CN112244025A - Sterile treatment agent for macromolecular protein and/or nucleic acid and preparation method and application thereof - Google Patents
Sterile treatment agent for macromolecular protein and/or nucleic acid and preparation method and application thereof Download PDFInfo
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- CN112244025A CN112244025A CN202011359365.5A CN202011359365A CN112244025A CN 112244025 A CN112244025 A CN 112244025A CN 202011359365 A CN202011359365 A CN 202011359365A CN 112244025 A CN112244025 A CN 112244025A
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- 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
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/48—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with two nitrogen atoms as the only ring hetero atoms
- A01N43/50—1,3-Diazoles; Hydrogenated 1,3-diazoles
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- 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
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
- A01N37/46—N-acyl derivatives
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Abstract
The invention is applicable to the technical field of disinfectants, and provides a sterile treatment agent for macromolecular proteins and/or nucleic acids, a preparation method and application thereof, wherein the sterile treatment agent comprises the following components in parts by weight: 0.2-1.5 parts of 5, 5-dimethylhydantoin, 0.2-1.5 parts of tween-800.2, 50.5-5 parts of human alpha defensin and 3-10 parts of buffer solution. The sterile treatment agent can thoroughly kill bacteria in macromolecular protein and/or nucleic acid solution, has no damage to protein and nucleic acid, and does not increase heat source substances in the solution while killing bacteria.
Description
Technical Field
The invention belongs to the technical field of disinfectants, and particularly relates to a sterile treatment agent for macromolecular proteins and/or nucleic acids, and a preparation method and application thereof.
Background
Aseptic processing of proteins and nucleic acids has been a difficult problem to solve. Such substances are not resistant to heat, and sterilization methods at high temperatures and pressures cause irreversible damage to the substance itself. Pasteurization is a tremendous advance in the sterilization of milk, but is only used for food products and does not have a long shelf life and meet medical requirements. Sterile filtration is also a common method, but for large molecules it is difficult to pass through the pores efficiently, resulting in significant loss of components and clogging.
An increasing number of macromolecular proteins and nucleic acids are being developed, and if a treatment agent that can be used for aseptic processing can be developed, it will be necessary to expand their application range, and it will also have great economic benefits.
Disclosure of Invention
An object of an embodiment of the present invention is to provide a sterile treatment agent for macromolecular proteins and/or nucleic acids, which is intended to solve the problems set forth in the background art.
The embodiment of the invention is realized by that the sterile treatment agent for the macromolecular protein and/or the nucleic acid comprises the following components in parts by weight: 0.2-1.5 parts of 5, 5-dimethylhydantoin, 0.2-1.5 parts of tween-800.2, 50.5-5 parts of human alpha defensin and 3-10 parts of buffer solution.
As a preferable scheme of the embodiment of the invention, the sterile treatment agent comprises the following components in parts by weight: 0.5-1 part of 5, 5-dimethylhydantoin, 0.5-1 part of tween-800.5, 51-3 parts of human alpha defensin and 5-8.5 parts of buffer solution.
As another preferable scheme of the embodiment of the invention, the sterile treatment agent comprises the following components in parts by weight: 0.7-0.8 part of 5, 5-dimethylhydantoin, 0.8 part of tween-800.7, 51.5-2.5 parts of human alpha defensin and 6.5-7 parts of buffer solution.
As another preferable aspect of the embodiment of the present invention, the buffer solution is a phosphate buffer solution.
Another object of the embodiments of the present invention is to provide a method for preparing the sterile treatment agent for macromolecular proteins and/or nucleic acids, which comprises the following steps:
weighing 5, 5-dimethylhydantoin, tween-80, human alpha defensin 5 and buffer solution according to the weight parts of the components;
and sequentially adding 5, 5-dimethylhydantoin, tween-80 and human alpha defensin 5 into the buffer solution, stirring and mixing to obtain the sterile treatment agent.
In another preferable embodiment of the present invention, in the step, the rotation speed of stirring and mixing is 500 to 1500 rpm.
Another object of the embodiments of the present invention is to provide an aseptic processing agent prepared by the above preparation method.
Another object of the embodiments of the present invention is to provide a use of the above-mentioned aseptic processing agent in aseptic processing of macromolecular proteins and/or nucleic acids.
It should be noted that the sterile treatment agent can be used for sterile treatment of a substance which is difficult to filter and cannot be sterilized at high temperature and high pressure, and is not limited to macromolecular proteins and nucleic acids.
In another preferred embodiment of the present invention, the mass ratio of the sterile treatment agent to the macromolecular protein and/or nucleic acid solution during sterile treatment is 1 (100-500).
In the invention, the 5, 5-dimethylhydantoin is a novel bactericide which has strong bactericidal effect and is widely applied to various aspects such as daily chemical industry (such as textile printing and dyeing, cosmetics), agriculture (plant and flower, seed disinfection and fruit preservation), animal husbandry, aquaculture, medical and sanitary disinfection, diet, drinking water, entertainment places, swimming pool disinfection, circulating cooling systems (algae killing) and the like. 5, 5-dimethylhydantoin has the effect of killing bacteria propagules, spores, viruses, fungi and algae, but when bacteria are killed, a large amount of endotoxin can be released, the endotoxin is also called a heat source, and the cascade reaction caused by the endotoxin causes organism sepsis, septic shock and systemic inflammatory response syndrome. Endotoxin contamination is a problem that is difficult to solve in the preparation process of protein biological products such as vaccines, antibody drugs and the like.
Human alpha defensin 5(HD5) is a cationic antimicrobial peptide, has broad-spectrum antimicrobial activity, and is not easy to generate drug resistance. But also can neutralize bacterial endotoxin, reduce the heat source in the sample and increase the safety in application. HD5 can cooperate with 5, 5-dimethylhydantoin to increase the ability to kill bacteria without increasing the heat source. However, HD5 is easily hydrolyzed in a solution, and the bactericidal activity is easily affected by salt ions and anionic substances. Tween-80 is a fusion agent of the system, and can form a uniform and stable solution of each component. Can make the sterilization effect lasting and stable. Meanwhile, the reagent has no irritation to the organism, so that the reagent is safer in use.
The sterile treatment agent for macromolecular protein and/or nucleic acid provided by the embodiment of the invention can thoroughly kill bacteria in a macromolecular protein/nucleic acid solution, does not damage protein and nucleic acid, does not increase heat source substances in the solution while killing the bacteria, and improves the use safety.
Detailed Description
In order to clearly and completely describe the technical solutions in the embodiments of the present invention in combination with the embodiments of the present invention, it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
This embodiment provides a sterile treatment agent for macromolecular proteins and/or nucleic acids, which is prepared by a method comprising the steps of:
s1, accurately weighing 0.2g of 5, 5-dimethylhydantoin, 800.2 g of Tween-5, 50.5 g of human alpha defensin and 10g of phosphate buffer solution for later use. The pH of the phosphate buffer solution was 7.2, which was obtained by dissolving 1.546g of disodium hydrogen phosphate, 0.262g of sodium dihydrogen phosphate, and 8.768g of sodium chloride in water and diluting to 1000 mL.
S2, sequentially adding 5, 5-dimethylhydantoin, Tween-80 and human alpha defensin 5 into the weighed phosphate buffer solution at room temperature, and stirring and mixing for 30min at the speed of 500 rpm by using a magnetic stirrer to obtain the uniform and stable sterile treatment agent.
Example 2
This embodiment provides a sterile treatment agent for macromolecular proteins and/or nucleic acids, which is prepared by a method comprising the steps of:
s1, accurately weighing 1.5g of 5, 5-dimethylhydantoin, 801.5 g of Tween-5, 55 g of human alpha defensin and 3g of phosphate buffer solution for later use. The pH of the phosphate buffer solution was 7.2, which was obtained by dissolving 1.546g of disodium hydrogen phosphate, 0.262g of sodium dihydrogen phosphate, and 8.768g of sodium chloride in water and diluting to 1000 mL.
S2, sequentially adding 5, 5-dimethylhydantoin, Tween-80 and human alpha defensin 5 into the weighed phosphate buffer solution at room temperature, and stirring and mixing for 30min at 1500 rpm by using a magnetic stirrer to obtain the uniform and stable sterile treatment agent.
Example 3
This embodiment provides a sterile treatment agent for macromolecular proteins and/or nucleic acids, which is prepared by a method comprising the steps of:
s1, accurately weighing 0.3g of 5, 5-dimethylhydantoin, 800.3 g of Tween-5, 54 g of human alpha defensin and 9g of phosphate buffer solution for later use. The pH of the phosphate buffer solution was 7.2, which was obtained by dissolving 1.546g of disodium hydrogen phosphate, 0.262g of sodium dihydrogen phosphate, and 8.768g of sodium chloride in water and diluting to 1000 mL.
S2, sequentially adding 5, 5-dimethylhydantoin, Tween-80 and human alpha defensin 5 into the weighed phosphate buffer solution at room temperature, and stirring and mixing for 30min at 800 rpm by using a magnetic stirrer to obtain the uniform and stable sterile treatment agent.
Example 4
This embodiment provides a sterile treatment agent for macromolecular proteins and/or nucleic acids, which is prepared by a method comprising the steps of:
s1, accurately weighing 1.4g of 5, 5-dimethylhydantoin, 801.3 g of Tween-5, 50.8 g of human alpha defensin and 4g of phosphate buffer solution for later use. The pH of the phosphate buffer solution was 7.2, which was obtained by dissolving 1.546g of disodium hydrogen phosphate, 0.262g of sodium dihydrogen phosphate, and 8.768g of sodium chloride in water and diluting to 1000 mL.
S2, sequentially adding 5, 5-dimethylhydantoin, Tween-80 and human alpha defensin 5 into the weighed phosphate buffer solution at room temperature, and stirring and mixing for 30min at 1200 rpm by using a magnetic stirrer to obtain the uniform and stable sterile treatment agent.
Example 5
This embodiment provides a sterile treatment agent for macromolecular proteins and/or nucleic acids, which is prepared by a method comprising the steps of:
s1, accurately weighing 0.5g of 5, 5-dimethylhydantoin, 800.5 g of Tween-5, 51 g of human alpha defensin and 8.5g of phosphate buffer solution for later use. The pH of the phosphate buffer solution was 7.2, which was obtained by dissolving 1.546g of disodium hydrogen phosphate, 0.262g of sodium dihydrogen phosphate, and 8.768g of sodium chloride in water and diluting to 1000 mL.
S2, sequentially adding 5, 5-dimethylhydantoin, Tween-80 and human alpha defensin 5 into the weighed phosphate buffer solution at room temperature, and stirring and mixing for 30min at the speed of 1000 rpm by using a magnetic stirrer to obtain the uniform and stable sterile treatment agent.
Example 6
This embodiment provides a sterile treatment agent for macromolecular proteins and/or nucleic acids, which is prepared by a method comprising the steps of:
s1, accurately weighing 1g of 5, 5-dimethylhydantoin, 801 g of Tween-801 g, 53 g of human alpha defensin and 5g of phosphate buffer solution for later use. The pH of the phosphate buffer solution was 7.2, which was obtained by dissolving 1.546g of disodium hydrogen phosphate, 0.262g of sodium dihydrogen phosphate, and 8.768g of sodium chloride in water and diluting to 1000 mL.
S2, sequentially adding 5, 5-dimethylhydantoin, Tween-80 and human alpha defensin 5 into the weighed phosphate buffer solution at room temperature, and stirring and mixing for 30min at the speed of 1000 rpm by using a magnetic stirrer to obtain the uniform and stable sterile treatment agent.
Example 7
This embodiment provides a sterile treatment agent for macromolecular proteins and/or nucleic acids, which is prepared by a method comprising the steps of:
s1, accurately weighing 0.7g of 5, 5-dimethylhydantoin, 800.7 g of Tween-5, 51.5 g of human alpha defensin and 7g of phosphate buffer solution for later use. The pH of the phosphate buffer solution was 7.2, which was obtained by dissolving 1.546g of disodium hydrogen phosphate, 0.262g of sodium dihydrogen phosphate, and 8.768g of sodium chloride in water and diluting to 1000 mL.
S2, sequentially adding 5, 5-dimethylhydantoin, Tween-80 and human alpha defensin 5 into the weighed phosphate buffer solution at room temperature, and stirring and mixing for 30min at the speed of 1000 rpm by using a magnetic stirrer to obtain the uniform and stable sterile treatment agent.
Example 8
This embodiment provides a sterile treatment agent for macromolecular proteins and/or nucleic acids, which is prepared by a method comprising the steps of:
s1, accurately weighing 0.8g of 5, 5-dimethylhydantoin, 800.8 g of Tween-5, 52.5 g of human alpha defensin and 6.5g of phosphate buffer solution for later use. The pH of the phosphate buffer solution was 7.2, which was obtained by dissolving 1.546g of disodium hydrogen phosphate, 0.262g of sodium dihydrogen phosphate, and 8.768g of sodium chloride in water and diluting to 1000 mL.
S2, sequentially adding 5, 5-dimethylhydantoin, Tween-80 and human alpha defensin 5 into the weighed phosphate buffer solution at room temperature, and stirring and mixing for 30min at the speed of 1000 rpm by using a magnetic stirrer to obtain the uniform and stable sterile treatment agent.
Example 9
This embodiment provides a sterile treatment agent for macromolecular proteins and/or nucleic acids, which is prepared by a method comprising the steps of:
s1, accurately weighing 0.5g of 5, 5-dimethylhydantoin, 800.5 g of Tween, 51 g of human alpha defensin and 8g of phosphate buffer solution for later use. The pH of the phosphate buffer solution was 7.2, which was obtained by dissolving 1.546g of disodium hydrogen phosphate, 0.262g of sodium dihydrogen phosphate, and 8.768g of sodium chloride in water and diluting to 1000 mL.
S2, sequentially adding 5, 5-dimethylhydantoin, Tween-80 and human alpha defensin 5 into the weighed phosphate buffer solution at room temperature, and stirring and mixing for 30min at the speed of 1000 rpm by using a magnetic stirrer to obtain the uniform and stable sterile treatment agent.
Example 10
This embodiment provides a sterile treatment agent for macromolecular proteins and/or nucleic acids, which is prepared by a method comprising the steps of:
s1, accurately weighing 1g of 5, 5-dimethylhydantoin, 801 g of Tween-801 g, 53 g of human alpha defensin and 5g of phosphate buffer solution for later use. The pH of the phosphate buffer solution was 7.2, which was obtained by dissolving 1.546g of disodium hydrogen phosphate, 0.262g of sodium dihydrogen phosphate, and 8.768g of sodium chloride in water and diluting to 1000 mL.
S2, sequentially adding 5, 5-dimethylhydantoin, Tween-80 and human alpha defensin 5 into the weighed phosphate buffer solution at room temperature, and stirring and mixing for 30min at the speed of 1000 rpm by using a magnetic stirrer to obtain the uniform and stable sterile treatment agent.
Example 11
This embodiment provides a sterile treatment agent for macromolecular proteins and/or nucleic acids, which is prepared by a method comprising the steps of:
s1, accurately weighing 0.5g of 5, 5-dimethylhydantoin, 801 g of Tween-801 g, 53 g of human alpha defensin and 5.5g of phosphate buffer solution for later use. The pH of the phosphate buffer solution was 7.2, which was obtained by dissolving 1.546g of disodium hydrogen phosphate, 0.262g of sodium dihydrogen phosphate, and 8.768g of sodium chloride in water and diluting to 1000 mL.
S2, sequentially adding 5, 5-dimethylhydantoin, Tween-80 and human alpha defensin 5 into the weighed phosphate buffer solution at room temperature, and stirring and mixing for 30min at the speed of 1000 rpm by using a magnetic stirrer to obtain the uniform and stable sterile treatment agent.
Example 12
This embodiment provides an aseptic processing method using the above aseptic processing agent, comprising the steps of:
the sterile treatment agents prepared in the above examples 9-11 are respectively added into a nucleic acid solution containing bacteria according to the mass ratio of 1:100 and 1:500, after the action for 1 hour, the sterile treatment agents are respectively transplanted into a culture medium, and a positive control and a negative control are additionally arranged. After 7 days of observation, the color of the added reagent group and the negative control is not changed (aseptic growth), and the color of the positive control is changed; then all the inoculation tubes are transplanted into a new culture medium again, after 7 days of observation, the color of the added reagent group and the negative control is not changed (aseptic growth), and the color of the positive control is changed. It can be seen that the sterile treatment agents prepared in the above examples 9 to 11 can completely kill bacteria. Specific results are shown in tables 1 and 2.
TABLE 1 Sterilization Effect of the reagents
Note: in Table 1, "-" represents no turbidity and aseptic growth; "+" indicates the appearance of turbidity with bacterial growth.
TABLE 2 bacterial growth after reimplantation inoculation
Note: in Table 2, "-" represents no turbidity and aseptic growth; "+" indicates the appearance of turbidity with bacterial growth.
Example 13
This example explores the effect of the above-described sterile treatment agents on nucleic acids, comprising the steps of:
the sterile treatment agent prepared in the above examples 9-11 is added into the polyinosinic solution according to the ratio of 1:100 and 1:500 respectively, and after 48 hours of action, relevant parameters are detected by an ultraviolet spectrophotometer. Results no significant difference was observed between the sterile treatment group and the no-addition group. It can be seen that the sterile treatment agents prepared in the above examples 9 to 11 have no influence on the nucleic acid structure, and the specific results are shown in Table 3.
TABLE 3 Effect of the respective Agents on polyinosinic cells
Example 14
This example explores the effect of the above-described sterile treatment on macromolecular proteins, comprising the steps of:
the sterile treatment agents prepared in the above examples 9-11 are added to an expressed macromolecular protein antigen (avian influenza) according to the ratio of the maximum concentration of 1:100, and after the action for 48 hours, vaccines are prepared to immunize SP F (specific pathogen free) chickens, wherein each group comprises 5 animals, and untreated protein antigens are used as a control immune group. Blood was collected 21 days after immunization to detect antibodies. Results no significant difference was observed between the sterile treatment group and the no-addition group. It can be seen that the sterile treatment agents prepared in the above examples 9 to 11 have no influence on the structure of the macromolecular protein, and the specific results are shown in table 4.
TABLE 4 Effect of the reagents on macromolecular proteins
In conclusion, the sterile treatment agent provided by the embodiment of the invention can thoroughly kill bacteria in a macromolecular protein/nucleic acid solution, does not damage protein and nucleic acid, does not increase heat source substances in the solution while killing the bacteria, and improves the use safety.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (9)
1. An aseptic treating agent for macromolecular proteins and/or nucleic acids is characterized by comprising the following components in parts by weight: 0.2-1.5 parts of 5, 5-dimethylhydantoin, 0.2-1.5 parts of tween-800.2, 50.5-5 parts of human alpha defensin and 3-10 parts of buffer solution.
2. The aseptic processing agent for macromolecular proteins and/or nucleic acids according to claim 1, characterized by comprising the following components in parts by weight: 0.5-1 part of 5, 5-dimethylhydantoin, 0.5-1 part of tween-800.5, 51-3 parts of human alpha defensin and 5-8.5 parts of buffer solution.
3. The aseptic processing agent for macromolecular proteins and/or nucleic acids according to claim 1, characterized by comprising the following components in parts by weight: 0.7-0.8 part of 5, 5-dimethylhydantoin, 0.8 part of tween-800.7, 51.5-2.5 parts of human alpha defensin and 6.5-7 parts of buffer solution.
4. A sterile treatment agent for macromolecular proteins and/or nucleic acids according to any one of claims 1 to 3, characterized in that the buffer solution is a phosphate buffer solution.
5. A method for preparing a sterile treatment agent for macromolecular proteins and/or nucleic acids according to any one of claims 1 to 4, comprising the steps of:
weighing 5, 5-dimethylhydantoin, tween-80, human alpha defensin 5 and buffer solution according to the weight parts of the components;
and sequentially adding 5, 5-dimethylhydantoin, tween-80 and human alpha defensin 5 into the buffer solution, stirring and mixing to obtain the sterile treatment agent.
6. A process for preparing an aseptic processing agent as defined in claim 5, wherein the rotation speed of stirring and mixing is 500-1500 rpm.
7. An aseptic treatment agent obtained by the production method as described in claim 5 or 6.
8. Use of the sterile treatment agent according to any one of claims 1 to 4 or 7 for the sterile treatment of macromolecular proteins and/or nucleic acids.
9. The use of an aseptic processing agent in the aseptic processing of macromolecular proteins and/or nucleic acids according to claim 8, wherein the mass ratio of the aseptic processing agent to the macromolecular protein and/or nucleic acid solution during the aseptic processing is 1 (100-500).
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Application publication date: 20210122 |