Shrimp hepatoenterocyte cryoprotectant and its preparation method and use

Abstract

The present invention is a cryoprotectant for shrimp hepatocystis and its preparation method and use, and relates to the technical field of cryopreservation of microsporidia. The cryoprotectant of the present invention is mainly made of glycerol, dimethyl sulfoxide and propylene glycol, with PBS buffer as the base liquid; the volume percentage of the raw materials dimethyl sulfoxide, glycerol, propylene glycol and PBS buffer is 10%:10%:10%:70%. The present invention optimizes the raw materials of the cryoprotectant, so that the obtained cryoprotectant has good effects on the mortality rate of shrimp hepatocystis, the load of infected healthy shrimp, polar tube protein 2 (EHP- ptp 2) Heat shock protein 70 ( hsp 70) has the best effect on indicators such as the impact. The present invention lays a foundation for the indoor storage of shrimp hepatoenteric cysts. It solves the technical problem that there is no cryoprotectant suitable for shrimp hepatoenteric cysts and no storage technology suitable for shrimp hepatoenteric cysts.

Description

Technical Field

[0001] The invention relates to the technical field of cryopreservation of microsporidia, and in particular to a protective agent material for cryopreservation of shrimp hepatoenterocystis, and a preparation method and application thereof. Background Art

[0002] Shrimp Enterocytosis Hepatocellularis EHP, also known as "Intestinal Epithelial Microsporidia", is an intracellular parasitic microsporidia. It mainly parasitizes the epithelial cells of the hepatic tubules of shrimps. It grows and reproduces by absorbing ATP in the host cells. It mainly infects shrimps and is highly contagious. There are no obvious symptoms in the early stage of infection. In the later stage, the shrimps in the group are of different sizes, which leads to slow growth of shrimps, reduced immunity, and easy invasion of other pathogens. They die after cross infection, affecting the production of shrimp farming and causing certain economic losses.

[0003] Since there is no suitable method for preserving EHP in the current microsporidia research laboratory, the experimental materials are mainly fed to healthy shrimp with frozen EHP-positive diseased shrimp, and then extracted from the hepatopancreas of live shrimp. The process is cumbersome and time-consuming, and the activity of spores in frozen diseased shrimp cannot be guaranteed. Therefore, choosing a suitable cryoprotectant can protect the spores from freezing damage, reduce metabolism, and avoid freezing damage to the muscles of diseased shrimp, which will lead to the inactivation of spores, and is expected to achieve long-term storage.

[0004] Therefore, it is of great significance to develop a cryoprotectant suitable for shrimp hepatoenteroides so that the frozen spores can remain active after thawing and removing the cryogen. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a new shrimp hepatoenteric cyst cryoprotectant in view of the deficiencies of the prior art, so as to solve the limiting factors of indoor storage of shrimp hepatoenteric cysts and ensure that the frozen spores remain active after thawing and removing the cryogen.

[0006] Another technical problem to be solved by the present invention is to provide a preparation method and use of the above-mentioned shrimp hepatoenteric cytozoon cryoprotectant.

[0007] The technical problem to be solved by the present invention is achieved through the following technical scheme. The present invention is a shrimp hepatocystis cryoprotectant, which is characterized in that the cryoprotectant is mainly made of glycerol, dimethyl sulfoxide and propylene glycol, with PBS buffer as the base liquid; the volume percentage of the raw materials dimethyl sulfoxide, glycerol, propylene glycol and PBS buffer is 10%: 10%: 10%: 70%.

[0008] A further preferred technical solution of the shrimp hepatoenteric cytozoon cryoprotectant of the present invention is that the freezing temperature of the cryoprotectant is -20°C to -80°C.

[0009] The invention also discloses a method for preparing the shrimp hepatoenteric cytozoon cryoprotectant as described in the above technical scheme, which is characterized by using a pipette to suck raw materials dimethyl sulfoxide, glycerol and propylene glycol into a container according to volume percentage and mixing them evenly to obtain the cryoprotectant.

[0010] The inventors found in experiments that EHP has a strong tolerance to extreme environments and can survive for a period of time in PBS buffer after leaving the host. When the appropriate environmental conditions (temperature, pH, changes in nutrients, light intensity, etc.) are reached, the spores will germinate. Therefore, the inventors have developed a cryoprotectant suitable for shrimp hepatocystis, which allows the frozen spores to remain active after thawing and removing the cryogen.

[0011] The inventors conducted experiments to compare the effects of various mixed reagents of glycerol, dimethyl sulfoxide and propylene glycol at different ratios on the mortality rate of hepatocystis in frozen shrimp, the load of infected healthy shrimp, and the polar tube protein 2 (EHP- ptp 2) Heat shock protein 70 ( hsp 70) to obtain the optimal formula of shrimp hepatoenteroides cryoprotectant.

[0012] Compared with the existing cryoprotectants, the present invention has the following advantages:

[0013] The present invention optimizes the raw materials of the cryoprotectant so that the obtained cryoprotectant has good performance in terms of mortality of shrimp hepatocystis, load of infected healthy shrimp, polar tube protein 2 (EHP- ptp 2) Heat shock protein 70 ( hsp 70) has the best effect on indicators such as impact.

[0014] The invention lays a foundation for the indoor storage of shrimp hepatoenteric cysts, and solves the technical problem that there is no cryoprotectant suitable for shrimp hepatoenteric cysts and no storage technology suitable for shrimp hepatoenteric cysts. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Figure 2 is the mortality rate of EHP spores under two low temperature conditions;

[0016] Figure 2 Figure 2 is the EHP loading diagram under two low temperature conditions;

[0017] Figure 3 This is the mortality graph of spores stored in mixed cryoprotectant group A for 1 month;

[0018] Figure 4 This is a comparison of the number of spores of Litopenaeus spinulosa infected by frozen spores of mixed cryoprotectant group A;

[0019] Figure 5 This is the mortality graph of spores stored in mixed cryoprotectant group B for 1 month;

[0020] Figure 6 This is a comparison of the number of spores of white shrimp infected with frozen spores of mixed cryoprotectant group B;

[0021] Figure 7 This is a graph showing the expression level of EHP spore wall protein 26 (wsp26) gene under mixed cryoprotectant conditions;

[0022] Figure 8 This is a graph of the expression level of the EHP spore polar tube protein 2 (ptp2) gene under mixed cryoprotectant conditions;

[0023] Fig. 9 This is a graph showing the expression level of heat shock protein 70 (hsp70) gene in EHP spores under mixed cryoprotectant conditions. DETAILED DESCRIPTION

[0024] The following examples will provide further illustration of the present invention.

[0025] The experiment was conducted in the Jiangsu Provincial Key Laboratory of Marine Biotechnology, Jiangsu Ocean University, and the experimental materials were obtained from this laboratory.

[0026] 1. Test materials and daily management

[0027] The white shrimps with an average body length of 6.2±0.5 cm, an average body weight of 2.5±0.5 g, intact body surface, and healthy and vigorous were selected. They were intensively infected with Enterocytozoa hepatica (EHP) for 3 days, and the diseased shrimps carrying EHP were fed twice a day (8:00 and 18:00), with the feeding amount of each time being 10% of the number of white shrimps, and metabolic waste and residual bait were removed by siphon.

[0028] 2. Selection of mixing ratio of cryoprotectant raw materials

[0029] Preparation of reagents:

[0030] Group A (mixture of two reagents)

[0031] 1. Dimethyl sulfoxide (10%), glycerol (10%): Use a pipette to pipette 20 μL of dimethyl sulfoxide, 20 μL of glycerol and 160 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0032] 2. Dimethyl sulfoxide (10%), glycerol (20%): Use a pipette to pipette 20 μL of dimethyl sulfoxide, 40 μL of glycerol and 140 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0033] 3. Dimethyl sulfoxide (10%), glycerol (30%): Use a pipette to pipette 20 μL of dimethyl sulfoxide, 60 μL of glycerol and 120 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0034] 4. Dimethyl sulfoxide (20%), glycerol (10%): Use a pipette to pipette 40 μL of dimethyl sulfoxide, 20 μL of glycerol and 140 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0035] 5. Dimethyl sulfoxide (20%), glycerol (20%): Use a pipette to pipette 40 μL of dimethyl sulfoxide, 40 μL of glycerol and 120 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0036] 6. Dimethyl sulfoxide (20%), glycerol (30%): Use a pipette to pipette 40 μL of dimethyl sulfoxide, 60 μL of glycerol and 100 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0037] 7. Dimethyl sulfoxide (30%), glycerol (10%): Use a pipette to pipette 60 μL of dimethyl sulfoxide, 20 μL of glycerol and 120 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0038] 8. Dimethyl sulfoxide (30%), glycerol (20%): Use a pipette to pipette 60 μL of dimethyl sulfoxide, 40 μL of glycerol, and 100 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0039] 9. Dimethyl sulfoxide (30%), glycerol (30%): Use a pipette to pipette 60 μL of dimethyl sulfoxide, 60 μL of glycerol and 80 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0040] 10. Dimethyl sulfoxide (10%), propylene glycol (10%): Use a pipette to pipette 20 μL of dimethyl sulfoxide, 20 μL of propylene glycol, and 160 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0041] 11. Dimethyl sulfoxide (10%), propylene glycol (20%): Use a pipette to pipette 20 μL of dimethyl sulfoxide, 40 μL of propylene glycol, and 140 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0042] 12. Dimethyl sulfoxide (10%), propylene glycol (30%): Use a pipette to pipette 20 μL of dimethyl sulfoxide, 60 μL of propylene glycol and 120 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0043] 13. Dimethyl sulfoxide (20%), propylene glycol (10%): Use a pipette to pipette 40 μL of dimethyl sulfoxide, 20 μL of propylene glycol, and 140 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0044] 14. Dimethyl sulfoxide (20%), propylene glycol (20%): Use a pipette to pipette 40 μL of dimethyl sulfoxide, 40 μL of propylene glycol, and 120 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0045] 15. Dimethyl sulfoxide (20%), propylene glycol (30%): Use a pipette to pipette 40 μL of dimethyl sulfoxide, 60 μL of propylene glycol and 100 μL of PBS buffer into a centrifuge tube and shake to mix evenly.

[0046] 16. Dimethyl sulfoxide (30%), propylene glycol (10%): Use a pipette to pipette 60 μL of dimethyl sulfoxide, 20 μL of propylene glycol, and 120 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0047] 17. Dimethyl sulfoxide (30%), propylene glycol (20%): Use a pipette to pipette 60 μL of dimethyl sulfoxide, 40 μL of propylene glycol and 100 μL of PBS buffer into a centrifuge tube and shake to mix evenly.

[0048] 18. Dimethyl sulfoxide (30%), propylene glycol (30%): Use a pipette to pipette 60 μL of dimethyl sulfoxide, 60 μL of propylene glycol and 80 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0049] 19. Glycerol (10%), propylene glycol (10%): Use a pipette to pipette 20 μL of glycerol, 20 μL of propylene glycol, and 160 μL of PBS buffer into a centrifuge tube and shake to mix evenly.

[0050] 20. Glycerol (10%), propylene glycol (20%): Use a pipette to pipette 20 μL of glycerol, 40 μL of propylene glycol, and 140 μL of PBS buffer into a centrifuge tube and shake to mix evenly.

[0051] 21. Glycerol (10%), propylene glycol (30%): Use a pipette to pipette 20 μL of glycerol, 60 μL of propylene glycol, and 120 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0052] 22. Glycerol (20%), propylene glycol (10%): Use a pipette to pipette 40 μL of glycerol, 20 μL of propylene glycol, and 140 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0053] 23. Glycerol (20%), propylene glycol (20%): Use a pipette to pipette 40 μL of glycerol, 40 μL of propylene glycol, and 120 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0054] 24. Glycerol (20%), propylene glycol (30%): Use a pipette to pipette 40 μL of glycerol, 60 μL of propylene glycol, and 100 μL of PBS buffer into a centrifuge tube and shake to mix evenly.

[0055] 25. Glycerol (30%), propylene glycol (10%): Use a pipette to pipette 60 μL of glycerol, 20 μL of propylene glycol, and 120 μL of PBS buffer into a centrifuge tube and shake to mix evenly.

[0056] 26. Glycerol (30%), propylene glycol (20%): Use a pipette to pipette 60 μL of glycerol, 40 μL of propylene glycol, and 100 μL of PBS buffer into a centrifuge tube and shake to mix evenly.

[0057] 27. Glycerol (30%), propylene glycol (30%): Use a pipette to pipette 60 μL of glycerol, 60 μL of propylene glycol, and 80 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0058] Group B (mixture of three reagents)

[0059] 1. Dimethyl sulfoxide (10%), glycerol (10%), propylene glycol (10%): Use a pipette to pipette 20 μL of dimethyl sulfoxide, 20 μL of glycerol, 20 μL of propylene glycol and 140 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0060] 2. Dimethyl sulfoxide (10%), glycerol (20%), propylene glycol (30%): Use a pipette to pipette 20 μL of dimethyl sulfoxide, 40 μL of glycerol, 60 μL of propylene glycol and 80 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0061] 3. Dimethyl sulfoxide (10%), glycerol (30%), propylene glycol (20%): Use a pipette to pipette 20 μL of dimethyl sulfoxide, 60 μL of glycerol, 40 μL of propylene glycol and 80 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0062] 4. Dimethyl sulfoxide (20%), glycerol (10%), propylene glycol (20%): Use a pipette to pipette 40 μL of dimethyl sulfoxide, 20 μL of glycerol, 40 μL of propylene glycol and 100 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0063] 5. Dimethyl sulfoxide (20%), glycerol (20%), propylene glycol (10%): Use a pipette to pipette 40 μL of dimethyl sulfoxide, 40 μL of glycerol, 20 μL of propylene glycol and 100 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0064] 6. Dimethyl sulfoxide (20%), glycerol (30%), propylene glycol (30%): Use a pipette to pipette 40 μL of dimethyl sulfoxide, 60 μL of glycerol, 60 μL of propylene glycol and 40 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0065] 7. Dimethyl sulfoxide (30%), glycerol (10%), propylene glycol (30%): Use a pipette to pipette 60 μL of dimethyl sulfoxide, 20 μL of glycerol, 60 μL of propylene glycol and 60 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0066] 8. Dimethyl sulfoxide (30%), glycerol (20%), propylene glycol (20%): Use a pipette to pipette 60 μL of dimethyl sulfoxide, 40 μL of glycerol, 40 μL of propylene glycol and 60 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0067] 9. Dimethyl sulfoxide (30%), glycerol (30%), propylene glycol (10%): Use a pipette to pipette 60 μL of dimethyl sulfoxide, 60 μL of glycerol, 20 μL of propylene glycol and 60 μL of PBS buffer into a centrifuge tube and vortex to mix evenly.

[0068] The above-prepared reagents were stored frozen for 30 days.

[0069] 3. Removal of cryopreservation solution

[0070] At room temperature, some cryoprotectants such as DMSO and glycerol are toxic to cells at high concentrations. After the sample is thawed, the cryoprotectant should be removed by multiple low-temperature centrifugation.

[0071] After adding 500 μL of sterile water to the thawed sample, centrifuge at 6000 rpm for 5 min. Discard the supernatant, add 500 μL of sterile water to the pellet, and vortex gently to suspend the spores. Repeat the centrifugation 3 to 4 times, and finally add 200 μL of sterile water to the pellet and suspend the spores.

[0072] Fluorescence detection and counting by light microscopy using DAPI, spore load test after infection, polar canal protein 2 (EHP- ptp 2) Spore wall protein 26 ( wsp 26) and heat shock protein 70 ( hsp 70) to determine the optimal mixed reagent formula for gene quantification.

[0073] Statistical analysis: The best formula in the above-mentioned groups A and B was screened out using analysis of variance and multiple comparisons.

[0074] 4. Experimental data and results.

[0075] Four chemical reagents and the commonly used basal liquid PBS buffer were used for spore freezing and infection experiments, and PBS buffer was used as the control group. Each reagent was prepared at a concentration of 10%-30%, and the experiments were conducted at -20℃ and -80℃ for 30 days.

[0076] 4.1. Figure 1 It can be seen that under the two low temperature conditions, the mortality rate at -20℃ is mostly lower than that at -80℃; the average mortality rate reached 59.835%. Under the condition of -80℃, the mortality rate of the control group (CG) was as high as 98%, and the mortality rate of the control group under the condition of -20℃ reached 72.22%; the mortality rate of spores preserved by 10%-30% DMSO (dimethyl sulfoxide) and G (glycerol) reagents at -20℃ was lower than that of 10%-30% DMSO and G at -80℃; among them, the mortality rate of spores preserved by EG (ethylene glycol) at -20℃ was higher than that of DMSO, G and PG (propylene glycol). The survival rate of spores preserved by 10%-30% PBS buffer under two low temperature conditions was not ideal, and the mortality rate was above 62.5%.

[0077] Comparison of Figure 2 shows that the infection capacity of spores stored at -20°C is significantly higher than that of spores stored at -80°C, with an average load of 10 6 (copies / mg). The infection ability of spores stored in 10%-30% EG and PBS buffer was poor, and the load after infecting healthy shrimp was 1.9×10 6 Below; -20℃10%DMSO (5.4×10 6 ) is significantly different from other groups of reagents ( P< 0.001). At the same time, 10%PG, 20%PG, 30%PG, 20%EG, 30%EG, 20%DMSO, 30%DMSO, 10%G, 20%G, 30%G, 10%PBS, 20%PBS, 30%PBS and the control group had significant or extremely significant differences under both low temperature conditions ( P< 0.01, P< 0.001), the loading capacity of DMSO, G, and PG groups was better.

[0078] From the above results, the temperature condition in the low-temperature freezing preservation experiment was selected as -20°C, and the three chemical reagents DMSO, G, and PG were selected as the mixed reagents.

[0079] 4.2 Experimental results on the effect of mixed cryoprotectant group A on the activity of EHP spores

[0080] Depend on Figure 3It can be seen that after the EHP spores were frozen at -20℃ for one month, the mixed groups of different cryoprotectants had different effects and significantly affected the spore mortality rate. The mortality rate of the control group was as high as 85.26%, and the average mortality rate of all groups reached 51.39%, which was lower than the mortality rate of spores preserved by a single cryoprotectant. Among them, the highest mortality rate was in the reagent group No. 6 (20% DMSO + 30% G), with a mortality rate of 77.94%, and the lowest was in the reagent group No. 24 (20% G + 30% PG), with a mortality rate of 31.24%; the mortality rate of the reagent group No. 2 (10% DMSO + 20% G) was 35.71%, which was close to the mortality rate of the reagent group No. 24, and the preservation effect was ideal.

[0081] Depend on Figure 4 It can be seen that the mixed groups of different cryoprotectants significantly affect the load of frozen spores after infection. After EHP spores infect white shrimp, there are statistical differences in the spore infection level after freezing and mixing with different reagents ( P< 0.05), the average loading was 10 7 (copies / mg). The spores in the control group infected healthy shrimp for 3 days, and the load was 4.76×10 5 (copies / mg); after 3 days of infection with 20% DMSO + 20% G, the EHP load reached 1.6439×10 8 (copies / mg), which was significantly higher than that of the other groups ( P< 0.001).

[0082] 4.3 Experimental results on the effect of mixed cryoprotectant group B on the activity of EHP spores

[0083] Depend on Figure 5 It can be calculated that the average mortality rate is 36.67%, which is lower than the 59.835% of the single agent group and the 51.39% of the mixed agent A group. The mortality rate of the control group reached 78.78%, which is significantly higher than that of the other groups ( P <0.01); the mortality rate of reagent No. 2 (10% DMSO + 20% G + 30% PG) was the lowest compared with the other 8 groups of mixed reagents, with a mortality rate of 25.81%; the mortality rate of reagent No. 1 (10% DMSO + 10% G + 10% PG) was 32.31%, the mortality rate of reagent No. 8 (30% DMSO + 20% G + 20% PG) was 32.17%, and the mortality rate of reagent No. 9 (30% DMSO + 30% G + 10% PG) was 32.81%. The mortality rates of these three mixed reagents were relatively low; the mortality rate of reagent No. 6 (20% DMSO + 30% G + 30% PG) was the highest, at 45.63%. At the same time, the mortality rates of the 9 mixed reagents were all lower than 45%, which shows that the freezing effect of the mixed reagent is better than that of a single cryoprotectant and a mixture of two cryoprotectants in terms of mortality.

[0084] Depend on Figure 6 The average load was calculated to be 10 8 (copies / mg), significantly higher than the 10 6 (copies / mg) and 10 of mixed reagent A 7 (copies / mg). The load of the control group was 5.3×10 5 , which was significantly lower than the other groups ( P <0.001); the spores stored in reagent group 1 (10% DMSO + 10% G + 10% PG) had the highest load after infecting healthy shrimp, which was 4.85×10 8 , which is significantly different from the other 8 mixed reagents ( P <0.01); the spore load of reagent group 4 (20% DMSO + 10% G + 20% PG) after infecting healthy shrimp was 3.84×10 8 The spores stored in reagent group 7 (30% DMSO + 10% G + 30% PG) had the lowest load after infecting healthy shrimp, which was 2.95×10 7 , which is significantly different from the other 8 mixed reagents ( P <0.01).

[0085] In summary, the effect of preserving spores is optimal when the three cryoprotectants are mixed at -20°C. Therefore, in order to further explore the optimal combination of the three reagents, this experiment was conducted to detect the shrimp hepatocystis-related vitality genes ( wsp 26. ptp 2) and stress protein genes ( hsp 70) related tests.

[0086] Depend on Figure 7 It can be seen that the spore wall protein 26 ( wsp 26) The gene expression level shows that the spores preserved by mixed reagent No. 1 wsp The expression level of 26 genes was the highest, which was significantly different from the other 8 groups of reagents ( P <0.01); frozen spores preserved with mixed reagent No. 2 and mixed reagent No. 3 wsp 26 Gene expression levels were relatively high; frozen spores preserved with mixed reagent No. 7 wsp The expression level of gene 26 was the lowest; the frozen spores preserved by mixed reagents No. 6, No. 8, and No. 9 wsp The expression levels of 26 genes were relatively low; the control group was only used as a reference and was not included in the experimental group for comparison.

[0087] Depend on Figure 8It can be seen that the polar tube protein 2 ( ptp 2) The gene expression level shows that the spores preserved by mixed reagent No. 1 ptp 2 gene expression level was the highest, with significant differences compared to the other 8 groups of reagents ( P <0.01); frozen spores preserved with mixed reagents No. 3, No. 4, and No. 5 ptp 2 The gene expression level is relatively high; the frozen spores preserved by mixed reagent No. 6 ptp 2 gene expression level was the lowest; frozen spores preserved by mixed reagents No. 7, No. 8, and No. 9 ptp 2 The gene expression level was relatively low; the control group was only used as a reference and was not included in the experimental group for comparison.

[0088] Depend on Fig. 9 It can be seen that the heat shock protein 70 ( hsp 70) The expression level of the gene can be seen that the spores preserved by mixed reagent No. 5 hsp The expression level of 70 genes was the lowest, which was significantly different from the other 8 groups of reagents ( P <0.01); frozen spores preserved by mixed reagents No. 1, No. 2, and No. 4 hsp The expression level of 70 genes was relatively low, and the frozen spores preserved by mixed reagents No. 1, No. 2, and No. 4 hsp There was no significant difference in the expression levels of 70 genes ( P >0.05); frozen spores preserved with mixed reagent No. 6 hsp The expression level of 70 genes was the highest; the frozen spores preserved by mixed reagent No. 7 hsp The expression levels of 70 genes were relatively high; the control group was only used as a reference and was not included in the experimental group for comparison.

[0089] Based on the above experimental results, the mortality rate is lower in mixed reagents No. 1, No. 2, No. 8, and No. 9; the loading capacity is higher in mixed reagents No. 1 and No. 4; frozen spores wsp The expression levels of 26 genes were higher in mixed reagents No. 1, No. 2, and No. 3; frozen spores ptp 2 The gene expression levels were higher in mixed reagents No. 1, No. 3, No. 4, and No. 5; frozen spores hsp The mixed reagents No. 1, No. 2, No. 4, and No. 5 had lower expression levels of 70 genes.

[0090] Based on the above experimental results, it was concluded that the first group of mixed reagents in Group B, namely 10% dimethyl sulfoxide plus 10% glycerol plus 10% propylene glycol, had the best results.

Claims

1. A shrimp hepato-enterocystis cryoprotectant, characterized in that: The cryoprotectant is mainly made of raw materials glycerol, dimethyl sulfoxide and propylene glycol, and uses PBS buffer as a base liquid; the volume percentages of the raw materials dimethyl sulfoxide, glycerol, propylene glycol and PBS buffer are 10%:10%:10%:70%.

2. The shrimp hepato-enteric cytozoon cryoprotectant according to claim 1, characterized in that: The freezing temperature of the cryoprotectant is -20°C to -80°C.

3. A method for preparing a shrimp hepato-enteric cytozoon cryoprotectant as claimed in claim 1 or 2, characterized in that: Use a pipette to draw the raw materials dimethyl sulfoxide, glycerol and propylene glycol into a container according to volume percentage, and mix them evenly.

4. A use of the shrimp hepato-enteric cytozoon cryoprotectant as claimed in claim 1, characterized in that: The application is to use the cryoprotectant as a cryoprotectant for shrimp hepatoenteric cysts.

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