Cryoprotectant for enterocytozoon hepatopenaei, and preparation method therefor and use thereof

Abstract

The present invention relates to the technical field of Microsporidia cryopreservation. Provided is a cryoprotectant for Enterocytozoon hepatopenaei. The cryoprotectant is mainly prepared from glycerol, dimethyl sulfoxide, and propylene glycol, with PBS buffer as a base solution. A volume percentage ratio of the raw materials dimethyl sulfoxide, glycerol, propylene glycol and PBS buffer is 10%:10%:10%:70%. The raw materials of the cryoprotectant are preferred, such that the obtained cryoprotectant achieves the optimal effect in indicators such as the mortality rate of Enterocytozoon hepatopenaei, the infection load in healthy shrimps, and the influence on polar tube protein 2 (EHP-ptp2) and heat shock protein 70 (hsp70). The present invention lays the foundation for achieving indoor preservation of Enterocytozoon hepatopenaei. The present invention solves the technical problem in the prior art that no cryoprotectant or preservation technique suitable for Enterocytozoon hepatopenaei is currently available.

Description

Freeze protective agent for hepatopanosoma sp. and preparation method and use thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of microsporidia cryopreservation, in particular to a freeze protective agent for hepatopanosoma sp. and a preparation method and use thereof. BACKGROUND

[0002] Hepatopanosoma sp. (EHP), also known as enterocyte epithelial microsporidium, is a kind of intracellular parasitic microsporidium, which mainly parasitizes in the liver small tube epithelial cells of shrimps, grows and reproduces by absorbing ATP in the host cells, mainly infects prawns, and has high infectivity. There is no obvious symptom in the early stage of infection, and in the late stage, the size of the shrimps in the group is different, which leads to slow growth of prawns, reduces the immunity, and is easy to be invaded by other pathogens, and after cross infection, the prawns die, which affects the yield of shrimp culture and causes certain economic loss.

[0003] At present, there is no suitable method for storing EHP in the laboratory for studying microsporidium, so the experiment is mainly carried out by feeding healthy shrimps with frozen EHP positive sick shrimps, and then extracting from the live shrimp hepatopancreas. The process is complicated and time-consuming, and the activity of the spores in the frozen sick shrimps cannot be guaranteed, so selecting a suitable freeze protective agent can protect the spores from freezing damage, reduce metabolism, avoid freezing damage to the muscles of the sick shrimps, and thus prevent the inactivation of the spores, which is expected to realize long-term storage.

[0004] Therefore, it is of great significance to develop a freeze protective agent suitable for hepatopanosoma sp. so that the spores after freezing and thawing still maintain activity after removing the freeze protective agent. SUMMARY

[0005] The present application solves the technical problems of the prior art by providing a new freeze protective agent for hepatopanosoma sp. to solve the limiting factors of indoor storage of hepatopanosoma sp. and to maintain the activity of the spores after freezing and thawing and removing the freeze protective agent.

[0006] The present application solves another technical problem by providing a preparation method and use of the aforementioned freeze protective agent for hepatopanosoma sp.

[0007] The present application solves the technical problems by the following technical solutions. The present application is a freeze protective agent for hepatopanosoma sp., which is characterized in that: the freeze protective agent is mainly made of glycerol, dimethyl sulfoxide and propylene glycol, and PBS buffer is used 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] The freeze protective agent for hepatopanosoma sp. according to the present application has a further preferred technical solution, in which the freezing temperature of the freeze protective agent is-20℃ to-80℃.

[0009] The application also discloses a preparation method of the hepatopancreatic ciliate of shrimps.

[0010] The inventors found in experiments that the EHP has strong tolerance to extreme environment, and can survive for a period of time in PBS buffer after leaving the host. When reaching suitable environmental conditions (temperature, pH, nutrient changes, light intensity, etc.), the spores will germinate. Therefore, the inventors have researched a cryoprotectant suitable for the hepatopancreatic ciliate of shrimps, so that the frozen spores still maintain activity after thawing and removing the cryoprotectant.

[0011] The inventors compare the effects of glycerol, dimethyl sulfoxide and propylene glycol in different proportions on the mortality of the hepatopancreatic ciliate of shrimps after freezing, the load of healthy shrimps infected, the effects of EHP-ptp2 and hsp70, so as to obtain the hepatopancreatic ciliate of shrimps cryoprotectant with the optimal formula.

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

[0013] The application optimizes the raw materials of the cryoprotectant, so that the obtained cryoprotectant has the optimal effect on the mortality of the hepatopancreatic ciliate of shrimps, the load of healthy shrimps infected, the effects of EHP-ptp2 and hsp70, etc.

[0014] The application lays a foundation for the indoor preservation of the hepatopancreatic ciliate of shrimps. The technical problems that there is no cryoprotectant suitable for the hepatopancreatic ciliate of shrimps and no preservation technology suitable for the hepatopancreatic ciliate of shrimps are solved. BRIEF DESCRIPTION OF DRAWINGS

[0015] Fig. 1 is a mortality graph of EHP spores under two kinds of low-temperature conditions;

[0016] Fig. 2 is an EHP load graph under two kinds of low-temperature conditions;

[0017] Fig. 3 is a mortality graph of spores preserved for 1 month by the mixed cryoprotectant A group;

[0018] Fig. 4 is a comparison graph of the number of spores of the mixed cryoprotectant A group of frozen spores invading the Palaemon carinicauda;

[0019] Fig. 5 is a mortality graph of spores preserved for 1 month by the mixed cryoprotectant B group;

[0020] Fig. 6 is a comparison graph of the number of spores of the mixed cryoprotectant B group of frozen spores invading the Palaemon carinicauda;

[0021] Figure 7 shows the gene expression level of EHP spore wall protein 26 (wsp26) under mixed cryoprotectant conditions;

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

[0023] Figure 9 shows the gene expression level of heat shock protein 70 (hsp70) in EHP spores under mixed cryoprotectant conditions. Detailed Implementation

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

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

[0026] 1. Experimental materials and daily management

[0027] Healthy and vigorous white shrimp with an average body length of 6.2±0.5 cm and an average weight of 2.5±0.5 g, and intact body surfaces, were selected. They were infected with Enterocytozoa hepatica (EHP) for 3 days. The infected shrimp were fed twice daily (8:00 AM and 6:00 PM), with each feeding amount being 10% of the total number of shrimp. Metabolic waste and uneaten feed were removed using a siphon.

[0028] 2. Selection of the 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 well.

[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 well.

[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 well.

[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 well.

[0035] 5, Dimethyl sulfoxide (20%), Glycerol (20%): 40 μL of dimethyl sulfoxide, 40 μL of glycerol and 120 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0036] 6, Dimethyl sulfoxide (20%), Glycerol (30%): 40 μL of dimethyl sulfoxide, 60 μL of glycerol and 100 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0037] 7, Dimethyl sulfoxide (30%), Glycerol (10%): 60 μL of dimethyl sulfoxide, 20 μL of glycerol and 120 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0038] 8, Dimethyl sulfoxide (30%), Glycerol (20%): 60 μL of dimethyl sulfoxide, 40 μL of glycerol and 100 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0039] 9, Dimethyl sulfoxide (30%), Glycerol (30%): 60 μL of dimethyl sulfoxide, 60 μL of glycerol and 80 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0040] 10, Dimethyl sulfoxide (10%), Propylene glycol (10%): 20 μL of dimethyl sulfoxide, 20 μL of propylene glycol and 160 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0041] 11, Dimethyl sulfoxide (10%), Propylene glycol (20%): 20 μL of dimethyl sulfoxide, 40 μL of propylene glycol and 140 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0042] 12, Dimethyl sulfoxide (10%), Propylene glycol (30%): 20 μL of dimethyl sulfoxide, 60 μL of propylene glycol and 120 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0043] 13, Dimethyl sulfoxide (20%), Propylene glycol (10%): 40 μL of dimethyl sulfoxide, 20 μL of propylene glycol and 140 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0044] 14, Dimethyl sulfoxide (20%), Propylene glycol (20%): 40 μL of dimethyl sulfoxide, 40 μL of propylene glycol and 120 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0045] 15, Dimethyl sulfoxide (20%), propylene glycol (30%): 40 μL of dimethyl sulfoxide, 60 μL of propylene glycol and 100 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0046] 16, Dimethyl sulfoxide (30%), propylene glycol (10%): 60 μL of dimethyl sulfoxide, 20 μL of propylene glycol and 120 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0047] 17, Dimethyl sulfoxide (30%), propylene glycol (20%): 60 μL of dimethyl sulfoxide, 40 μL of propylene glycol and 100 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0048] 18, Dimethyl sulfoxide (30%), propylene glycol (30%): 60 μL of dimethyl sulfoxide, 60 μL of propylene glycol and 80 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0049] 19, Glycerol (10%), propylene glycol (10%): 20 μL of glycerol, 20 μL of propylene glycol and 160 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0050] 20, Glycerol (10%), propylene glycol (20%): 20 μL of glycerol, 40 μL of propylene glycol and 140 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0051] 21, Glycerol (10%), propylene glycol (30%): 20 μL of glycerol, 60 μL of propylene glycol and 120 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0052] 22, Glycerol (20%), propylene glycol (10%): 40 μL of glycerol, 20 μL of propylene glycol and 140 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0053] 23, Glycerol (20%), propylene glycol (20%): 40 μL of glycerol, 40 μL of propylene glycol and 120 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0054] 24, Glycerol (20%), propylene glycol (30%): 40 μL of glycerol, 60 μL of propylene glycol and 100 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0055] 25, Glycerol (30%), propylene glycol (10%): 60 μL of glycerol, 20 μL of propylene glycol and 120 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0056] 26, Glycerol (30%), Propylene glycol (20%): 60 μL of glycerol, 40 μL of propylene glycol and 100 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0057] 27, Glycerol (30%), Propylene glycol (30%): 60 μL of glycerol, 60 μL of propylene glycol and 80 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0058] Group B (three reagents mixed)

[0059] 1, Dimethyl sulfoxide (10%), Glycerol (10%), Propylene glycol (10%): 20 μL of dimethyl sulfoxide, 20 μL of glycerol, 20 μL of propylene glycol and 140 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0060] 2, Dimethyl sulfoxide (10%), Glycerol (20%), Propylene glycol (30%): 20 μL of dimethyl sulfoxide, 40 μL of glycerol, 60 μL of propylene glycol and 80 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0061] 3, Dimethyl sulfoxide (10%), Glycerol (30%), Propylene glycol (20%): 20 μL of dimethyl sulfoxide, 60 μL of glycerol, 40 μL of propylene glycol and 80 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0062] 4, Dimethyl sulfoxide (20%), Glycerol (10%), Propylene glycol (20%): 40 μL of dimethyl sulfoxide, 20 μL of glycerol, 40 μL of propylene glycol and 100 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0063] 5, Dimethyl sulfoxide (20%), Glycerol (20%), Propylene glycol (10%): 40 μL of dimethyl sulfoxide, 40 μL of glycerol, 20 μL of propylene glycol and 100 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0064] 6, Dimethyl sulfoxide (20%), Glycerol (30%), Propylene glycol (30%): 40 μL of dimethyl sulfoxide, 60 μL of glycerol, 60 μL of propylene glycol and 40 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0065] 7, Dimethyl sulfoxide (30%), Glycerol (10%), Propylene glycol (30%): 60 μL of dimethyl sulfoxide, 20 μL of glycerol, 60 μL of propylene glycol and 60 μL of PBS buffer were pipetted into a centrifuge tube, and mixed well by shaking.

[0066] 8, Dimethyl sulfoxide (30%), glycerol (20%), propylene glycol (20%): 60 μL of dimethyl sulfoxide, 40 μL of glycerol, 40 μL of propylene glycol and 60 μL of PBS buffer were taken in a centrifuge tube using a pipette, and mixed uniformly by shaking.

[0067] 9, Dimethyl sulfoxide (30%), glycerol (30%), propylene glycol (10%): 60 μL of dimethyl sulfoxide, 60 μL of glycerol, 20 μL of propylene glycol and 60 μL of PBS buffer were taken in a centrifuge tube using a pipette, and mixed uniformly by shaking.

[0068] The above reagents were prepared and stored at -80°C for 30 days.

[0069] 3, Removal of cryopreservation solution

[0070] Some cryoprotectants such as DMSO and glycerol are toxic to cells at high concentrations at room temperature, and the cryoprotectant should be removed after thawing the sample by multiple low-temperature centrifugation.

[0071] The thawed sample was immediately centrifuged at 6000 rpm for 5 min after adding 500 μL of sterile water. The supernatant was discarded, the precipitate was re-added with 500 μL of sterile water, and the spores were suspended by gentle shaking. Repeat the centrifugation for 3-4 times, and finally add 200 μL of sterile water to the precipitate and suspend the spores.

[0072] Optimal mixed reagent formula was determined by fluorescence detection counting using DAPI, post-infection spore load test, gene quantification of polar tube protein 2 (EHP-ptp2), spore wall protein 26 (wsp26) and heat shock protein 70 (hsp70) using optical microscopy.

[0073] Statistical analysis: variance analysis and multiple comparison method were used to screen the best formula in the above A and B groups.

[0074] 4, Experimental data and results.

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

[0076] 4.1、From Figure 1, under two low temperature conditions, the mortality rate at -20℃ was mostly lower than that at -80℃; the average mortality rate reached 59.835%. The mortality rate of the control group (CG) at -80℃ was as high as 98%, and that at -20℃ reached 72.22%. The mortality rate of spores preserved with 10%-30% DMSO and G at -20℃ was lower than that at -80℃. The mortality rate of spores preserved with EG at -20℃ was higher than that with DMSO, G and PG. The survival rate of spores preserved with 10%-30% PBS buffer was not ideal under two low temperature conditions, and the mortality rate was more than 62.5%.

[0077] From the comparison of Figure 2, the infection capacity of spores preserved at -20℃ was significantly higher than that of spores preserved at -80℃, and the average load reached 10 6 (copies / mg). The infection capacity of spores preserved with 10%-30% EG and PBS buffer was poor, and the load after infecting healthy shrimps was 1.9×10 6 below; 10% DMSO (5.4×10 6 ) at -20℃ had a very significant difference (P<0.001) with other groups of reagents. 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 very significant differences (P<0.01, P<0.001) under two low temperature conditions, and the load 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℃, and the mixed reagents were selected as DMSO, G and PG.

[0079] 4.2 Effects of mixed cryoprotectant group A on the activity of EHP spores and the results

[0080] As shown in Fig. 3, the mixed groups of different cryoprotectants have different effects on the mortality of EHP spores after being frozen at -20℃ for one month, and significantly affect the mortality of spores. The mortality of the control group is as high as 85.26%, and the average mortality of all groups is 51.39%, which is lower than the mortality of spores preserved by single cryoprotectant. The mortality of the No. 6 reagent group (20% DMSO + 30% G) is the highest, reaching 77.94%, and the mortality of the No. 24 reagent group (20% G + 30% PG) is the lowest, reaching 31.24%; the mortality of the No. 2 reagent group (10% DMSO + 20% G) is 35.71%, which is close to the mortality of the No. 24 reagent group, and the preservation effect is ideal.

[0081] As shown in Fig. 4, the mixed groups of different cryoprotectants significantly affect the load after the frozen spores infect. After the EHP spores infect the healthy Exopalaemon modestus, there is a statistically significant difference (P<0.05) in the infection level of spores preserved by the mixed groups of different reagents, and the average load is 10 7 copies / mg. The load of spores preserved by the control group is 4.76x10 5 copies / mg after 3 days of infection; the load of EHP spores preserved by 20% DMSO + 20% G reaches 1.6439x10 8 copies / mg after 3 days of infection, which is significantly higher than that of the other groups (P<0.001).

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

[0083] As shown in Fig. 5, the average mortality is 36.67%, which is lower than 59.835% of the single reagent group and 51.39% of the mixed reagent A group. The mortality of the control group reaches 78.78%, which is significantly higher than that of the other groups (P<0.01); the mortality of the No. 2 reagent (10% DMSO + 20% G + 30% PG) is the lowest among the other 8 mixed reagents, which is 25.81%; the mortality of the No. 1 reagent group (10% DMSO + 10% G + 10% PG) is 32.31%, the mortality of the No. 8 reagent group (30% DMSO + 20% G + 20% PG) is 32.17%, and the mortality of the No. 9 reagent group (30% DMSO + 30% G + 10% PG) is 32.81%, which are relatively low among the three groups of mixed reagents; the mortality of the No. 6 reagent (20% DMSO + 30% G + 30% PG) is the highest, which is 45.63%. At the same time, the mortality of the 9 groups of mixed reagents is lower than 45%, which indicates that the freezing effect of the mixed reagents is better than that of the single cryoprotectant and the mixed two cryoprotectants.

[0084] As shown in Fig. 6, the average load is 10 8(copies / mg), significantly higher than 10 6 (copies / mg) with 10 7 (copies / mg). The control group had a load of 5.3 x 10 5 , extremely significantly lower than the rest of the groups (P <0.001); the 1st reagent group (10% DMSO + 10% G + 10% PG) preserved spores with the highest load after infecting healthy shrimps, 4.85 x 10 8 , with extremely significant differences (P <0.01) relative to the other 8 mixed reagents; the 4th reagent group (20% DMSO + 10% G + 20% PG) preserved spores with a load of 3.84 x 10 8 (copies / mg), relatively high. The 7th reagent group (30% DMSO + 10% G + 30% PG) preserved spores with the lowest load after infecting healthy shrimps, 2.95 x 10 7 , with extremely significant differences (P <0.01) relative to the other 8 mixed reagents.

[0085] In summary, under the condition of -20℃, the effect of preserving spores by mixing three cryoprotectants is the best, therefore, to further explore the optimal combination of the three reagents, the experiment carried out related activity genes (wsp26, ptp2) and stress protein genes (hsp70) of hepatopancreatic coccidium in shrimps.

[0086] As can be seen from Figure 7, the expression level of spore wall protein 26 (wsp26) gene of frozen spores can be seen that the wsp26 gene expression level of spores preserved by the 1st mixed reagent is the highest, with extremely significant differences (P <0.01) relative to the other 8 reagents; the wsp26 gene expression level of frozen spores preserved by the 2nd and 3rd mixed reagents is relatively high; the wsp26 gene expression level of frozen spores preserved by the 7th mixed reagent is the lowest; the wsp26 gene expression level of frozen spores preserved by the 6th, 8th and 9th mixed reagents is relatively low; the control group is only for reference and is not included in the comparison of the test groups.

[0087] As can be seen from Figure 8, the expression level of polar tube protein 2 (ptp2) gene of frozen spores can be seen that the ptp2 gene expression level of spores preserved by the 1st mixed reagent is the highest, with significant differences (P <0.01) relative to the other 8 reagents; the ptp2 gene expression level of frozen spores preserved by the 3rd, 4th and 5th mixed reagents is relatively high; the ptp2 gene expression level of frozen spores preserved by the 6th mixed reagent is the lowest; the ptp2 gene expression level of frozen spores preserved by the 7th, 8th and 9th mixed reagents is relatively low; the control group is only for reference and is not included in the comparison of the test groups.

[0088] As shown in Figure 9, the expression level of heat shock protein 70 (hsp70) gene of the cryopreserved spores can be seen, the expression level of hsp70 gene of the spores preserved by the No. 5 mixed reagent is the lowest, which has a significant difference (P < 0.01) relative to the other 8 groups of reagents; the expression level of hsp70 gene of the cryopreserved spores by the No. 1, No. 2 and No. 4 mixed reagents is relatively low, and the expression level of hsp70 gene of the cryopreserved spores by the No. 1, No. 2 and No. 4 mixed reagents has no significant difference (P > 0.05); the expression level of hsp70 gene of the cryopreserved spores by the No. 6 mixed reagent is the highest; the expression level of hsp70 gene of the cryopreserved spores by the No. 7 mixed reagent is relatively high; the control group is only for reference and is not included in the comparison of the test groups.

[0089] According to the above experimental results, the No. 1, No. 2, No. 8 and No. 9 mixed reagents have lower mortality; the No. 1 and No. 4 mixed reagents have higher load; the No. 1, No. 2 and No. 3 mixed reagents have higher expression level of wsp26 gene of the cryopreserved spores; the No. 1, No. 3, No. 4 and No. 5 mixed reagents have higher expression level of ptp2 gene of the cryopreserved spores; the No. 1, No. 2, No. 4 and No. 5 mixed reagents have lower expression level of hsp70 gene of the cryopreserved spores.

[0090] According to the above experimental results, it is concluded that the No. 1 group of the B group of mixed reagents, i.e. 10% dimethyl sulfoxide plus 10% glycerol plus 10% propylene glycol, is the best.

Claims

1. A hepatopanosoma sp. cryoprotectant, characterized in that: The cryoprotectant is mainly made of glycerol, dimethyl sulfoxide and propylene glycol, and PBS buffer is used as a base solution; the volume percentage of the raw materials dimethyl sulfoxide, glycerol, propylene glycol and PBS buffer is 10:10:10:

70.

2. The hepatopanosoma sp. cryoprotectant of claim 1, wherein: The freezing temperature of the cryoprotectant is -20℃ to -80℃.

3. A method of preparing a hepatopancreatic ciliate cryoprotectant as claimed in claim 1 or 2, characterized in that: The raw materials dimethyl sulfoxide, glycerol and propylene glycol are taken by a pipette according to the volume percentage, mixed uniformly in a container, and the cryoprotectant is obtained.

4. Use of a hepatopancreatic ciliate cryoprotectant according to claim 1, characterized in that: The use is that the cryoprotectant is used as a cryoprotectant for Enterocytozoon hepatopenaei.

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