A low-temperature freezing protection solution for koi herpes virus and its preparation method and application

Abstract

The present invention provides a cryopreservation solution for Koi herpesvirus, as well as its preparation method and application, relating to the field of virus preservation technology. The solution comprises 2-8 w / v% hydroxyethyl starch, 3-5 w / v% sucrose, and 1-3 w / v% bovine serum albumin. The Koi herpesvirus has an activity of ≥90% after resuscitation, can withstand five freeze-thaw cycles, can be stably frozen at -20°C, and has a shelf life of up to two years. This solution solves the technical problems in the prior art, such as the need to store cryopreservation solutions at ultra-low temperatures, the risk of osmotic pressure imbalance and viral envelope rupture, and low activity after cryopreservation and resuscitation.

Description

Technical Field

[0001] The present invention relates to the technical field of virus preservation, in particular to a low-temperature freezing protection solution for koi herpes virus, a preparation method and an application thereof. Background Art

[0002] Koi herpesvirus (KHV) belongs to the genus Herpesvirus ( Herpesviridae ), Fish Herpesviridae ( Alloherpesviridae Cyprinid herpesvirus 3 (CyHV-3) is a spherical virus with a diameter of approximately 170-230 nm and a double lipid envelope. Its genome consists of linear double-stranded DNA (~295 kbp) encoding over 150 proteins. Its high pathogenicity and infectivity have caused significant losses to common carp and koi aquaculture both domestically and internationally. Inactivated vaccines are ineffective, making them difficult to commercialize. Live vaccines are highly effective and can be used for large-scale immersion immunization, but their commercialization requires extremely high preservation requirements for the live virus.

[0003] Currently, KHV is cryopreserved using a generic formula based on fetal bovine serum, dimethyl sulfoxide, or glycerol, and is typically stored at ultra-low temperatures of -80°C. Due to the significant difference in osmotic pressure between freshwater and marine fish fluids, existing buffers (such as PBS) are difficult to adapt, resulting in osmotic pressure imbalance. Furthermore, traditional gradient cooling methods can easily rupture the viral envelope, resulting in data showing that KHV activity is less than 50% after cryopreservation.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a low-temperature cryopreservation protective solution for Koi Herpes Virus, so as to solve the technical problems in the prior art that the cryopreservation solution needs to be stored under ultra-low temperature conditions, suffers from osmotic pressure imbalance and viral envelope rupture, and has low activity after cryopreservation and recovery.

[0006] A second object of the present invention is to provide a method for preparing the above-mentioned low-temperature cryopreservation protective solution.

[0007] The third object of the present invention is to provide an application of the above-mentioned low-temperature freezing protection solution.

[0008] A fourth object of the present invention is to provide a product for freezing Koi herpes virus.

[0009] A fifth object of the present invention is to provide a method for freezing Koi herpes virus.

[0010] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0011] In a first aspect, the present invention provides a low-temperature cryopreservation protective solution for koi herpes virus, comprising 2-8 w / v % hydroxyethyl starch, 3-5 w / v % sucrose, and 1-3 w / v % bovine serum albumin.

[0012] Furthermore, the concentration of hydroxyethyl starch is 5w / v%, the concentration of sucrose is 4w / v%, and the concentration of bovine serum albumin is 2w / v%.

[0013] Furthermore, the pH is 7.0~8.0.

[0014] Furthermore, the salinity of the solvent of the low-temperature cryopreservation protection solution is 7-12‰;

[0015] The solvent includes 6-12 g / L of NaCl, 0.2-0.5 g / L of KCl, and 0.1-0.3 g / L of CaCl2.

[0016] In a second aspect, the present invention provides a method for preparing the above-mentioned cryopreservation protective solution, comprising dissolving sucrose and hydroxyethyl starch in a solvent, and adding bovine serum albumin to prepare the cryopreservation protective solution.

[0017] Furthermore, before dissolving the sucrose and hydroxyethyl starch in the solvent, the process further includes preheating the solvent to 32-37° C.;

[0018] The method further comprises sterilizing the filter membrane after adding bovine serum albumin.

[0019] In a third aspect, the present invention provides the use of the above-mentioned low-temperature cryopreservation protection solution or the low-temperature cryopreservation protection solution prepared by the above-mentioned preparation method in the cryopreservation of koi herpes virus or in the preparation of products for the cryopreservation of koi herpes virus.

[0020] In a fourth aspect, the present invention provides a product for freezing Koi Herpes Virus, comprising the above-mentioned low-temperature freezing protection solution or the low-temperature freezing protection solution prepared by the above-mentioned preparation method.

[0021] In a fifth aspect, the present invention provides a method for freezing Koi herpes virus, comprising mixing a cryopreservation protection solution with a virus suspension, precooling at 4°C, cooling to -20°C, and then deep freezing with liquid nitrogen;

[0022] The low-temperature cryopreservation protection liquid is the same as the above-mentioned low-temperature cryopreservation protection liquid or the low-temperature cryopreservation protection liquid prepared by the above-mentioned preparation method.

[0023] Furthermore, the volume ratio of the low-temperature freezing protection solution to the virus suspension is 1:1;

[0024] The precooling time is 20 to 40 minutes;

[0025] The cooling rate from 4°C to -20°C is 0.8~1.2°C / min;

[0026] The liquid nitrogen deep freezing time is ≥2h.

[0027] The present invention provides a low-temperature cryopreservation protective solution for koi herpes virus, which combines hydroxyethyl starch and low-concentration sucrose to reduce ice crystal formation, stabilize the virus envelope, and avoid rupture of the virus envelope. Experiments have shown that compared with traditional formulas, the koi herpes virus preserved in the low-temperature cryopreservation protective solution provided by the present invention has an activity of ≥90% after recovery, can withstand 5 freeze-thaw cycles, can be stably frozen at -20°C, and the shelf life is extended to 2 years. This solves the technical problems in the prior art that the freezing solution needs to be stored under ultra-low temperature conditions, and there are osmotic pressure imbalances and virus envelope ruptures, and low activity after freezing and recovery. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 This is a comparison of electron microscopic observations of viral envelope protection by different cryopreservation protection solutions provided in Experiment 2 of the present invention;

[0030] Figure 2 This is a curve showing the effect of different salinity cryopreservation protective solutions on virus survival rate provided in Experiment 3 of the present invention. DETAILED DESCRIPTION

[0031] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, the use of "or" means "and / or" unless otherwise stated. In addition, the use of the term "including" and other forms is non-limiting.

[0032] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0033] On one hand, the present invention provides a low-temperature freezing protection solution for koi herpes virus, comprising 2-8 w / v % of hydroxyethyl starch, 3-5 w / v % of sucrose and 1-3 w / v % of bovine serum albumin.

[0034] The combination of hydroxyethyl starch and low-concentration sucrose can reduce the formation of ice crystals to stabilize the viral envelope and avoid rupture of the viral envelope. Experiments have shown that compared with traditional formulas, the koi herpes virus preserved in the low-temperature cryopreservation protective solution provided by the present invention has an activity of ≥90% after recovery, can withstand 5 freeze-thaw cycles, can be stably frozen at -20°C, and the shelf life is extended to 2 years. This solves the technical problems in the prior art that the cryopreservation solution needs to be stored at ultra-low temperature conditions, and there are osmotic pressure imbalances and viral envelope ruptures, and low activity after cryopreservation recovery.

[0035] The concentration of hydroxyethyl starch may be, but is not limited to, 2 w / v%, 3 w / v%, 4 w / v%, 5 w / v%, 6 w / v%, 7 w / v% or 8 w / v%, and may be any value between 2 and 8 w / v%, preferably 5 w / v%.

[0036] The concentration of the sucrose may be, but is not limited to, 3 w / v%, 3.3 w / v%, 3.5 w / v%, 3.8 w / v%, 4 w / v%, 4.3 w / v%, 4.5 w / v%, 4.8 w / v% or 5 w / v%, and may be any value between 3 and 5 w / v%, preferably 4 w / v%.

[0037] The concentration of the bovine serum albumin can be, but is not limited to, 1 w / v%, 1.3 w / v%, 1.5 w / v%, 1.8 w / v%, 2 w / v%, 2.3 w / v%, 2.5 w / v%, 2.8 w / v% or 3 w / v%, and can also be any value between 1 and 3 w / v%, preferably 2 w / v%.

[0038] In order to further improve the effect of the cryopreservation protective solution, in some specific embodiments, the concentration of hydroxyethyl starch is 5 w / v%, the concentration of sucrose is 4 w / v%, and the concentration of bovine serum albumin is 2 w / v%.

[0039] In some specific embodiments, the pH is 7.0-8.0, preferably 7.4.

[0040] In some specific embodiments, the salinity of the solvent of the cryopreservation solution is 7-12‰ to adapt to the osmotic pressure of seawater or freshwater fish body fluids to avoid osmotic pressure imbalance. In some specific embodiments, the solvent includes 6-12 g / L NaCl, 0.2-0.5 g / L KCl, and 0.1-0.3 g / L CaCl2;

[0041] The salinity of the solvent may be, but is not limited to, 7‰, 7.5‰, 8‰, 8.5‰, 9‰, 9.5‰, 10‰, 10.5‰, 11‰, 11.5‰ or 12‰, or any value between 7‰ and 12‰. The specific salinity may be adjusted based on freshwater fish viruses or marine fish viruses.

[0042] The concentration of NaCl may be, but is not limited to, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L or 12 g / L, or any value between 6 and 12 g / L.

[0043] The concentration of KCl may be, but is not limited to, 0.2 g / L, 0.25 g / L, 0.3 g / L, 0.35 g / L, 0.4 g / L, 0.45 g / L or 0.5 g / L, or any value between 0.2 and 0.5 g / L.

[0044] The concentration of CaCl2 can be, but is not limited to, 0.1 g / L, 0.13 g / L, 0.15 g / L, 0.18 g / L, 0.2 g / L, 0.23 g / L, 0.25 g / L, 0.28 g / L or 0.3 g / L, and can also be any value between 0.1 and 0.3 g / L.

[0045] According to another aspect of the present invention, a method for preparing the above-mentioned cryopreservation protection solution is also provided, comprising dissolving sucrose and hydroxyethyl starch in a solvent, and adding bovine serum albumin to prepare the cryopreservation protection solution.

[0046] In some specific embodiments, the step of preheating the solvent to 32-37° C. before dissolving sucrose and hydroxyethyl starch in the solvent to improve dissolution efficiency, preferably 37° C. In some specific embodiments, the step of sterilizing the filter membrane after adding bovine serum albumin is further performed.

[0047] According to another aspect of the present invention, there is also provided the use of the above-mentioned low-temperature cryopreservation protection solution or the low-temperature cryopreservation protection solution prepared by the above-mentioned preparation method in the cryopreservation of koi herpes virus or the preparation of a product for the cryopreservation of koi herpes virus.

[0048] According to another aspect of the present invention, a product for freezing Koi Herpes Virus is provided, comprising the above-mentioned cryopreservation protection solution or the cryopreservation protection solution prepared by the above-mentioned preparation method.

[0049] According to another aspect of the present invention, a method for freezing koi herpes virus is also provided, comprising mixing a low-temperature freezing protection liquid with a virus suspension, pre-cooling the mixture to 4°C, cooling the mixture to -20°C, and then deep-freezing the mixture with liquid nitrogen; the low-temperature freezing protection liquid is the above-mentioned low-temperature freezing protection liquid or the low-temperature freezing protection liquid prepared by the above-mentioned preparation method.

[0050] Use the above-mentioned low-temperature cryopreservation protection solution, pre-cool at 4°C, cool to -20°C, and then enter the two-step gradient freezing method of liquid nitrogen deep freezing, so that the activity of the virus after recovery is ≥90%. The virus can be stably frozen at -20°C, and the shelf life can be extended to 2 years.

[0051] To improve freezing, in some embodiments, the volume ratio of the cryopreservation solution to the virus suspension is 1:1. In some embodiments, the pre-cooling time is 20-40 minutes, preferably 30 minutes. In some embodiments, the cooling rate from 4°C to -20°C is 0.8-1.2°C / min, preferably 1°C / min.

[0052] In some specific embodiments, the liquid nitrogen deep freezing time is ≥2 h, and then the sample can be stored at -20°C or in liquid nitrogen for a long term.

[0053] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0054] 1. Low temperature cryopreservation protection solution for Koi herpes virus

[0055] The present invention provides cryopreservation protective solutions with different component contents. The components and contents of each embodiment and comparative example are shown in Table 1 and Table 2.

[0056] Table 1

[0057]

[0058] Table 2

[0059]

[0060] 2. Freezing Koi Herpes Virus

[0061] Koi herpesvirus: The Koi herpesvirus strain used was isolated from the viscera of infected Koi (freshwater fish). The specific steps are as follows:

[0062] 1. Organization and processing procedures:

[0063] 1) Homogenate preparation: mince the visceral tissues of the collected diseased koi, add them to DMEM medium (1:10, w / v), and grind them thoroughly using a tissue homogenizer.

[0064] 2) Centrifugation and filtration: Centrifuge at 8000 × g for 15 minutes at 4°C. Filter the supernatant through a 0.45 μm filter to remove cell debris.

[0065] 3) Aliquoting and storage: Aliquot the filtrate into sterile cryovials and store at -80°C until use.

[0066] 2. Inoculation and Culture: Inoculate the tissue filtrate onto a monolayer of CCB cells and culture at 25°C. Observe the cytopathic effect (CPE) daily. Amplify the isolated virus, preserve it, and identify it for later use.

[0067] Examples 8 to 14

[0068] The cryopreservation protection solution provided in Examples 1 to 7 was used to freeze the Koi Herpes Virus. The following steps were performed: the cryopreservation protection solution was mixed with the virus suspension in a ratio of 1:1, pre-cooled at 4°C for 30 minutes, cooled to -20°C at a cooling rate of 1°C / min, and then placed in liquid nitrogen for deep freezing for 2 hours, and stored at -20°C for a long time. The virus titer of the virus suspension was 10 6.8 TCID 50 / mL.

[0069] Comparative Examples 8-14

[0070] The difference from Example 8 is that the low-temperature freezing protection solutions provided in Comparative Examples 1 to 7 were used to freeze the Koi Herpes Virus.

[0071] Comparative Example 15

[0072] The low-temperature cryopreservation protection solution of Example 1 was used to freeze the koi herpes virus, and the following steps were performed: the low-temperature cryopreservation protection solution and the virus suspension were mixed in a ratio of 1:1, and the cryopreservation box was directly transferred to a liquid nitrogen tank for ultra-low temperature freezing for 2 hours according to the traditional freezing method. After being frozen solid, it was placed at -20°C for long-term storage.

[0073] Comparative Example 16: Different from Example 8, no low-temperature cryopreservation protection solution was added.

[0074] Experiment 1: Freezing Stability Test

[0075] This experiment uses the virus titer of Koi Herpes Virus samples frozen for 1 day, 7 days, 1 month, 3 months, 6 months, 9 months, 12 months, 15 months, 20 months and 24 months to evaluate their stability. Specifically, the following steps were followed:

[0076] 1. Thawing: Thaw the frozen sample in a 37°C water bath until completely dissolved (about 1-3 minutes), then place it in an ice bath for later use.

[0077] 2. Virus titer detection: The recovered sample was diluted 10-fold in serum-free DMEM medium. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6Six dilutions were inoculated into 96-well cell culture plates with well-grown CCB cell monolayers. Each dilution was repeated in 6 wells, 0.1 ml / well. Six wells of normal cells were also set up as controls. Cultured in a 25°C, 5% CO2 incubator for 7 days. TCID was calculated using the Reed-Muench method based on the number of wells showing cytopathic effects. 50 .

[0078] Table 3

[0079]

[0080] The results are shown in Table 3. The virus titer after resuscitation in Example 8 is much higher than that in Comparative Example 8 and Comparative Example 16. It can be seen that the virus titer after resuscitation of the virus frozen in the low-temperature freezing preservation solution provided in Example 1 is higher than that in Comparative Example 1. The virus titer after resuscitation has not changed significantly after storage for 24 months. The low-temperature freezing preservation solution provided in the example has good stability.

[0081] Experiment 2 Virus activity detection

[0082] Koi herpes virus samples frozen for 1 day and 12 months in Examples 8 to 14 and Comparative Examples 8 to 16 were taken respectively and revived according to the method of Experiment 1. The activity was detected by the virus titer detection method in Experiment 1, and the virus envelope was observed under an electron microscope. The proportion of intact envelopes was counted and the envelope integrity estimation data was obtained. The results are shown in Table 4. Figure 1 As shown, the viral envelope is shown using the microscopic observation images of Example 8 and Comparative Example 8 as examples, wherein A is Example 8, the scale bar is 1 μm, and B is Comparative Example 8, the scale bar is 500 nm.

[0083] Table 4

[0084]

[0085] Experiment 3: Detection of virus survival rate in solvents with different salinity

[0086] The low-temperature cryopreservation protection solution provided in Example 1 was selected and only its salinity was adjusted to obtain low-temperature cryopreservation protection solutions with salinity of 0 to 15. The koi herpes virus was cryopreserved according to the method of Example 8, and the koi herpes virus was tested for viral activity after being frozen for 1 month. The results were as follows: Figure 2 The solvent components corresponding to different salinities are shown in Table 5.

[0087] Table 5

[0088]

[0089] Depend on Figure 2It can be seen that KHV virus activity is best when the salinity is between 7‰ and 12‰. For viruses isolated from marine fish, the salinity of the freezing solution can be appropriately increased.

[0090] Experiment 4 Freeze-thaw cycle tolerance test

[0091] Example 8, Comparative Example 8 and Comparative Example 16 were selected to conduct repeated freeze-thaw experiments, specifically according to the following steps:

[0092] 1. Thawing: Thaw the frozen sample in a 37°C water bath until completely dissolved (about 1-3 minutes), then place it in an ice bath for later use.

[0093] 2. Refreeze: Thawed samples were directly frozen at -20°C. The samples thawed again were called freeze-thawed twice. Similarly, samples frozen and thawed once, twice, three times, five times, and seven times were taken and viral titers were tested according to the procedures in Experiment 1. The experimental results are shown in Table 6.

[0094] Table 6

[0095]

[0096] As shown in Table 6, compared with Comparative Example 8 and Comparative Example 16, it can be seen that the low-temperature cryopreservation protection solution used in Example 8 enables the frozen sample to withstand at least 5 freeze-thaw cycles without affecting its virus titer.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A low-temperature freezing protection solution for Koi herpes virus, characterized in that: It is composed of 2~8w / v% hydroxyethyl starch, 3~5w / v% sucrose and 1~3w / v% bovine serum albumin, with a pH of 7.4; The salinity of the solvent of the low-temperature freezing protection solution is 7-12‰; The solvent consists of 6-12 g / L NaCl, 0.2-0.5 g / L KCl and 0.1-0.3 g / L CaCl2.

2. The cryogenic freezing protection solution according to claim 1, characterized in that The concentration of hydroxyethyl starch is 5w / v%, the concentration of sucrose is 4w / v%, and the concentration of bovine serum albumin is 2w / v%.

3. The method for preparing the cryopreservation protective solution according to claim 1 or 2, characterized in that: The method comprises dissolving sucrose and hydroxyethyl starch in a solvent and adding bovine serum albumin to prepare a low-temperature freezing protection solution.

4. The preparation method according to claim 3, characterized in that Before dissolving the sucrose and hydroxyethyl starch in the solvent, the solvent is preheated to 32-37° C.; The method further includes sterilizing the filter membrane after adding bovine serum albumin.

5. Use of the low-temperature freezing protection solution according to claim 1 or 2 or the low-temperature freezing protection solution prepared by the preparation method according to claim 3 or 4 in freezing Koi herpes virus or in preparing a product for freezing Koi herpes virus.

6. A product for freezing Koi herpes virus, characterized in that: The invention comprises the low-temperature cryopreservation protection solution according to claim 1 or 2 or the low-temperature cryopreservation protection solution prepared by the preparation method according to claim 3 or 4.

7. A method for freezing Koi herpes virus, characterized in that: The process includes mixing the cryopreservation protection solution with the virus suspension, precooling at 4°C, cooling to -20°C and then deep freezing with liquid nitrogen; The low-temperature cryopreservation protection liquid is the low-temperature cryopreservation protection liquid according to claim 1 or 2 or the low-temperature cryopreservation protection liquid prepared by the preparation method according to claim 3 or 4.

8. The cryopreservation method according to claim 7, characterized in that: The volume ratio of the low-temperature freezing protection solution to the virus suspension is 1:1; The precooling time is 20 to 40 minutes; The cooling rate from 4°C to -20°C is 0.8~1.2°C / min; The liquid nitrogen deep freezing time is ≥2h.

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