3 The cell lesions observed under a microscope during the three digestion harvests are shown. The cell lesions were uniform during the three digestion harvests.

[0061] S6. Freezing of virus harvest solution: Centrifuge the cell suspension collected three times at 8°C and 4000rpm for 10 minutes. After centrifugation, take the precipitate and add virus protection solution to resuspend and disperse evenly by blowing, which is the virus harvest solution, and freeze it below -60°C;

[0062] S7. Cell disruption and virus clarification: Take out the virus harvest and place it in a room temperature running water bath to thaw naturally. When the ice begins to separate from the inner wall of the container, break the cells by freeze-breaking. After the ice surface is completely melted by freeze-breaking, use a stainless steel mesh to clarify and filter. Finally, obtain the virus filtrate and take samples for virus titer detection.

[0063] Comparative Example 1

[0064] This comparative example proposes a method for increasing the yield of varicella live attenuated vaccine virus. The method provided in this comparative example is a conventional preparation method, which differs from Example 2 in that MEM culture medium is used instead of mixed culture medium, and secondary culture, multiple digestion, multiple culture, and multiple harvesting of the virus are not performed.

[0065] Specifically, the virus was directly harvested once after inoculation and culture: when the lesion reached 75%, the cells were harvested by digestion with 0.3 mg / ml recombinant trypsin, and the harvested cell suspension was centrifuged and frozen. Figure 4 The cytopathic effect observed under a microscope during virus digestion and harvesting in this comparative example is shown. Since only MEM culture medium was used, virus culture was performed once and harvested once, the cytopathic effect was uneven and the cells were obviously detached.

[0066] Comparative Example 2

[0067] This comparative example proposes a method for increasing the yield of live attenuated varicella vaccine virus. The difference between this comparative example and Example 2 is that in step S1, MEM medium is used instead of mixed medium. During the experiment, it was found that when only MEM medium was used, the peeling of layers during digestion was not obvious. When the recombinant trypsin was used for the first digestion, the cells basically fell off together, and it was difficult to separate them during the experiment. The second and third digestions could not be performed, that is, the virus could only be harvested once. Figure 5 The cytopathic effect observed under a microscope when the virus of this comparative example was harvested is shown. The cytopathic effect is uniform but the virus is obviously shedding.

[0068] Comparative Example 3

[0069] This comparative example proposes a method for increasing the yield of live attenuated varicella vaccine virus, which differs from Example 2 in that in step S1, the mixed culture medium is replaced by a mixture of MEM culture medium and Biote D126C culture medium in a volume ratio of 7:3. Figure 6 The figure shows the cell pathological changes observed under a microscope when the virus of this comparative example was harvested for the first time. After the addition of Biote D126C, the cell density increased, but the cell maintenance was still poor, and some cells fell off. When the recombinant trypsin was used for the first digestion, the cells fell off at the same time, making it difficult to peel off layer by layer for subsequent harvesting, so the second and third digestions were not performed.

[0070] Comparative Example 4

[0071] This comparative example proposes a method for increasing the yield of live attenuated varicella vaccine virus, which differs from Example 2 in that in step S1, the mixed culture medium is replaced by a mixture of MEM culture medium and source culture HDC culture medium in a volume ratio of 8:2. Figure 7 The figure shows the cell pathological changes observed under a microscope during the first digestion and harvest of the virus in this comparative example. After the HDC culture medium was added, although the cell maintenance degree was improved, it was still difficult to support multiple digestions and harvests due to the low cell density. The cells fell off at the same time during the first digestion with recombinant trypsin, so the second and third digestions, cultures and harvests were not performed.

[0072] Comparative Example 5

[0073] This comparative example proposes a method for increasing the yield of live attenuated varicella vaccine virus, which differs from Example 2 only in that the virus is harvested only once.

[0074] Specifically, a single harvest was performed after the secondary virus culture: when the lesion reached 75%, the cells were harvested by digestion with 0.3 mg / ml recombinant trypsin, and the harvested cell suspension was centrifuged and frozen. Figure 8 The cytopathic effect observed under a microscope when the virus was digested and harvested in this comparative example is shown.

[0075] Comparative Example 6

[0076] This comparative example proposes a method for increasing the yield of live attenuated varicella vaccine virus, which differs from Example 2 only in that the virus is not cultured a second time.

[0077] Specifically, multiple virus digestion, cultivation and harvesting steps are performed directly after virus inoculation and initial cultivation. Fig. 9 The cytopathic condition observed under a microscope when the virus of this comparative example was harvested is shown, and the picture shows that the cells are unevenly distributed.

[0078] Test example

[0079] 1. The cells harvested from the above examples and comparative examples were observed under a microscope. The results are shown in Table 1:

[0080] Table 1: Observation results of cell harvesting in Examples and Comparative Examples

[0081]

[0082]

[0083] 2. The virus filtrates obtained in the above examples and comparative examples were tested for virus titer, and the results are shown in Table 2:

[0084] Table 2: Virus harvest titer results of Examples and Comparative Examples

[0085]

[0086] From the results in Table 2, it can be seen that the preparation methods of Examples 1-3 using mixed culture medium combined with secondary virus culture and multiple digestion, culture and harvesting have an increase of more than 5 times in virus harvest yield compared with the conventional method, and the virus yield obtained by the methods of Comparative Examples 2-6 is also significantly improved.

[0087] Obviously, the embodiments described above are only some embodiments of the present application, rather than all embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application is described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions recorded in the aforementioned specific implementation methods, or to perform equivalent replacement of some of the technical features therein. Any equivalent structure made using the contents of the specification and drawings of this application, directly or indirectly used in other related technical fields, is similarly within the scope of patent protection of this application.

Claims

1. A method for increasing the yield of live attenuated varicella vaccine virus, characterized in that: The following steps are involved: A mixed culture medium is used to culture human diploid cells with high density and long maintenance time, and the varicella-zoster virus Oka strain is inoculated for the first culture. When the cells are cultured to the early stage of lesions, a high concentration of recombinant trypsin is used to re-digest the virus-carrying cells, and the lesion cells are evenly distributed and then cultured for the second time. According to the degree of cell lesions, different concentrations of recombinant trypsin are used for multiple culture and digestion harvesting, and finally the virus harvesting liquid is frozen; The culture medium of the human diploid cells and the cells inoculated with the virus are both composed of mixed culture medium and bovine serum; The mixed culture medium is composed of MEM culture medium, Biot D126C culture medium and Source Culture HDC culture medium in a volume percentage of 40% to 60%: 25 to 35%: 15% to 25%; In the steps of first culture of cells after inoculation of varicella-zoster virus Oka strain, second culture after re-digestion in the early stage of lesions, and multiple digestion and culture according to the degree of lesions, the culture medium is composed of mixed culture medium and bovine serum in a volume ratio of 97% to 99%: 1% to 3%; When the secondary cultured cells have a lesion of 40% to 60%, a low concentration of recombinant trypsin is used for the first digestion, and when the cell surface is 30% to 40% peeled off, the first harvest is made; the cells that have not shed off are continued to be cultured until the lesion reaches 40% to 60%, a low concentration of recombinant trypsin is used for the second digestion, and when the cell surface is 45% to 55% peeled off, the second harvest is made; the cells that have not shed off are continued to be cultured until the lesion reaches more than 75%, and a high concentration of recombinant trypsin is used for the third digestion and harvest; the human diploid cells are MRC-5 cells, and the passage ratio is 1:2 to 1:4; The culture medium for subculturing the human diploid cell MRC-5 is composed of a mixed culture medium and bovine serum in a volume ratio of 88% to 92%: 8% to 12%; Inoculate the MRC-5 cells with the varicella-zoster virus Oka strain at an MOI of 0.01 to 0.1 and perform the first culture. When the cell lesion rate reaches 5% to 10%, re-digest with high-concentration recombinant trypsin to make the lesion cells evenly distributed and then perform the second culture. The concentration of the high-concentration recombinant trypsin is 0.2-0.4 mg / ml; The cell suspension harvested after multiple digestions according to the degree of cytopathic effect is centrifuged at 2-8°C and 3000-4000 rpm for 8-10 minutes, the lower layer of sediment is removed, virus protection solution is added to resuspend and cryopreserved below -60°C.

2. A method for preparing a varicella attenuated live vaccine virus for increasing its yield according to claim 1, characterized in that: The concentration of the low-concentration recombinant trypsin is 0.1-0.2 mg / ml.

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