Low-temperature cryopreservation protection liquid for koi herpesvirus as well as preparation method and application of low-temperature cryopreservation protection liquid

Abstract

The invention provides a low-temperature cryopreservation protective solution for koi herpesvirus as well as a preparation method and application thereof, and relates to the technical field of virus preservation, the low-temperature cryopreservation protective solution comprises 2-8w / v% of hydroxyethyl starch, 3-5w / v% of sucrose and 1-3w / v% of bovine serum albumin, the activity of the koi herpesvirus after recovery is greater than or equal to 90%, the koi herpesvirus can tolerate five freeze-thaw cycles, the koi herpesvirus can be stably frozen at-20 DEG C, and the storage life is prolonged to 2 years. The technical problems that in the prior art, cryopreservation liquid needs to be stored under the ultralow temperature condition, osmotic pressure is unbalanced, virus envelopes are broken, and the activity is low after cryopreservation recovery are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of virus preservation, and particularly relates to a cryopreservation protective solution for koi herpesvirus, a preparation method thereof, and an application thereof. Background Art

[0002] Koi herpesvirus (KHV) belongs to the genus Herpesviridae ), the family Ictaluridae Alloherpesviridae ), also known as Cyprinid herpesvirus 3 (CyHV-3); the virus particles are spherical, with a diameter of about 170-230 nm, having a double-layer lipid envelope, and the genome is linear double-stranded DNA (~295 kbp), encoding more than 150 proteins. Its high pathogenicity and infectivity have caused huge losses to the breeding of common carp and koi at home and abroad. The inactivated vaccine has poor effects and is difficult to industrialize. The live vaccine has significant effects and can achieve large-scale immersion immunization, but its industrialization has extremely high requirements for the preservation of live virus.

[0003] At present, the cryopreservation of KHV mostly adopts a general formula based on fetal bovine serum, dimethyl sulfoxide or glycerol, and basically needs to be stored under ultra-low temperature conditions of -80 °C. Due to the large difference in the osmotic pressure of fresh water or seawater fish body fluids, it is difficult for existing buffers (such as PBS) to adapt, resulting in osmotic pressure imbalance; and traditional gradient cooling easily leads to the rupture of the virus envelope. Data shows that the activity of KHV after cryopreservation is <50%.

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

[0005] One of the purposes of the present invention is to provide a cryopreservation protective solution for koi herpesvirus, so as to solve the technical problems in the prior art that the cryopreservation solution needs to be stored under ultra-low temperature conditions, and there are problems of osmotic pressure imbalance, rupture of the virus envelope, and low activity after cryopreservation and recovery.

[0006] Another purpose of the present invention is to provide a preparation method of the above cryopreservation protective solution.

[0007] Another purpose of the present invention is to provide an application of the above cryopreservation protective solution.

[0008] Another purpose of the present invention is to provide a product for cryopreserving koi herpesvirus.

[0009] Another purpose of the present invention is to provide a cryopreservation method for koi herpesvirus.

[0010] In order to achieve the above purposes of the present invention, the following technical solutions are specifically adopted: In a first aspect, the present invention provides a cryopreservation protective solution for koi herpesvirus, comprising 2-8 w / v% hydroxyethyl starch, 3-5 w / v% sucrose, and 1-3 w / v% bovine serum albumin.

[0011] Further, 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%.

[0012] Further, the pH is 7.0-8.0.

[0013] Further, the salinity of the solvent of the cryopreservation protective solution is 7-12‰; The solvent comprises 6-12 g / L of NaCl, 0.2-0.5 g / L of KCl, and 0.1-0.3 g / L of CaCl2.

[0014] In a second aspect, the present invention provides a method for preparing the above cryopreservation protective solution, comprising dissolving sucrose and hydroxyethyl starch in a solvent, and adding bovine serum albumin to obtain the cryopreservation protective solution.

[0015] Further, before dissolving sucrose and hydroxyethyl starch in the solvent, the solvent is preheated to 32-37°C; After adding bovine serum albumin, membrane filtration sterilization is further included.

[0016] In a third aspect, the present invention provides an application of the above cryopreservation protective solution or the cryopreservation protective solution prepared by the above preparation method in cryopreserving koi herpesvirus or preparing a product for cryopreserving koi herpesvirus.

[0017] In a fourth aspect, the present invention provides a product for cryopreserving koi herpesvirus, comprising the above cryopreservation protective solution or the cryopreservation protective solution prepared by the above preparation method.

[0018] In a fifth aspect, the present invention provides a method for cryopreserving koi herpesvirus, comprising mixing the cryopreservation protective solution with a virus suspension, pre-cooling at 4°C, cooling to -20°C, and then deep-freezing in liquid nitrogen; The cryopreservation protective solution is the above cryopreservation protective solution or the cryopreservation protective solution prepared by the above preparation method.

[0019] Further, the volume ratio of the cryopreservation protective solution to the virus suspension is 1:1; The pre-cooling time is 20-40 min; The cooling rate from 4°C to -20°C is 0.8-1.2°C / min; The time of deep-freezing in liquid nitrogen is ≥2 h.

[0020] A cryopreservation protective solution for koi herpesvirus provided by the present invention combines hydroxyethyl starch and low-concentration sucrose to reduce ice crystal formation and stabilize the virus envelope, preventing the rupture of the virus envelope. Experiments have shown that compared with traditional formulations, the koi herpesvirus preserved in the cryopreservation protective solution provided by the present invention has an activity ≥ 90% after resuscitation, can withstand 5 freeze-thaw cycles, can be stably cryopreserved at -20 °C, and the preservation period is extended to 2 years. This solves the technical problems in the prior art that the cryopreservation solution needs to be stored under ultra-low temperature conditions, there are problems of osmotic pressure imbalance and virus envelope rupture, and the activity after cryopreservation and resuscitation is low. BRIEF DESCRIPTION OF THE DRAWINGS In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0021] Figure 1 It is a comparative electron microscopy observation diagram of the protection of the virus envelope by different cryopreservation protective solutions provided in Experiment 2 of the present invention; Figure 2 It is an influence curve of cryopreservation protective solutions with different salinities on the virus survival rate provided in Experiment 3 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] 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 meanings and scopes of the terms should be clear. However, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or extrinsic definition. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms is non-restrictive.

[0023] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0024] On the one hand, the present invention provides a cryopreservation protective solution for koi herpesvirus, which includes 2 - 8 w / v% hydroxyethyl starch, 3 - 5 w / v% sucrose, and 1 - 3 w / v% bovine serum albumin.

[0025] Combining hydroxyethyl starch and low-concentration sucrose reduces ice crystal formation, stabilizes the virus envelope, and prevents the virus envelope from rupturing. Experiments have shown that compared with traditional formulations, the koi herpesvirus preserved in the cryopreservation solution provided by the present invention has an activity ≥90% after resuscitation, can withstand 5 freeze-thaw cycles, can be stably stored at -20°C, and the storage period is extended to 2 years. This solves the technical problems in the prior art that the cryopreservation solution needs to be stored under ultra-low temperature conditions, there are problems of osmotic pressure imbalance and virus envelope rupture, and the activity after cryopreservation and resuscitation is low.

[0026] Among them, the concentration of the hydroxyethyl starch can 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 can also be any value between 2 and 8 w / v%, preferably 5 w / v%.

[0027] The concentration of the sucrose can 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 can also be any value between 3 and 5 w / v%, preferably 4 w / v%.

[0028] 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%.

[0029] In order to further improve the effect of the cryopreservation solution, in some specific embodiments, it includes hydroxyethyl starch at a concentration of 5 w / v%, sucrose at a concentration of 4 w / v%, and bovine serum albumin at a concentration of 2 w / v%.

[0030] In some specific embodiments, the pH is 7.0 - 8.0, preferably 7.4.

[0031] 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 and avoid osmotic pressure imbalance. In some specific embodiments, 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; Among them, the salinity of the solvent can be, but is not limited to, 7‰, 7.5‰, 8‰, 8.5‰, 9‰, 9.5‰, 10‰, 10.5‰, 11‰, 11.5‰ or 12‰, and can also be any value between 7 and 12‰. Specifically, it can be adjusted according to freshwater fish viruses or seawater fish viruses.

[0032] The concentration of the NaCl can 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, and can also be any value between 6 and 12 g / L.

[0033] The concentration of the KCl can 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, and can also be any value between 0.2 and 0.5 g / L.

[0034] The concentration of the 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.

[0035] According to another aspect of the present invention, there is also provided a method for preparing the above cryopreservation protective solution, which includes dissolving sucrose and hydroxyethyl starch in a solvent, and adding bovine serum albumin to obtain the cryopreservation protective solution.

[0036] In some specific embodiments, before dissolving the sucrose and hydroxyethyl starch in the solvent, the solvent is preheated to 32 - 37 °C to improve its dissolution efficiency, preferably 37 °C. In some specific embodiments, after adding the bovine serum albumin, membrane filtration sterilization is also included.

[0037] According to another aspect of the present invention, there is also provided the application of the above cryopreservation protective solution or the cryopreservation protective solution prepared by the above preparation method in the cryopreservation of koi herpesvirus or the preparation of products for the cryopreservation of koi herpesvirus.

[0038] According to another aspect of the present invention, there is also provided a product for the cryopreservation of koi herpesvirus, which includes the above cryopreservation protective solution or the cryopreservation protective solution prepared by the above preparation method.

[0039] According to another aspect of the present invention, there is also provided a method for cryopreserving koi herpesvirus, which includes mixing the cryopreservation protective solution with the virus suspension, pre-cooling at 4 °C, and then deep-freezing in liquid nitrogen after cooling to -20 °C; the cryopreservation protective solution is the above cryopreservation protective solution or the cryopreservation protective solution prepared by the above preparation method.

[0040] Using the above cryopreservation protective solution and combining with the two-step gradient cryopreservation method of pre-cooling at 4 °C and then deep-freezing in liquid nitrogen after cooling to -20 °C, the activity of the virus after resuscitation is ≥90%, the virus can be stably cryopreserved at -20 °C, and the storage period is extended to 2 years.

[0041] To improve the cryopreservation effect, in some specific embodiments, the volume ratio of the cryopreservation solution to the virus suspension is 1:1. In some specific embodiments, the pre-cooling time is 20 - 40 min, preferably 30 min. In some specific embodiments, the cooling rate from 4°C to -20°C is 0.8 - 1.2°C / min, preferably 1°C / min.

[0042] In some specific embodiments, the time for deep freezing in liquid nitrogen is ≥2 h, and then it can be stored at -20°C or in liquid nitrogen for a long time.

[0043] The present invention will be further described below through examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0044] I. Cryopreservation solution for koi herpesvirus The present invention provides cryopreservation solutions with different component contents. The components and contents of each example and comparative example are shown in Tables 1 and 2.

[0045] Table 1

[0046] Table 2

[0047] II. Cryopreservation of koi herpesvirus Koi herpesvirus: The used koi herpesvirus strain was isolated from the internal organs of diseased koi (freshwater fish). The specific operation steps are as follows: 1. Tissue processing process: 1) Homogenate preparation: The internal organ tissues of the collected diseased koi were minced and added to DMEM medium (1:10, w / v), and then thoroughly ground using a tissue homogenizer.

[0048] 2) Centrifugation and filtration: Centrifuge at 8000×g for 15 minutes at 4°C, and take the supernatant and filter it through a 0.45 μm filter membrane to remove cell debris.

[0049] 3) Sub-packaging and storage: The filtrate was sub-packaged into sterile cryotubes and stored at -80°C for later use.

[0050] 2. Inoculation and culture: Inoculate the tissue filtrate into monolayer CCB cells and culture at 25°C. Observe the cytopathic effect (CPE) every day. The isolated virus was amplified, preserved, and identified for later use.

[0051] Examples 8 - 14 Cryopreserve koi herpesvirus using the cryopreservation protective solutions provided in Examples 1 to 7 respectively, and the specific operation is as follows: Mix the cryopreservation protective solution and the virus suspension at a ratio of 1:1, pre-cool at 4°C for 30 min, cool down to -20°C at a rate of 1°C / min, then place it in liquid nitrogen for deep freezing for 2 h, and store it at -20°C for long-term preservation. Among them, the virus titer of the virus suspension is 10 6.8 TCID 50 / mL.

[0052] Comparative Examples 8 to 14 Different from Example 8, cryopreserve koi herpesvirus using the cryopreservation protective solutions provided in Comparative Examples 1 to 7 respectively.

[0053] Comparative Example 15 Select the cryopreservation protective solution of Example 1 to cryopreserve koi herpesvirus, and the specific operation is as follows: Mix the cryopreservation protective solution and the virus suspension at a ratio of 1:1, and according to the traditional cryopreservation method, directly transfer the cryopreservation box to the ultra-low temperature of the liquid nitrogen tank for freezing for 2 h. After freezing solid, store it at -20°C for long-term preservation.

[0054] Comparative Example 16: Different from Example 8, no cryopreservation protective solution is added.

[0055] Experiment 1 Detection of cryopreservation stability In this experiment, the virus titers of the koi herpesvirus samples cryopreserved in Example 8, Comparative Example 8, and Comparative Example 16 for 1 day, 7 days, 1 month, 3 months, 6 months, 9 months, 12 months, 15 months, 20 months, and 24 months were used to evaluate their stability. The specific operation is as follows: 1. Thawing: Thaw the cryopreserved sample in a 37°C water bath until completely dissolved (about 1 - 3 min), and then insert it into an ice bath for standby.

[0056] 2. Virus titer detection: Serially dilute the thawed sample 10-fold with serum-free DMEM medium. Take 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 and 10 -6 A total of 6 dilution degrees, inoculate them into a 96-well cell culture plate with a well-grown monolayer of CCB cells, repeat 6 wells for each dilution degree, 0.1 ml / well, and at the same time set up 6 wells of normal cell control. Incubate in a 25°C, 5% CO2 incubator for 7 days, and calculate the TCID 50 .

[0057] Table 3

[0058] 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 cryopreserved with the cryopreservation solution provided in Example 1 is higher than that in Comparative Example 1. After being stored for 24 months, the virus titer after resuscitation has not changed significantly. The cryopreservation solution provided in the example has good stability.

[0059] Experiment 2 Virus Activity Detection Take the koi herpesvirus samples cryopreserved for 1 day and 12 months in Examples 8 - 14 and Comparative Examples 8 - 16 respectively, resuscitate them according to the method of Experiment 1, detect their activities by the method of virus titer detection in Experiment 1, and observe the virus envelope under an electron microscope, take pictures and count the proportion of the complete envelope to obtain the estimated data of its envelope integrity. The results are shown in Table 4, as Figure 1 shown. Taking the microscopic observation pictures of Example 8 and Comparative Example 8 as an example to show the virus envelope. Among them, A is Example 8, the scale bar is 1μm, and B is Comparative Example 8, the scale bar is 500nm.

[0060] Table 4

[0061] Experiment 3 Detection of Virus Survival Rate in Different Salinity Solvents Select the cryopreservation solution provided in Example 1 and only adjust its salinity to obtain cryopreservation solutions with salinities of 0 - 15 respectively. Cryopreserve the koi herpesvirus according to the method of Example 8, and take the koi herpesvirus cryopreserved for 1 month to detect the virus activity. The results are as Figure 2 shown. Among them, the solvent components corresponding to different salinities are shown in Table 5.

[0062] Table 5

[0063] It can be Figure 2 seen that when the salinity is 7 - 12‰, the KHV virus has the best activity. For the virus isolated from marine fish, the salinity of the cryopreservation solution can be appropriately increased.

[0064] Experiment 4 Experiment on the Number of Freeze - Thaw Cycles Tolerance Select Example 8, Comparative Example 8 and Comparative Example 16 to conduct repeated freeze - thaw experiments, and the specific operations are as follows: 1. Resuscitation: After thawing the cryopreserved sample in a 37°C water bath until it is completely dissolved (about 1 - 3 min), insert it into an ice bath for later use.

[0065] 2. Re-freezing: After directly freezing the thawed sample at -20°C, the sample after re-thawing is called the sample that has been freeze-thawed twice. And so on. Samples that have been freeze-thawed once, twice, three times, five times, and seven times are taken respectively, and the virus titer is detected according to the operation of Experiment 1. The experimental results are shown in Table 6.

[0066] Table 6

[0067] As shown in Table 6, compared with Comparative Example 8 and Comparative Example 16, it can be seen that the cryopreservation protective solution used in Example 8 enables the frozen sample to withstand at least 5 freeze-thaw cycles without affecting its virus titer.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions 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 includes 2~8w / v% hydroxyethyl starch, 3~5w / v% sucrose and 1~3w / v% bovine serum albumin.

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 cryogenic freezing protection solution according to claim 1, characterized in that: The pH is 7.0~8.

0.

4. The cryopreservation protection solution according to any one of claims 1 to 3, characterized in that: The salinity of the solvent of the low temperature freezing protection solution is 7-12‰; The solvent includes 6-12 g / L NaCl, 0.2-0.5 g / L KCl and 0.1-0.3 g / L CaCl2.

5. The method for preparing the low temperature freezing protection solution according to any one of claims 1 to 4, characterized in that: The method comprises dissolving sucrose and hydroxyethyl starch in a solvent and adding bovine serum albumin to prepare a low-temperature cryopreservation protection solution.

6. The preparation method according to claim 5, characterized in that: Before dissolving sucrose and hydroxyethyl starch in the solvent, the step further includes preheating the solvent to 32-37° C.; The method further comprises sterilizing the filter membrane after adding bovine serum albumin.

7. Use of the low-temperature freezing protection solution described in any one of claims 1 to 4 or the low-temperature freezing protection solution prepared by the preparation method described in claim 5 or 6 in freezing Koi herpes virus or in preparing products for freezing Koi herpes virus.

8. A product for freezing Koi herpes virus, characterized in that: The invention comprises the low temperature cryopreservation protection solution as described in any one of claims 1 to 4 or the low temperature cryopreservation protection solution prepared by the preparation method as described in claim 5 or 6.

9. A method for freezing Koi herpes virus, characterized in that: The process includes mixing a cryopreservation protection solution with a 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 any one of claims 1 to 4 or the low-temperature cryopreservation protection liquid prepared by the preparation method according to claim 5 or 6.

10. The cryopreservation method according to claim 9, 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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