CN112400864A - Oocyst type biological wall breaking agent composition and preparation method and application thereof - Google Patents
Oocyst type biological wall breaking agent composition and preparation method and application thereof Download PDFInfo
- Publication number
- CN112400864A CN112400864A CN202011355601.6A CN202011355601A CN112400864A CN 112400864 A CN112400864 A CN 112400864A CN 202011355601 A CN202011355601 A CN 202011355601A CN 112400864 A CN112400864 A CN 112400864A
- Authority
- CN
- China
- Prior art keywords
- oocyst
- percent
- agent composition
- type biological
- breaking agent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 210000003250 oocyst Anatomy 0.000 title claims abstract description 128
- 239000000203 mixture Substances 0.000 title claims abstract description 55
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims abstract description 42
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 39
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 29
- KMUONIBRACKNSN-UHFFFAOYSA-N potassium dichromate Chemical compound [K+].[K+].[O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O KMUONIBRACKNSN-UHFFFAOYSA-N 0.000 claims abstract description 28
- 108010019160 Pancreatin Proteins 0.000 claims abstract description 15
- 229940055695 pancreatin Drugs 0.000 claims abstract description 15
- 239000001509 sodium citrate Substances 0.000 claims abstract description 15
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims abstract description 15
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims abstract description 14
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims abstract description 14
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims abstract description 14
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims abstract description 14
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims abstract description 13
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims abstract description 13
- 235000019799 monosodium phosphate Nutrition 0.000 claims abstract description 13
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims abstract description 13
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 12
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 12
- 239000008215 water for injection Substances 0.000 claims abstract description 11
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 14
- 239000007853 buffer solution Substances 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 11
- 238000003756 stirring Methods 0.000 claims description 11
- 229940093429 polyethylene glycol 6000 Drugs 0.000 claims description 9
- 238000002347 injection Methods 0.000 claims description 7
- 239000007924 injection Substances 0.000 claims description 7
- 238000001556 precipitation Methods 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 4
- 239000000645 desinfectant Substances 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- 230000008595 infiltration Effects 0.000 claims description 2
- 238000001764 infiltration Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 230000004660 morphological change Effects 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 239000013049 sediment Substances 0.000 claims description 2
- 238000003260 vortexing Methods 0.000 claims 1
- 238000005138 cryopreservation Methods 0.000 abstract description 20
- 238000007710 freezing Methods 0.000 abstract description 13
- 230000008014 freezing Effects 0.000 abstract description 13
- 238000004017 vitrification Methods 0.000 abstract description 10
- 239000011148 porous material Substances 0.000 abstract description 8
- 239000013078 crystal Substances 0.000 abstract description 7
- 239000000463 material Substances 0.000 abstract description 7
- 230000035699 permeability Effects 0.000 abstract description 5
- 102000003886 Glycoproteins Human genes 0.000 abstract description 4
- 108090000288 Glycoproteins Proteins 0.000 abstract description 4
- 102000011782 Keratins Human genes 0.000 abstract description 4
- 108010076876 Keratins Proteins 0.000 abstract description 4
- 150000003904 phospholipids Chemical class 0.000 abstract description 4
- 239000000872 buffer Substances 0.000 abstract description 2
- 230000002708 enhancing effect Effects 0.000 abstract description 2
- 239000004480 active ingredient Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 16
- 238000000034 method Methods 0.000 description 13
- 241000224483 Coccidia Species 0.000 description 12
- 229960005486 vaccine Drugs 0.000 description 9
- 241000223935 Cryptosporidium Species 0.000 description 8
- 238000004321 preservation Methods 0.000 description 8
- 241000223934 Eimeria maxima Species 0.000 description 7
- 239000008118 PEG 6000 Substances 0.000 description 7
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 7
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 238000003860 storage Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 241000287828 Gallus gallus Species 0.000 description 4
- 239000011232 storage material Substances 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 206010061598 Immunodeficiency Diseases 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 239000013543 active substance Substances 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 241000223924 Eimeria Species 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 230000003071 parasitic effect Effects 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 239000012466 permeate Substances 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000002335 preservative effect Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000223931 Eimeria acervulina Species 0.000 description 1
- 241000499566 Eimeria brunetti Species 0.000 description 1
- 241000499563 Eimeria necatrix Species 0.000 description 1
- 241000499544 Eimeria praecox Species 0.000 description 1
- 241000223932 Eimeria tenella Species 0.000 description 1
- 208000029462 Immunodeficiency disease Diseases 0.000 description 1
- 208000002720 Malnutrition Diseases 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 241000702670 Rotavirus Species 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 239000003096 antiparasitic agent Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 229940031567 attenuated vaccine Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000007813 immunodeficiency Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000001071 malnutrition Effects 0.000 description 1
- 235000000824 malnutrition Nutrition 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 208000015380 nutritional deficiency disease Diseases 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 229960004063 propylene glycol Drugs 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 208000026775 severe diarrhea Diseases 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 238000002660 stem cell treatment Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/10—Preservation of living parts
- A01N1/12—Chemical aspects of preservation
- A01N1/122—Preservation or perfusion media
- A01N1/125—Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/10—Preservation of living parts
- A01N1/12—Chemical aspects of preservation
- A01N1/122—Preservation or perfusion media
- A01N1/124—Disinfecting agents, e.g. antimicrobials
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/10—Preservation of living parts
- A01N1/12—Chemical aspects of preservation
- A01N1/122—Preservation or perfusion media
- A01N1/126—Physiologically active agents, e.g. antioxidants or nutrients
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Medicinal Preparation (AREA)
Abstract
本发明公开了一种卵囊型生物破壁剂组合物及其制备方法与应用，本发明卵囊型生物破壁剂组合物组份包括有丙二醇、胰酶、聚乙二醇、柠檬酸钠、脯氨酸、海藻糖、重铬酸钾、磷酸二氢钠、磷酸氢二钠、标准PBS缓冲液和注射用水，本发明卵囊型生物破壁剂组合物中的有效成份能与卵囊壁结构中的角质蛋白、糖蛋白、磷脂等主要成份相互作用并破坏其致密排列的刚性结构，使之形成允许冷冻保存材料通过的孔道结构，实现仅在轻度破坏卵囊壁结构的基础上增加卵囊渗透性，从而增强卵囊内玻璃化能力和抑制结冰能力，减少冰晶损害，延长低温保存时间，有效扩展了冷冻保存方案在含有卵囊的生物的领域的可应用性。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
Technical Field
The invention relates to the technical field of low-temperature cryopreservation, in particular to an oocyst type biological wall breaking agent composition and a preparation method and application thereof.
Background
In recent years, the cryopreservation of cells, tissues and organs is rapidly developed, and the breakthrough of the cryopreservation technology provides powerful theoretical and experimental support for the development of the fields of regenerative medicine, embryonic stem cell treatment, slice pathological detection, preservation of products such as vaccines and cell medicines and the like. Active substances such as cells and the like which are separated from the primary maternal environment consume nutrients in the cells due to life activities such as metabolism and the like in the preservation process, and the activity of the active substances inevitably weakens along with the prolonging of the preservation time until the active substances are completely inactivated. The extremely low temperature, usually 77K (-196 ° C, the boiling point of liquid nitrogen), can prolong the life of cells by effectively slowing down or even stopping the vital activities such as metabolism in cells, and realize long-term preservation of the cells and other substances.
At present, a vitrification freezing method is generally adopted, and biological materials such as cells and the like are put into a vitrification freezing preservation reagent for balance and then directly put into liquid nitrogen, so that the liquid inside and outside the cells rapidly enters a vitrification state. The vitrification cryopreservation reagent usually contains high-concentration small molecular substances such as DMSO, glycerol and the like, and the substances can enter the inside of cells through cell membranes to enhance the vitrification capacity in the cells, so that the cells can directly enter a glass state in the rapid cooling process, and the damage of freezing to the cells is avoided. In addition, in addition to the vitrification freezing method, a set of new freezing preservation strategy is gradually developed in recent years, namely, the main component of the freezing preservative is a bionic freezing material with an ice control protein structure and function, the freezing preservative has the effects of controlling freezing, inhibiting recrystallization of ice crystals, regulating the appearance of the ice crystals, reducing the growth rate of the ice crystals and the like, and after entering the cells, the substances such as the cells and the like are preserved for a long time at low temperature by regulating the evolution of the ice crystals. At present, biological materials which have been subjected to cryopreservation include various animal cells and tissues such as blood cells, sperms, ova, embryos, stem cells and the like, but no significant progress has been made on cryopreservation of organisms containing oocysts, wherein the key concern lies in that the compact structure of the oocyst wall prevents the cryopreservation materials from entering the inside of the oocysts.
Coccidia and cryptosporidium are parasitic protozoa of recent interest that contain stages of oocyst development, which are critical to their infectious development. Coccidian is a protozoa which can parasitize in epithelial cells of intestinal tracts of animals, has strong infectivity and great harmfulness, and seriously threatens the life health of poultry and livestock. At present, the coccidian vaccine is an effective prevention and control method, overcomes the defects of high cost, long administration time, easy generation of drug resistance and the like of using chemical drugs, and has the advantage of one-time immune lifelong protection. One of the key links is to ensure the quality stability of the vaccine in the storage and transportation processes to fully play the role of the coccidium vaccine. The international leading chicken coccidian precocity breeding attenuated vaccine has been researched and produced in China, but the popularization and application process is limited by short preservation time and difficult transportation. The main component of the coccidium chicken vaccine is sporulated oocysts of a plurality of coccidium, the coccidium chicken vaccine has the characteristic of low temperature resistance, and the coccidium chicken vaccine is usually stored and transported at 2-8 ℃, so that the storage life is only 7 months at most, and the interspecific transfer and large-scale production of the vaccine are seriously influenced.
Cryptosporidium is a parasitic protozoan of a smaller volume than coccidia, and like coccidia, also infects humans and animals with sporulated oocysts. They are widely present in a variety of vertebrates, and are an enteropathogen, second only to rotavirus, that can cause severe diarrhea. Cryptosporidium is also an important opportunistic pathogenic parasite, which is in a recessive infection in a immunocompromised host, but when the host is immunocompromised, it proliferates abnormally rapidly, causing life threatening persistent diarrhea and wasting. Current antiparasitic drugs are largely ineffective in people with immunodeficiency and malnutrition and treatment methods are in urgent need of improvement. The research on the pathogenic protozoan cryptosporidium is therefore urgent, but the cryptosporidium oocysts have a short shelf life, coupled with the difficulty of obtaining oocysts with genetic characteristics due to the lack of suitable cryopreservation methods. At present, laboratory strains of cryptosporidium must be kept 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 the sharing of oocysts between different laboratories and the evaluation of the efficacy of related vaccines and therapeutic methods.
The difficulty of cryopreservation of oocysts such as coccidium, cryptosporidium and the like is that the oocysts have different structures from cells, the oocyst walls are compact and have certain rigidity, and a cryopreservation material is difficult to permeate into the oocysts through the oocyst walls and cannot play a role in increasing vitrification capacity or inhibiting icing, so that damage of the oocysts caused by icing in a low-temperature preservation process cannot be avoided. Therefore, a novel oocyst wall breaking agent is urgently needed, which can slightly break the wall structure of the oocyst to form a certain pore passage, and a frozen storage material enters the inside of the oocyst through the pore passage, so that the low-temperature long-term storage of the oocyst-class active organisms is realized. At present, the research and the patent of cryopreservation of the organisms containing the oocysts are less. The low-temperature cryopreservation of the organisms containing the oocysts is a great challenge and an urgent problem to be solved in the field, and the key is to find a way of slightly damaging the wall structure of the oocysts so that cryopreservation materials can smoothly enter the oocysts. There is currently no mature solution to this problem for the time being.
Disclosure of Invention
In view of the above-mentioned disadvantages, an object of the present invention is to provide a reasonable formulation of a oocyst type biological wall breaking agent composition, which can form a pore passage allowing a cryopreservation material to enter the composition by only slightly damaging the structure of the oocyst wall, thereby effectively prolonging the cryopreservation time.
The second purpose of the invention is to provide a preparation method of the oocyst type biological wall-breaking agent composition.
The invention also aims to provide application of the oocyst type biological wall-breaking agent composition.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
an oocyst type biological wall breaking agent composition comprises 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.
A preparation method of an oocyst type biological wall breaking agent composition 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 a standard PBS buffer solution, sodium dihydrogen phosphate and disodium hydrogen phosphate into the third mixture, and adjusting the pH value to 6.8-7.4 to prepare the oocyst type biological wall breaking agent composition.
As a preferred embodiment of the present invention, the polyethylene glycol is preferably polyethylene glycol 6000(PEG 6000).
The key components such as propylene glycol, pancreatin, polyethylene glycol 6000(PEG6000), sodium citrate and the like can respectively interact with the main components such as keratin, glycoprotein, phospholipid and the like in the oocyst wall structure and destroy the densely arranged rigid structure, so that a certain pore structure is formed.
The proline, the trehalose and other components mainly have the effects of providing nutrient components for the sporangium in the oocyst and buffering the damage of the components such as propylene glycol, protease and the like to the sporangium.
The potassium dichromate is a common disinfectant, and mainly has the main effects of killing bacteria in the oocyst type biological wall breaking agent composition and inhibiting the bacteria from damaging oocysts.
The sodium dihydrogen phosphate, the disodium hydrogen phosphate, the standard PBS buffer solution and other components have the function of adjusting the pH value of the oocyst type biological wall breaking agent composition to be 6.8-7.4 suitable for cryopreservation of oocysts.
The application of the composition of the oocyst type biological wall breaking agent in oocysts comprises the following application steps:
(S1) obtaining the purified oocyst solution, adding the oocyst type biological wall breaking agent composition, stirring or mixing in a vortex mode, and standing;
(S2) finally, observing the state of the oocysts, taking the oocyst suspension which is kept still for different time periods and placing the oocyst suspension under a microscope, observing the morphological change of the oocysts and analyzing the infiltration situation by comparing with a blank group.
The purification step in the 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, preferably three times, and finally obtaining the oocyst solution without disinfectant components.
The invention has the beneficial effects that: the oocyst type biological wall breaking agent composition has a reasonable formula, wherein the propylene glycol, the pancreatin, the polyethylene glycol, the sodium citrate and other key components can interact with the main components of keratin, glycoprotein, phospholipid and the like in the oocyst wall structure and destroy the densely arranged rigid structure of the oocyst wall structure to form a channel for allowing the frozen storage material to pass through, namely, on the basis of slightly damaging the oocyst wall structure, a pore passage for the frozen storage material to pass through is formed to achieve the purpose of increasing the permeability of the oocyst, so that the vitrification capability and the icing inhibition capability in the oocyst are enhanced, the damage of ice crystals is reduced, and the low-temperature long-term storage is realized. The preparation method of the oocyst type biological wall breaking agent composition provided by the invention has simple steps and is easy to realize, and the oocyst type biological wall breaking agent composition which can form a pore passage allowing a cryopreservation material to enter can be quickly prepared only by slightly damaging the structure of the oocyst wall.
The present invention will be further described with reference to the following examples.
Detailed Description
Example 1: this example illustrates the increase in the permeability of Eimeria maxima oocysts, but is not intended to limit the scope of the invention. Such as Eimeria oocysts, Eimeria tenella oocysts, Eimeria acervulina oocysts, Eimeria brunetti oocysts, Eimeria necatrix oocysts, Eimeria praecox oocysts, Eimeria varians oocysts, and Cryptosporidium oocysts, and the like.
Preparing an oocyst type biological wall breaking agent composition: taking 80ml of injection water, putting into a beaker placed in a 4-degree-C water bath, adding a stirrer, and adjusting the rotation speed to 500 rpm; adding pancreatin 0.02g, polyethylene glycol 6000(PEG6000)1.2g and sodium citrate 1.5g, and stirring for 10 min; adding 3.5g of proline, 0.7g of trehalose and 0.8g of potassium dichromate in sequence; adding 25g of propylene glycol, and stirring for 15 minutes; then, 3ml of a standard PBS buffer, 0.3g of sodium dihydrogenphosphate and 0.7g of disodium hydrogenphosphate were added thereto, and the mixture was stirred for 30 minutes.
Secondly, pretreating oocysts, and carrying out the following purification pretreatment before adding the E.maxima oocysts:
1. centrifuging 2ml of Eimeria maxima oocyst solution (3000rpm, 5min) for precipitation;
2. removing supernatant, adding 2ml saturated sodium chloride aqueous solution, and centrifuging (3000rpm, 5 min);
3. taking out supernatant containing Eimeria maxima oocyst liquid, transferring into a centrifuge tube filled with 2ml of injection water, and centrifuging again (3000rpm for 5min) to obtain oocyst precipitation liquid;
4. transferring the oocyst precipitation solution into a centrifuge tube filled with 2ml of injection water, centrifuging (3000rpm for 5min), and repeating the centrifuging step for three times to obtain the coccidia oocyst solution without disinfectant component.
And (3) dividing the treated coccidian oocyst solution into equal parts, respectively adding a freezing tube (group A) containing 3ml of the oocyst type biological wall breaking agent composition and a freezing tube (group B) containing 3ml of isotonic sodium chloride solution, performing vortex stirring for 10, 30 and 60 minutes, taking out, placing under a microscope, observing the morphology of the coccidian oocysts, counting the volume of the coccidian oocysts, and analyzing the permeability change of the coccidian oocysts.
The results show that: the volume of the oocysts of the eimeria maxima is obviously reduced after being soaked by the oocyst type biological wall breaking agent composition, and the volume of the oocysts of the eimeria maxima in the isotonic sodium chloride solution is not obviously changed, which shows that the oocyst type biological wall breaking agent composition can effectively destroy the walls of the oocysts, so that the components in the oocysts can permeate to the outside of the oocyst walls, in other words, the components outside the oocyst walls can also enter the insides of the oocysts.
Referring to Table 1, Table 1 shows the change in volume of E.maxima oocysts over time after soaking in the oocyst type biological wall breaking agent composition (A) and isotonic sodium chloride solution (B). The data in Table 1 is a statistical average of the volume of 50 coccidial oocysts, here taken as the volume of fresh coccidia of 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: the present example is substantially the same as example 1, except that the oocyst type biological wall breaking agent composition comprises the following components by mass percent: 10% of propylene glycol, 0.2% of pancreatin, 5% of polyethylene glycol 6000(PEG6000), 6% of sodium citrate, 3% of proline, 7% of trehalose, 0.5% of potassium dichromate, 0.1% of sodium dihydrogen phosphate, 1% of disodium hydrogen phosphate, 2% of a standard PBS buffer solution and 65.2% of water for injection.
Example 3: the present example is substantially the same as example 1, except that the oocyst type biological wall breaking agent composition comprises the following components by mass percent: 30% of propylene glycol, 0.03% of pancreatin, 2% of polyethylene glycol 6000(PEG6000), 3% of sodium citrate, 5% of proline, 6% of trehalose, 5% of potassium dichromate, 0.5% of sodium dihydrogen phosphate, 0.5% of disodium hydrogen phosphate, 5% of a standard PBS buffer solution and 42.97% of water for injection.
Example 4: the present example is substantially the same as example 1, except that the oocyst type biological wall breaking agent composition comprises the following components by mass percent: 45% of propylene glycol, 0.01% of pancreatin, 0.6% of polyethylene glycol 6000(PEG6000), 0.8% of sodium citrate, 0.5% of proline, 10% of trehalose, 8% of potassium dichromate, 0.8% of sodium dihydrogen phosphate, 1% of disodium hydrogen phosphate, 2% of standard PBS buffer solution and 31.29% of water for injection.
Example 5: the present example is substantially the same as example 1, except that the oocyst type biological wall breaking agent composition comprises the following components by mass percent: 15% of propylene glycol, 0.07% of pancreatin, 4% of polyethylene glycol 6000(PEG6000), 5% of sodium citrate, 5% of proline, 0.5% of trehalose, 5% of potassium dichromate, 0.6% of sodium dihydrogen phosphate, 0.2% of disodium hydrogen phosphate, 4% of standard PBS buffer solution and 60.63% of water for injection.
The above examples are only preferred embodiments of the present invention, and the present invention is not limited to all embodiments, and any technical solution using one of the above examples or equivalent changes made according to the above examples is within the scope of the present invention.
When in use, the key components of propylene glycol, pancreatin, polyethylene glycol, sodium citrate and the like in the oocyst type biological wall breaking agent composition can interact with main components of keratin, glycoprotein, phospholipid and the like in the oocyst wall structure and destroy the densely arranged rigid structure of the proteins, so that a pore passage structure allowing the frozen storage material to pass is formed, and the aim of increasing the permeability of the oocysts on the basis of only slightly damaging the oocyst wall structure is fulfilled, thereby enhancing the vitrification capability and the freezing inhibition capability in the oocysts, reducing the damage of ice crystals and prolonging the low-temperature storage time.
Variations and modifications to the above-described embodiments may occur to those skilled in the art, which fall within the scope and spirit of the above description. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and variations of the present invention should fall within the scope of the claims of the present invention. In addition, although specific terms are used herein, they are used for convenience of description and do not limit the present invention in any way, and other compositions obtained by the same or similar methods, and methods of making and using the same are within the 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 oocyst type biological wall breaking agent composition of claim 3, wherein: the polyethylene glycol is polyethylene glycol 6000.
5. The oocyst type biological wall breaking agent composition of claim 1, wherein: and (3) adding proline, trehalose and potassium dichromate in sequence from front to back.
6. Use of an oocyst type biological wall breaking agent composition of any one of claims 1 to 2 or 3 to 5 in oocysts.
7. The application of the oocyst type biological wall breaking agent composition to oocysts according to 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 any one of claims 1 to 2 or 3 to 5, 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011355601.6A CN112400864B (en) | 2020-11-27 | 2020-11-27 | Oocyst-type biological wall-breaking agent composition and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011355601.6A CN112400864B (en) | 2020-11-27 | 2020-11-27 | Oocyst-type biological wall-breaking agent composition and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112400864A true CN112400864A (en) | 2021-02-26 |
| CN112400864B CN112400864B (en) | 2022-05-03 |
Family
ID=74842759
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011355601.6A Active CN112400864B (en) | 2020-11-27 | 2020-11-27 | Oocyst-type biological wall-breaking agent composition and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112400864B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030143717A1 (en) * | 2001-08-30 | 2003-07-31 | Hutchins James E. | Improved Methods for producing oocysts |
| CN1837349A (en) * | 2006-04-04 | 2006-09-27 | 浙江大学 | Isolation and purification of coccidia oocysts and preparation method of sporozoites |
| CN111670898A (en) * | 2020-07-16 | 2020-09-18 | 松山湖材料实验室 | Coccidia oocyst cryopreservation agent and preparation method and application thereof |
-
2020
- 2020-11-27 CN CN202011355601.6A patent/CN112400864B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030143717A1 (en) * | 2001-08-30 | 2003-07-31 | Hutchins James E. | Improved Methods for producing oocysts |
| CN1837349A (en) * | 2006-04-04 | 2006-09-27 | 浙江大学 | Isolation and purification of coccidia oocysts and preparation method of sporozoites |
| CN111670898A (en) * | 2020-07-16 | 2020-09-18 | 松山湖材料实验室 | Coccidia oocyst cryopreservation agent and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 吴亮: "隐孢子虫虫种鉴定及安氏隐孢子虫体外培养研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 田永清等: "柔嫩艾美耳球虫体外培养方法与影响因素", 《中国动物检疫》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112400864B (en) | 2022-05-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | Cultivation of fish ciliate parasites: Progress and prospects | |
| CN111602648B (en) | A kind of immune cell serum-free cryopreservation solution and cryopreservation method | |
| Citarasu et al. | Isolation of Aeromonas hydrophila from infected ornamental fish hatchery during massive disease outbreak | |
| CN104585164A (en) | Improved diluent for long-term preservation of semen of ruminant | |
| CN107347873B (en) | Eimeria coccidium cryopreservation agent and Eimeria coccidium cryopreservation method | |
| CN104322483A (en) | Boar antiviral long-acting semen diluent formula and preparation method | |
| CN104818241A (en) | Skin tissue cell line of Epinephelus lanceolatus and establishing method thereof | |
| CN110463686A (en) | A kind of cell-preservation liquid that can effectively save cell for a long time | |
| CN112400864A (en) | Oocyst type biological wall breaking agent composition and preparation method and application thereof | |
| Ercan et al. | Reducing bacterial density in the semen of rainbow trout (Oncorhynchus mykiss) by applying gradient centrifugation and swim‐up washing methods | |
| CN112400865A (en) | Wall-breaking agent composition, preparation method thereof and application thereof in oocysts | |
| CN112369410B (en) | Oocyst wall breaking agent composition and preparation method and application thereof | |
| TW202227043A (en) | Antibiotic-free compositions for the prevention or control of coccidiosis | |
| CN116286371B (en) | Freeze-drying protective agent formula for improving freeze-drying effect of strain | |
| KOMIYA et al. | SURVIVAL OF ANCYLOSTOMA CANINUM IN VITRO (I) 1 | |
| CN110585223B (en) | Flavonoid inhibitor for resisting fish viruses and preparation method and application thereof | |
| CN114667998A (en) | A kind of sheep semen cryopreservation antioxidant diluent and preparation method and application thereof | |
| CN110711190B (en) | A kind of terpene inhibitor against fish disease and its preparation method and application | |
| RU2303631C1 (en) | Method for production of mesenchymal stem cells and biotransplant using the same | |
| CN108124853A (en) | A kind of store method of mescenchymal stem cell | |
| RU2323702C2 (en) | Synthetic medium for ram sperm dilution and cryopreservation | |
| CN117084231A (en) | Optimized formula and preparation and cryopreservation method of a cell cryopreservation solution | |
| CN114984232B (en) | Vaccine heat-resistant freeze-drying protective agent and preparation method and application thereof | |
| US20240057609A1 (en) | Biological Control Method For Ciliate Disease | |
| CN110367240A (en) | A kind of sheep sperm cryo-conservation dilution and method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |