CN111117976B - Method for inactivating vesicular stomatitis virus - Google Patents
Method for inactivating vesicular stomatitis virus Download PDFInfo
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- CN111117976B CN111117976B CN201911335164.9A CN201911335164A CN111117976B CN 111117976 B CN111117976 B CN 111117976B CN 201911335164 A CN201911335164 A CN 201911335164A CN 111117976 B CN111117976 B CN 111117976B
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- hemin
- vesicular stomatitis
- stomatitis virus
- inactivating
- alkali
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- 241000711975 Vesicular stomatitis virus Species 0.000 title claims abstract description 19
- 230000000415 inactivating effect Effects 0.000 title claims abstract description 16
- 238000000034 method Methods 0.000 title claims abstract description 15
- BTIJJDXEELBZFS-QDUVMHSLSA-K hemin Chemical compound CC1=C(CCC(O)=O)C(C=C2C(CCC(O)=O)=C(C)\C(N2[Fe](Cl)N23)=C\4)=N\C1=C/C2=C(C)C(C=C)=C3\C=C/1C(C)=C(C=C)C/4=N\1 BTIJJDXEELBZFS-QDUVMHSLSA-K 0.000 claims abstract description 26
- 229940025294 hemin Drugs 0.000 claims abstract description 26
- 239000003513 alkali Substances 0.000 claims abstract description 14
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- 230000002779 inactivation Effects 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 4
- 231100000956 nontoxicity Toxicity 0.000 abstract description 4
- 239000002904 solvent Substances 0.000 abstract description 3
- 210000003501 vero cell Anatomy 0.000 description 13
- 241000700605 Viruses Species 0.000 description 10
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 238000001690 micro-dialysis Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- BMUDPLZKKRQECS-UHFFFAOYSA-K 3-[18-(2-carboxyethyl)-8,13-bis(ethenyl)-3,7,12,17-tetramethylporphyrin-21,24-diid-2-yl]propanoic acid iron(3+) hydroxide Chemical compound [OH-].[Fe+3].[N-]1C2=C(C)C(CCC(O)=O)=C1C=C([N-]1)C(CCC(O)=O)=C(C)C1=CC(C(C)=C1C=C)=NC1=CC(C(C)=C1C=C)=NC1=C2 BMUDPLZKKRQECS-UHFFFAOYSA-K 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 150000003278 haem Chemical class 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229940109738 hematin Drugs 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/20011—Rhabdoviridae
- C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
- C12N2760/20261—Methods of inactivation or attenuation
- C12N2760/20263—Methods of inactivation or attenuation by chemical treatment
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Virology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to a method for inactivating vesicular stomatitis virus, comprising contacting hemin with vesicular stomatitis virus. The invention also relates to a reagent for inactivating vesicular stomatitis virus, which comprises alkali, hemin and a solvent, wherein the mass percentage concentration of the alkali is 0.01-0.1%, and the mass percentage concentration of the hemin is 0.001-0.1%. The reagent has no toxicity to cells, and can well inactivate vesicular stomatitis virus.
Description
Technical Field
The invention belongs to the field of virus inactivation in the biomedical industry, and particularly relates to a method for inactivating vesicular stomatitis virus.
Background
The virus inactivating method in the existing biochemical medicines mainly comprises a low pH method, an ethanol treatment method, a heating method and the like. The existing chemical method is used for inactivating viruses and simultaneously denaturing and inactivating proteins in a sample to a certain degree, so that the yield of nucleic acid or protein in the sample is possibly low.
Disclosure of Invention
It is an object of the present invention to provide a method of inactivating vesicular stomatitis virus which is not toxic to cells.
In order to solve the technical problems, the invention adopts the following technical scheme:
in one aspect of the invention, a method is provided for inactivating vesicular stomatitis virus by contacting hemin with the vesicular stomatitis virus.
Preferably, the hemin is obtained by alkali treatment of hemin.
More preferably, the alkali is a sodium hydroxide solution with a feeding mass percentage concentration of 0.01-0.1%.
More preferably, the charging mass percentage concentration of the hemin in the alkali solution is 0.001-0.1%.
Preferably, the inactivation is controlled for 30min and above.
In a second aspect, the invention provides the use of hematin for inactivating vesicular stomatitis virus in a biological product.
The third aspect of the invention provides a reagent for inactivating vesicular stomatitis virus, which comprises alkali, hemin and a solvent, wherein the mass percentage concentration of the alkali is 0.01-0.1%, and the mass percentage concentration of the hemin is 0.001-0.1%.
Preferably, the base is sodium hydroxide and the solvent is water.
In a fourth aspect of the invention, there is provided a method of inactivating vesicular stomatitis virus by contacting a product comprising vesicular stomatitis virus with an agent as described above.
Preferably, the contact time is controlled to 30min or more.
In the invention, the contact can be carried out by soaking, spraying and the like.
The present invention utilizes hemin dissolved in dilute alkali solution to form hemin, which has no toxicity to cells and has the function of inactivating vesicular stomatitis virus. In addition, as hemin is a heme crystal purified from animal tissues and animal blood, can be used as a raw material of medicines and can be used for preparing anticancer specific medicines, and has no toxic or side effect on bodies, so that after virus inactivation is completed by using the hemin, the hemin does not need to be removed by a physical or chemical method.
Due to the implementation of the technical scheme, compared with the prior art, the invention has the following advantages:
the reagent has no toxicity to cells, and can well inactivate vesicular stomatitis virus.
Drawings
FIG. 1 is a micrograph of negative Vero cells;
FIG. 2 is a microscopic image of Vero cells after solution treatment of example 1;
FIG. 3 is a microscopic image of Vero cells after solution treatment of example 2;
FIG. 4 is a graph showing the trend of the virus titer of example 1 with time;
FIG. 5 is a graph showing the trend of the virus titer of example 2 with time.
Detailed Description
The present invention will be described in further detail with reference to specific examples. It is to be understood that these embodiments are provided to illustrate the basic principles, essential features and advantages of the present invention, and the present invention is not limited by the following embodiments. The implementation conditions used in the examples can be further adjusted according to specific requirements, and the implementation conditions not indicated are generally the conditions in routine experiments.
Example 1
Preparing 0.04% NaOH-0.003% hemin solution: hemin is dissolved in aqueous NaOH.
Taking 100 mu L of prepared solution, inoculating the prepared solution into Vero cells, incubating the Vero cells in a 5% carbon dioxide incubator at 37 ℃ for 72h, observing the Vero cells by using a microscope, and obtaining a result shown in figure 2, wherein the Vero cells are normal in shape, and the solution of the embodiment is nontoxic to the Vero cells.
Adding VSV into the solution at a mass ratio of 50:1, standing at room temperature for 60min, and determining virus titer from 6.62lgTCID by microdialysis50Down to 3.25lgTCID50. The trend of the virus titer decrease at 60min is shown in FIG. 4.
Example 2
Preparing 0.04% NaOH-0.03% hemin solution: hemin is dissolved in aqueous NaOH.
Taking 100 mu L of prepared solution, inoculating the solution into Vero cells, incubating the Vero cells in a 5% carbon dioxide incubator at 37 ℃ for 72h, observing the Vero cells by using a microscope, wherein the result is shown in figure 3, the Vero cells are normal in shape, and the solution of the embodiment has no toxicity to the Vero cells.
Adding VSV into the solution at a mass ratio of 50:1, standing at room temperature for 60min, and determining virus titer from 6.62lgTCID by microdialysis50Down to 2.38lgTCID50. The trend of the virus titer decrease at 60min is shown in FIG. 5.
The above embodiments are merely illustrative of the technical concept and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the content of the present invention and implement the invention, and not to limit the scope of the invention, and all equivalent changes or modifications made according to the spirit of the present invention should be covered by the scope of the present invention.
Claims (2)
1. A method of inactivating vesicular stomatitis virus, comprising: the method comprises the steps of contacting hemin with vesicular stomatitis virus, wherein the hemin is obtained by treating hemin with alkali, the alkali is a sodium hydroxide solution with a feeding mass percentage concentration of 0.04%, the feeding mass percentage concentration of the hemin in the alkali solution is 0.003-0.3%, and the inactivation time is controlled to be 30min or more.
2. The application of the hemin in inactivating vesicular stomatitis virus in biological products is characterized in that: the hemin is obtained by treating hemin with alkali, wherein the alkali is a sodium hydroxide solution with a feeding mass percentage concentration of 0.04%, the feeding mass percentage concentration of the hemin in the alkali solution is 0.003-0.3%, and the inactivation time is controlled to be 30min or more.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911335164.9A CN111117976B (en) | 2019-12-23 | 2019-12-23 | Method for inactivating vesicular stomatitis virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911335164.9A CN111117976B (en) | 2019-12-23 | 2019-12-23 | Method for inactivating vesicular stomatitis virus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111117976A CN111117976A (en) | 2020-05-08 |
| CN111117976B true CN111117976B (en) | 2022-01-11 |
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|---|---|---|---|
| CN201911335164.9A Active CN111117976B (en) | 2019-12-23 | 2019-12-23 | Method for inactivating vesicular stomatitis virus |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101392457A (en) * | 2008-09-24 | 2009-03-25 | 东华大学 | Fibrilia containing hemin and preparation method thereof |
| CN103285016A (en) * | 2013-05-23 | 2013-09-11 | 中山大学 | Application of chlorhematin in preparing drug for resisting porcine reproductive and respiratory syndrome virus |
| CN104267193A (en) * | 2014-09-15 | 2015-01-07 | 中国农业科学院兰州兽医研究所 | Primer and kit for detecting vesicular stomatitis viruses and preparation method of kit |
| CN107821739A (en) * | 2017-11-22 | 2018-03-23 | 河南邑鸿善成生物技术有限公司 | The application of composite feed additive, the preparation method of antivirotic and antivirotic |
| CN110302192A (en) * | 2018-06-22 | 2019-10-08 | 华东师范大学 | New antimicrobial compositions and its preparation method and application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6627426B2 (en) * | 1997-02-14 | 2003-09-30 | Invitrogen Corporation | Methods for reducing adventitious agents and toxins and cell culture reagents produced thereby |
-
2019
- 2019-12-23 CN CN201911335164.9A patent/CN111117976B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101392457A (en) * | 2008-09-24 | 2009-03-25 | 东华大学 | Fibrilia containing hemin and preparation method thereof |
| CN103285016A (en) * | 2013-05-23 | 2013-09-11 | 中山大学 | Application of chlorhematin in preparing drug for resisting porcine reproductive and respiratory syndrome virus |
| CN104267193A (en) * | 2014-09-15 | 2015-01-07 | 中国农业科学院兰州兽医研究所 | Primer and kit for detecting vesicular stomatitis viruses and preparation method of kit |
| CN107821739A (en) * | 2017-11-22 | 2018-03-23 | 河南邑鸿善成生物技术有限公司 | The application of composite feed additive, the preparation method of antivirotic and antivirotic |
| CN110302192A (en) * | 2018-06-22 | 2019-10-08 | 华东师范大学 | New antimicrobial compositions and its preparation method and application |
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| CN111117976A (en) | 2020-05-08 |
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Application publication date: 20200508 Assignee: Jiangsu Jinmao Finance Leasing Co.,Ltd. Assignor: LIANGCHEN BIO (SUZHOU) Corp. Contract record no.: X2023320010004 Denomination of invention: A method for inactivating vesicular stomatitis virus Granted publication date: 20220111 License type: Exclusive License Record date: 20230106 |
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| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A method for inactivating vesicular stomatitis virus Effective date of registration: 20230109 Granted publication date: 20220111 Pledgee: Jiangsu Jinmao Finance Leasing Co.,Ltd. Pledgor: LIANGCHEN BIO (SUZHOU) Corp. Registration number: Y2023320010019 |
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| EC01 | Cancellation of recordation of patent licensing contract | ||
| EC01 | Cancellation of recordation of patent licensing contract |
Assignee: Jiangsu Jinmao Finance Leasing Co.,Ltd. Assignor: LIANGCHEN BIO (SUZHOU) Corp. Contract record no.: X2023320010004 Date of cancellation: 20240203 |
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| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Granted publication date: 20220111 Pledgee: Jiangsu Jinmao Finance Leasing Co.,Ltd. Pledgor: LIANGCHEN BIO (SUZHOU) Corp. Registration number: Y2023320010019 |