CN112400864B - Oocyst-type biological wall-breaking agent composition and preparation method and application thereof - Google Patents

Abstract

The invention discloses an oocyst-type biological wall-breaking agent composition and a preparation method and application thereof. The components of the oocyst-type biological wall-breaking agent composition of the invention include propylene glycol, pancreatin, polyethylene glycol and sodium citrate , proline, trehalose, potassium dichromate, sodium dihydrogen phosphate, disodium hydrogen phosphate, standard PBS buffer and water for injection, the active ingredients in the oocyst type biological wall-breaking agent composition of the present invention can be combined with oocysts. The main components such as keratin, glycoprotein, and phospholipid in the wall structure interact and destroy the densely arranged rigid structure, forming a pore structure that allows the passage of cryopreservation materials, and only slightly destroys the oocyst wall structure. The permeability of oocysts is increased, thereby enhancing the vitrification ability and the ability to inhibit freezing in the oocysts, reducing the damage of ice crystals, prolonging the cryopreservation time, and effectively expanding the applicability of the cryopreservation scheme in the field of organisms containing oocysts.

Description

Oocyst-type biological wall-breaking agent composition and preparation method and application thereof

technical field

The invention relates to the technical field of cryopreservation, in particular to an oocyst-type biological wall-breaking agent composition and a preparation method and application thereof.

Background technique

In recent years, the cryopreservation of cells, tissues and organs has developed rapidly, and breakthroughs in cryopreservation technology will provide a strong theoretical and theoretical basis for the development of regenerative medicine, embryonic stem cell therapy, pathological examination of slices, preservation of vaccines and cell drugs and other fields. Experimental support. Active substances that are separated from the original maternal environment, such as cells, consume their own nutrients in the cells due to metabolism and other life activities during the preservation process, and their activities inevitably weaken with the prolongation of preservation time until they are completely inactivated. Extremely low temperature, usually 77K (-196゜C, the boiling point of liquid nitrogen), can prolong the life of cells and achieve long-term preservation of cells and other substances by effectively slowing down or even stopping life activities such as intracellular metabolism.

At present, the vitrification method is commonly used. After equilibrating biological materials such as cells in a vitrification cryopreservation reagent, they are directly put into liquid nitrogen, so that the liquid inside and outside the cells quickly enters a vitrified state. Vitrification cryopreservation reagents usually contain relatively high concentrations of DMSO, glycerol and other small molecular substances, which can enter the cell through the cell membrane, enhance the intracellular vitrification ability, and make the cells directly enter the glass state during the rapid cooling process to avoid freezing damage to cells. In addition, in addition to the vitrification method, a new set of cryopreservation strategies has been gradually developed in recent years, that is, the main component of the cryopreservation agent is a biomimetic freezing material with ice-controlling protein structure and function. It can inhibit the recrystallization of ice crystals, regulate the morphology of ice crystals, and reduce the growth rate of ice crystals. At present, biological materials that have been cryopreserved include a variety of animal cells and tissues, such as blood cells, sperm, egg cells, embryos, stem cells, etc., but no significant progress has been made in cryopreservation of active organisms containing oocysts. The key shackle is that The dense structure of the oocyst wall prevents cryopreservation material from entering the interior of the oocyst.

Coccidia and Cryptosporidium are parasitic protozoa that have attracted much attention in recent years and contain oocysts, which are crucial to their infectious development. Coccidia is a protozoa that can parasitize in the intestinal epithelial cells of animals. It is highly infectious and harmful, and seriously threatens the life and health of poultry and livestock. At present, the use of coccidiosis vaccine is an effective prevention and control method, which overcomes the disadvantages of using chemical drugs, such as high cost, long medication time, and easy generation of drug resistance, and has the advantage of one-time immunity for life-long protection. To give full play to the role of chicken coccidiosis vaccine, one of the key links is to ensure the quality stability of the vaccine during storage and transportation. my country has developed and produced the world's leading chicken coccidia early-maturing attenuated vaccine, but its popularization and application are limited by short storage time and difficult transportation. The main component of chicken coccidiosis vaccine is the sporulated oocysts of several species of coccidioides, which are not resistant to low temperature and usually need to be stored and transported at 2 to 8 °C, resulting in a storage period of at most 7 months, which seriously affects the vaccine. intergenerational transmission and mass production.

Cryptosporidium is a parasitic protozoan smaller than coccidia, similar to coccidia, and also infects humans and animals with sporulated oocysts. They are widespread in a variety of vertebrates and are an enteric pathogen that is second only to rotavirus and can cause severe diarrhea. Cryptosporidium is also an important opportunistic pathogenic protozoa that is in a recessive state of infection in an immunocompetent host, but when the host is immunocompromised, it proliferates abnormally rapidly, causing life-threatening persistent diarrhea and weight loss. Current antiparasitic drugs are largely ineffective in immunocompromised and malnourished people, and treatments are in urgent need of improvement. Therefore, the research on the pathogenic protozoa Cryptosporidium is very urgent, but the preservation period of Cryptosporidium oocysts is short, and due to the lack of suitable cryopreservation methods, it is very difficult to obtain oocysts with genetic characteristics. Currently, laboratory strains of Cryptosporidium must be maintained in live animals (mice, calves, and piglets) and subcultured every 6-8 weeks, which is not only an expensive and time-consuming process, but also limits egg production Sharing of sacs between different laboratories and evaluation of the efficacy of related vaccines and treatments.

The difficulty of cryopreservation of oocysts such as coccidia and cryptosporidium is that their structure is different from that of cells, and the oocyst wall is dense and rigid. Or the effect of inhibiting freezing, therefore, the damage to oocysts caused by freezing during cryopreservation cannot be avoided. Therefore, a new type of oocyst wall-breaking agent is urgently needed, which can slightly break the oocyst wall structure to form a certain channel, and allow the cryopreservation material to enter the oocyst through the channel, so as to achieve low-temperature and long-term low temperature containing oocyst-like active organisms. save. At present, there are few researches and patents on cryopreservation of active organisms containing oocysts. The cryopreservation of active organisms containing oocysts is a major challenge and an urgent problem to be solved in this field. It is the key to find a way to slightly disrupt the structure of the oocyst wall so that the cryopreservation material can enter the oocyst smoothly. At present, there is no mature solution to solve this problem.

SUMMARY OF THE INVENTION

In view of the above-mentioned deficiencies, one of the objects of the present invention is to provide a reasonable formula, which only needs to slightly damage the oocyst wall structure to form a pore allowing the cryopreservation material to enter therein, and effectively prolong the cryopreservation time of eggs. Capsule-type biological wall-breaking agent composition.

Another object of the present invention is to provide a preparation method of the above-mentioned oocyst-type biological wall-breaking agent composition.

The third object of the present invention is to provide an application of the above-mentioned oocyst-type biological wall-breaking agent composition.

For achieving the above object, the technical scheme provided by the present invention is:

An oocyst-type biological wall-breaking agent composition, comprising the following components by mass percentage: 10%-45% of propylene glycol, 0.01%-0.2% of pancreatin, 0.6%-5% of polyethylene glycol, citric acid Sodium 0.8%～6%, Proline 0.5%～5%, Trehalose 0.5%～10%, Potassium dichromate 0.5%～8%, Sodium dihydrogen phosphate 0.1%～0.8%, Disodium hydrogen phosphate 0.2% ~ 1%, standard PBS buffer 2% ~ 5%, and the rest are water for injection.

A preparation method of an oocyst-type biological wall-breaking agent composition, comprising the following steps:

(1) Prepare the following components by mass percentage: propylene glycol 10%-45%, pancreatin 0.01%-0.2%, polyethylene glycol 0.6%-5%, sodium citrate 0.8%-6%, proline 0.5%～5%, trehalose 0.5%～10%, potassium dichromate 0.5%～8%, sodium dihydrogen phosphate 0.1%～0.8%, disodium hydrogen phosphate 0.2%～1%, standard PBS buffer 2% ~5%, the rest is water for injection;

(2) add pancreatin, polyethylene glycol and sodium citrate to described water for injection, after stirring and dissolving, obtain the first mixture;

(3) add proline, trehalose and potassium dichromate to described first mixture, after stirring and dissolving, obtain second mixture;

(4) adding isopropanol to the second mixture, after stirring and dissolving, obtain the third mixture;

(5) adding standard PBS buffer, sodium phosphate dihydrogen phosphate and disodium hydrogen phosphate to the third mixture, and adjusting the pH to 6.8-7.4 to prepare an oocyst-type biological wall-breaking agent composition.

As a preferred solution of the present invention, the polyethylene glycol is preferably polyethylene glycol 6000 (PEG6000).

The key components such as propylene glycol, pancreatin, polyethylene glycol 6000 (PEG6000) and sodium citrate can respectively interact with the main components such as keratin, glycoprotein, and phospholipid in the oocyst wall structure and destroy the rigidity of its dense arrangement. structure to form a certain pore structure.

The main functions of the components such as proline and trehalose are to provide nutrients for the sporangia in the oocyst and to buffer the destruction of the sporangia by components such as propylene glycol and protease.

The potassium dichromate is a common disinfectant, and its main function is to kill the bacteria in the oocyst-type biological wall-breaking agent composition, and to inhibit the bacteria from causing damage to the oocysts.

The components such as sodium dihydrogen phosphate, disodium hydrogen phosphate and standard PBS buffer are used to adjust the pH value of the oocyst-type biological wall-breaking agent composition, so that the pH value is between 6.8 and 7.4, which is suitable for cryopreservation of oocysts. .

A kind of application of above-mentioned oocyst type biological wall-breaking agent composition in oocyst, its application steps are as follows:

(S1) obtain the oocyst fluid after the purification treatment, then add the above-mentioned oocyst-type biological wall-breaking agent composition, mix by stirring or vortexing, and then leave standstill;

(S2) Finally, the state of the oocysts was observed, and the oocyst suspensions that had been left standing for different time periods were taken and placed under a microscope to observe the morphological changes of the oocysts and compared with the blank group to analyze the infiltration situation.

The purification step in the step (S1) is as follows:

(I) take oocyst fluid, carry out centrifugation and precipitation;

(II) Remove the supernatant, add saturated saline to resuspend the precipitate, and centrifuge;

(III) moving the upper layer liquid containing the oocyst fluid into a centrifuge tube filled with water for injection, and centrifuging again to obtain the oocyst sedimentation fluid;

(IV) Transfer the oocyst precipitation liquid into a centrifuge tube containing injection water, repeat the previous step several times, preferably three times, and finally obtain the oocyst liquid that does not contain the disinfectant composition.

The beneficial effects of the present invention are as follows: the formulation of the oocyst-type biological wall-breaking agent composition of the present invention is reasonable, and the key ingredients such as propylene glycol, pancreatin, polyethylene glycol and sodium citrate can interact with the keratin, The main components such as glycoproteins and phospholipids interact and destroy their densely arranged rigid structures to form channels that allow cryopreservation materials to pass through, that is, on the basis of mildly destroying the oocyst wall structure, forming channels for cryopreservation materials to pass through. The purpose of increasing the permeability of oocysts, thereby enhancing the ability of vitrification and inhibiting freezing in the oocysts, reducing the damage of ice crystals, and realizing long-term storage at low temperature. The preparation method of the oocyst-type biological wall-breaking agent composition provided by the present invention has simple steps, is easy to realize, and can quickly prepare the oocyst wall structure that only needs to be slightly damaged to form a oocyst that allows cryopreservation materials to enter it. Oocyst-type biowall-breaking agent composition for pores.

Below in conjunction with embodiment, the present invention is further described.

Detailed ways

Example 1: This example is illustrated by increasing the permeability of Eimeria giant oocysts as an example, but is not intended to limit the scope of the present invention. For example, Eimeria tenella oocysts, Eimeria tenacious oocysts, Eimeria pilosa oocysts, Eimeria brucei oocysts, Eimeria poisoning oocysts, Eimeria precocious oocysts, Metamorphosis Eimeria oocysts and Cryptosporidium oocysts, etc.

(1) preparation of oocyst-type biological wall-breaking agent composition: get 80ml water for injection and put it into a beaker placed in a 4°C water bath, put into a stirrer, and adjust the rotating speed to 500rpm; add pancreatic enzyme 0.02g, polyethylene glycol Alcohol 6000 (PEG6000) 1.2g, sodium citrate 1.5g, stir for 10 minutes; add proline 3.5g, trehalose 0.7g, potassium dichromate 0.8g in turn; add propylene glycol 25g, stir for 15 minutes; then add standard PBS 3 ml of buffer solution, 0.3 g of sodium dihydrogen phosphate and 0.7 g of disodium hydrogen phosphate, and stirred for 30 minutes.

(2) Pretreatment of oocysts, the following purification pretreatment is required before the addition of Eimeria giant oocysts:

1. Take 2ml of Eimeria giant oocyst fluid and centrifuge (3000rpm, 5min) for precipitation;

2. Remove the supernatant, add 2ml of saturated aqueous sodium chloride solution, and centrifuge (3000rpm, 5min);

3. The supernatant liquid containing Eimeria giant oocyst liquid was taken out, moved into a centrifuge tube containing 2ml of water for injection, and centrifuged again (3000rpm, 5min) to obtain oocyst sedimentation liquid;

4. Transfer the oocyst sediment into a centrifuge tube containing 2 ml of water for injection, centrifuge (3000 rpm, 5 min), and repeat the centrifugation process three times to finally obtain coccidial oocysts that do not contain disinfectant components.

After the above-mentioned treated coccidial oocyst fluid was divided into equal parts, the cryopreservation tube (group A) containing 3ml oocyst-type biological wall-breaking agent composition and the cryopreservation tube (group B) containing 3ml isotonic sodium chloride solution were added respectively. ), and vortexed for 10, 30, and 60 minutes, then taken out and placed under a microscope to observe the shape of coccidial oocysts, count the volume of coccidial oocysts, and analyze changes in permeability.

The results showed that the volume of Eimeria maxima oocysts was significantly reduced after soaking in the oocyst-type biological wall-breaking agent composition, while the volume of Eimeria maxima oocysts in isotonic sodium chloride solution did not change significantly, indicating that The oocyst-type biological wall-breaking agent composition of the present invention can effectively destroy the oocyst wall, so that the components in the oocyst penetrate outside the oocyst wall, in other words, the components outside the oocyst wall can also enter into the oocyst.

See Table 1. Table 1 shows the change in volume of Eimeria maxima oocysts with time after soaking with the oocyst-type biological wall-breaking agent composition (A) and isotonic sodium chloride solution (B). The data in Table 1 is the statistical average of the volume of 50 coccidia oocysts, where the volume of fresh coccidia is taken as 1.

Table 1

0min. 10min. 30min. 60min. A 1 0.92 0.79 0.53 B 1 0.99 0.98 0.96

Example 2: This example is basically the same as Example 1, except that the oocyst-type biological wall-breaking agent composition includes the following components by mass: 10% of propylene glycol, 0.2% of pancreatin, polyethylene glycol Alcohol 6000 (PEG6000) 5%, sodium citrate 6%, proline 3%, trehalose 7%, potassium dichromate 0.5%, sodium dihydrogen phosphate 0.1%, disodium hydrogen phosphate 1%, standard PBS buffer 2%, water for injection 65.2%.

Example 3: This example is basically the same as Example 1, except that the oocyst-type biological wall-breaking agent composition includes the following components by mass: 30% of propylene glycol, 0.03% of pancreatin, polyethylene glycol Alcohol 6000 (PEG6000) 2%, sodium citrate 3%, proline 5%, trehalose 6%, potassium dichromate 5%, sodium dihydrogen phosphate 0.5%, disodium hydrogen phosphate 0.5%, standard PBS buffer 5%, water for injection 42.97%.

Example 4: This example is basically the same as Example 1, except that the oocyst-type biological wall-breaking agent composition includes the following components by mass: 45% of propylene glycol, 0.01% of pancreatin, polyethylene glycol Alcohol 6000 (PEG6000) 0.6%, sodium citrate 0.8%, proline 0.5%, trehalose 10%, potassium dichromate 8%, sodium dihydrogen phosphate 0.8%, disodium hydrogen phosphate 1%, standard PBS buffer 2%, water for injection 31.29%.

Example 5: This example is basically the same as Example 1, except that the oocyst-type biological wall-breaking agent composition includes the following components by mass: 15% of propylene glycol, 0.07% of pancreatin, polyethylene glycol Alcohol 6000 (PEG6000) 4%, sodium citrate 5%, proline 5%, trehalose 0.5%, potassium dichromate 5%, sodium dihydrogen phosphate 0.6%, disodium hydrogen phosphate 0.2%, standard PBS buffer 4%, water for injection 60.63%.

The above embodiment is only a better embodiment of the present invention, and the present invention cannot enumerate all the embodiments one by one. Any technical solution that adopts one of the above embodiments, or equivalent changes made according to the above embodiments, are all included in the present invention. within the scope of protection of the invention.

During use, the key components such as propylene glycol, pancreatin, polyethylene glycol and sodium citrate in the oocyst-type biological wall-breaking agent composition of the present invention can interact with the main components such as keratin, glycoprotein, and phospholipid in the oocyst wall structure. It interacts and destroys its densely arranged rigid structure to form a pore structure that allows the passage of cryopreservation materials, and achieves the purpose of increasing the permeability of the oocyst on the basis of only mildly destroying the structure of the oocyst wall, thereby enhancing the inner glass of the oocyst It can improve the ability of melting and inhibiting freezing, reduce the damage of ice crystals, and prolong the storage time at low temperature.

Based on the disclosure and teaching of the above specification, those skilled in the art to which the present invention pertains can also make changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and changes to the present invention should also fall within the protection scope of the claims of the present invention. In addition, although some specific terms are used in this specification, these terms are only for the convenience of description and do not constitute any limitation to the present invention. Other compositions obtained by the same or similar methods and their preparation methods and applications are all in within the protection scope of the present invention.

Claims (9)

1. An egg sac type biological wall breaking agent composition is characterized by comprising the following components in percentage by mass: 10 to 45 percent of propylene glycol, 0.01 to 0.2 percent of pancreatin, 0.6 to 5 percent of polyethylene glycol, 0.8 to 6 percent of sodium citrate, 0.5 to 5 percent of proline, 0.5 to 10 percent of trehalose, 0.5 to 8 percent of potassium dichromate, 0.1 to 0.8 percent of sodium dihydrogen phosphate, 0.2 to 1 percent of disodium hydrogen phosphate, 2 to 5 percent of standard PBS buffer solution and the balance of water for injection.

2. The oocyst type biological wall breaking agent composition of claim 1, wherein: the polyethylene glycol is polyethylene glycol 6000.

3. A preparation method of an oocyst type biological wall breaking agent composition is characterized by comprising the following steps: which comprises the following steps:

(1) preparing the following components in percentage by mass: 10 to 45 percent of propylene glycol, 0.01 to 0.2 percent of pancreatin, 0.6 to 5 percent of polyethylene glycol, 0.8 to 6 percent of sodium citrate, 0.5 to 5 percent of proline, 0.5 to 10 percent of trehalose, 0.5 to 8 percent of potassium dichromate, 0.1 to 0.8 percent of sodium dihydrogen phosphate, 0.2 to 1 percent of disodium hydrogen phosphate, 2 to 5 percent of standard PBS buffer solution and the balance of water for injection;

(2) adding pancreatin, polyethylene glycol and sodium citrate into the water for injection, and stirring and dissolving to obtain a first mixture;

(3) adding proline, trehalose and potassium dichromate into the first mixture, and stirring to dissolve to obtain a second mixture;

(4) adding isopropanol into the second mixture, and stirring to dissolve to obtain a third mixture;

(5) and adding standard PBS buffer solution, sodium dihydrogen phosphate and disodium hydrogen phosphate into the third mixture, and adjusting the pH value to be 6.8-7.4.

4. The method for preparing an oocyst type biological wall breaking agent composition according to claim 3, wherein: the polyethylene glycol is polyethylene glycol 6000.

5. The method for preparing an oocyst type biological wall breaking agent composition according to claim 3, wherein: and (3) adding proline, trehalose and potassium dichromate in sequence from front to back.

6. Use of the oocyst type biological wall breaking agent composition of claim 1 or 2 in oocysts.

7. The application of the oocyst type biological wall breaking agent composition to oocysts as claimed in claim 6, wherein the application steps are as follows:

(S1) obtaining the purified oocyst solution, adding the oocyst type biological wall breaking agent composition of claim 1 or 2, mixing by stirring or vortexing, and then standing;

(S2) placing the oocyst suspension which is kept still for different time periods under a microscope, observing the morphological change of the oocysts and analyzing the infiltration situation by comparing with a blank group.

8. The use of an oocyst type biological wall breaking agent composition according to claim 7, wherein the purification step in step (S1) is as follows:

taking an oocyst solution, centrifuging and precipitating;

(II) removing the supernatant, adding saturated saline water for resuspension and precipitation, and centrifuging;

(III) transferring the supernatant liquid containing the oocyst liquid into a centrifuge tube filled with injection water, and centrifuging again to obtain an oocyst precipitation liquid;

(IV) transferring the oocyst sediment solution into a centrifuge tube filled with injection water, repeating the previous step for several times, and finally obtaining the oocyst solution without disinfectant components.

9. The use of an oocyst type biological wall breaking agent composition according to claim 8, wherein the step repeated in step (IV) is three times.